Gene silencing

Description

FIELD OF THE INVENTION

The present invention relates to unique constructs for producing a nucleic acid product that downregulates or prevents expression of a desired target polynucleotide.

State of the art

[0002] Suppression of gene expression can be accomplished by constructs that effect post-transcriptional or transcriptional gene silencing. These silencing mechanisms can down-regulate the expression of a desired polynucleotide or gene by chromatin modification, RNA attachment, translation repression, or by previously unknown mechanisms, see Meister G. and Tuschl T., Nature, vol. 431, pp. 343-349, 2004. .

[0003] A construct typically used in the art comprises a desired polynucleotide that has sequence identity with at least a portion of a target gene that is operably linked to a promoter and terminator. As is known, the promoter initiates transcription and the terminator terminates transcription at a specific site and then mediates polyadenylation. Such transcript processing is essential for the stability of the transcript and its transport from the nucleus and in the cytoplasm.

[0004] Accordingly, the terminator plays an important role in conventional gene silencing constructs. For example, WO 99/53050 describes a construct comprising a promoter, a polynucleotide containing a first sequence with homology to the target gene, a second sequence which is inversely complementary to the target gene, and a terminator. The terminator of a conventional construct need not be placed immediately downstream of the desired polynucleotide. For example, Mette et al. Described a plasmid containing a desired polynucleotide that is separated from a terminator operably linked by the hygromycin gene (Mette et aL, EMBO J 18: 241-8,1999; Mette et aL, EMBO J 19: 5194-201, 2000) .

[0005] Other conventional constructs designed to silence genes between a promoter and a terminator contain the polynucleotide in a sense or antisense orientation. Such a classical gene silencing construct typically produces RNA transcripts that are of similar size, determined by the distance from the transcription initiation site to the terminal cleavage and the polyadenylated tail

The present invention relates to novel gene silencing strategies and constructs that are generally more effective than conventional constructs. In addition, the present invention relates to novel gene silencing strategies and constructs using a polynucleotide that is not operably linked to a promoter and terminator but is operably linked to two convergent oriented promoters.

Summary of the invention

[0007] Strategies and constructs may be characterized by certain functions. The construct, for example, can contain a region of DNA, such as a terminator, which is involved in 3'-terminus formation and polyadenylation. Alternatively, the construct may contain a non-functional terminator that is naturally non-functional or that has been modified or mutated to become non-functional.

[0008] The construct may also form a promoter system on both sides of the desired polynucleotide. Accordingly, the construct may contain two or more promoters that flank one or more of the desired polynucleotides or that flank copies of the desired polynucleotide such that both strands of the desired polynucleotide are transcribed. That is, one promoter may be oriented to initiate transcription from the 5 'end of the desired polynucleotide, while the other promoter may be functionally oriented to initiate transcription from the 3' end of the same desired polynucleotide. The oppositely oriented promoters can flank multiple copies of the desired polynucleotide. Since the number of copies can vary, the construct can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more than 100 copies, or any intermediate integer copies, of a desired polynucleotide, flanked by promoters that are oriented to effect convergent transcription.

[0009] Alternatively, the first promoter may be operably linked to a first polynucleotide in case A, for example, and a second promoter may be operably linked to a second polynucleotide, e.g., in case B. The polynucleotides of each cassette may or may not include one. nucleotide sequence itself, but may also share a certain percentage of sequence identity with the target nucleic acid sequence of interest. Cassettes can be arranged in tandem, i.e. so that they are adjacent to each other in the structure. Moreover, cassette B, for example, may be directed in the reverse direction complementary to cassette A. Therefore, in this arrangement, transcription from the cassette B promoter will continue towards the promoter of cassette A. Thus, these cassettes are arranged in to induce convergent transcription.

[0010] If no cassette contains a terminator sequence, such a construct, due to the convergent transcription pattern, can produce RNA transcripts that are of different lengths.

[0011] In this situation, therefore, there may be subpopulations of partially or fully transcribed RNA transcripts that contain partial or full-length sequences of the transcribed desired polynucleotide from the appropriate cassette. Alternatively, in the absence of a functional terminator, the transcription machinery may continue to be active beyond the end of the desired polynucleotide to produce a transcript that is longer than the length of the desired polynucleotide.

[0012] Thus, in a construct that includes two copies of the desired polynucleotide, where one of the polynucleotides may or may not be oriented in the reverse complementary direction of the other, and where the polynucleotides are operably linked to promoters that induce convergent transcription, and the construct has no functional terminator, transcription machinery, that initiates from one polynucleotide of interest may continue to transcribe to another copy of the desired polynucleotide, and vice versa. Multiple copies of a desired polynucleotide may be oriented with different permutations: in the case where two copies of the desired polynucleotide are present in the construct, both copies may, for example, be oriented in the same direction, in the opposite direction to each other or in the opposite direction complementary to yourself, for example.

[0013] In an arrangement in which one of the desired polynucleotides is oriented in the reverse complementary direction of another polynucleotide, an RNA transcript may be produced that includes not only the sense sequence of the first polynucleotide but also the antisense sequence of the second polynucleotide. Where the first and second polynucleotides comprise the same or substantially the same DNA sequences, a single RNA transcript can include two regions that are complementary to each other and that can, therefore, hybridize. Hence, the single RNA transcript which is so transformed may form part or all of the duplex hairpin structure.

[0014] On the other hand, if two copies of such a long transcript have been made, one from each promoter, then there will be two RNA molecules with common sequence complementarity regions. Therefore, the sense region of the first RNA transcript can hybridize to the antisense region the second RNA transcript and vice versa. Accordingly, in this system, another RNA duplex can be made which is composed of two separate RNA transcripts as opposed to a hairpin duplex which is made up of a single internally complementary RNAi transcript.

[0015] Alternatively, the two copies of a desired polynucleotide may be oriented in the same direction, such that when transcribed by reading, a long RNA transcript that is produced from one promoter may include, for example, the sense sequence of the first copy of the desired polynucleotide, and also the sense sequence of the second copy of the desired polynucleotide. An RNA transcript that is produced from a different tapered promoter may thus include the antisense sequence of the second copy of the desired polynucleotide as well as the antisense sequence of the first polynucleotide. Accordingly, it is likely that no RNA transcript will contain regions of exact complementarity, and therefore no RNA transcript is likely to fold to form a hairpin structure. On the other hand, two separate RNA transcripts can hybridize to each other to form an RNA duplex.

[0016] In a first aspect, the invention features a construct comprising an expression cassette that comprises (i) a first promoter operably linked to a first polynucleotide and (ii) a second promoter operably linked to a second polynucleotide, wherein neither the first nor the second polynucleotide is operably linked to terminator, the construct produces nucleic acid molecules that prevent or reduce the expression of a gene or gene product. wherein (a) the first polynucleotide comprises a sequence of at least 23 contiguous nucleotides of sequence identity with a target gene regulatory element which is associated with a target gene, an exon of a given target gene, an intron from a target gene, the 5 'untranslated region of the target gene and / or untranslated region of the target gene translation of the 3 'region of the target gene, (b) a second polynucleotide, positioned as an inverted repeat copy of the first polynucleotide; and (c) the first promoter and the second promoter are oriented to initiate convergent transcription of the first polynucleotide and the second polynucleotide.

[0017] In a second aspect, the invention provides a transformation plasmid comprising a construct according to claim 1. The method of claim 1, wherein the transformation plasmid comprises an expression cassette that comprises in the 5 'to 3' direction (1) a first promoter that is operably linked to (2) in the first desired polynucleotide that is adjacent to (3) at least one optional polynucleotide. spacer, wherein the 3'-end of one of the spacer polynucleotides adjoins (4) a second desired polynucleotide that is operably linked to (5) a second promoter. wherein none of the desired polynucleotides in the expression cassette is operably linked to any transcription terminator, wherein the construct produces nucleic acid molecules that prevent or reduce expression of the gene or gene product, wherein the second desired polynucleotide is oriented in the reverse direction of the first desired polynucleotide polynucleotide.

In a third aspect, the invention provides a method for reducing expression of a gene normally expressed in a plant cell, wherein the plant cell is exposed to a construct according to claim 1, wherein the construct is maintained in a bacterial strain selected from the group consisting of Agrobacterium tumefaciens, Rhizobium. trifolii, Rhizobium leg in my nose rum Phyllobacterium myrsirzacearum, Sinorhizobium meliloti and Mesorhizobium loti, and wherein the desired polynucleotide comprises a sequence identical to a target sequence in the genome of the plant cell.

[0019] In a fourth aspect, the invention features a method of reducing cold-induced tuber sweetness, comprising expressing a construct according to claim 1 in tuber cells, wherein (a) the first polynucleotide comprises a sequence that is identical to at least a portion of the R1 gene or to at least a portion of the R1 gene. the promoter of the R1 gene, (c) one or both of the first and second promoters are GBSS or AGP, and (d) expression of the construct in the cell reduces the transcription and / or translation of the R1 gene in the genome of tuber cells, reducing tuber coldness caused by coldness.

According to a fifth aspect, the invention provides a method for increasing the tolerance to darkening of tubers comprising expressing a construct according to claim 1 in tuber cells, wherein (a) the first polynucleotide comprises a sequence identical to at least a portion of a tuber-expressed polyphenol oxidase gene of the promoter a polyphenol oxidase gene expressed in a tuber, (c) one or both of the first and second promoters are GBSS or AGP, and (d) expressing the construct in the cell reduces the transcription and / or translation of the polyphenol oxidase gene in the genome of tuber cells, which increases the tolerance of the tuber to darkening.

[0021] In a sixth aspect, the invention provides a method of increasing the concentration of oleic acid in an oil plant comprising expressing a construct according to claim 1 in an oilseed cell, wherein (a) the first polynucleotide comprises a sequence identical to at least a portion of the Fad2 gene or the promoter of the Fad2 gene , (c) one or both of the first and second promoters are the napin gene, the Fad2 gene or the promoters of the stearoyl-ACP desaturase genes; and (d) expression of the construct in a cell reduces the transcription and / or translation of the Fad2 gene in an oilseed cell and thus increases the oil content of the seed.

[0022] In a seventh aspect, the invention provides a method of reducing lignin content in a plant comprising expressing a construct according to claim 1 in plant cells, wherein (a) the first polynucleotide comprises a sequence identical to at least a portion of a promoter sequence associated with a gene selected from the group consisting of is derived from the caffeic acid / 5-hydroxyferulic acid 3 / 5-0-methyltransferase (COMT) gene, the caffeoyl CoA 3-0-methyltransferase (CCOMT) gene (c) one or both, the first and second promoters are the petE or Pal gene promoter and (d) expression of the construct in a cell reduces the transcription and / or translation of the COMT or CCOMT gene in the plant cell and thus reduces the lignin content of the plant.

[0023] In an eighth aspect, the invention provides a method of reducing pectin degradation in a fruit of a plant comprising expressing a construct according to claim 1 in a plant fruit cell, wherein (a) the first polynucleotide comprises the sequence of part of a polygalacturonase gene, (c) both promoters are specific promoters for fruit, and (d) expression of the construct in a fruit cell reduces the transcription and / or translation of the polygalacturonase gene in plant cells, thus reducing pectin degradation in the fruit.

[0024] In a ninth aspect the invention provides a method for reducing allergenicity of a plant-produced food comprising expressing a construct according to claim 1 in plant cells, wherein (a) the first polynucleotide comprises the sequence of a portion of a gene that encodes an allergen, and (c) expression of the construct reduces transcription and / or translation of the allergen and thus reduces the allergenicity of food produced by plants.

[0025] In a tenth aspect, the invention provides a method for reducing the expression of multiple genes in a plant comprising expressing in a plant cell (1) a construct comprising a sequence identical to at least a portion of the sequence set out in SEQ ID NO: 40 that down regulates the expression of a polyphenol oxidase gene. , L phosphorylase, and the R1 gene in a plant cell; or (2) a construct comprising the sequence set out in SEQ ID NO: 42 that downregulates the expression of the polyphenol oxidase gene, phosphorylase L, and the R1 gene in a plant cell.

[0026] Preferred embodiments of the present invention, in terms of various aspects, are as described below or as defined in the claims.

[0027] The present invention features a construct that has no or no terminator that is preceded by a self-truncating region of DNA encoding a ribozyme, but that includes a first promoter that is operably linked to the first polynucleotide and a second promoter that is operably linked to a second polynucleotide, wherein (1) the first and second polynucleotides have at least partially identical sequences, (2) the first promoter is oriented such that the direction of transcription initiated by that promoter is towards the second promoter and vice versa, and (3) this convergent arrangement results in a series of RNA transcripts that differ substantially in length. [0028] The desired polynucleotides may be perfect and imperfect repeats, or perfect or imperfect inverse complement repeats. For a construct that includes a first polynucleotide and a second polynucleotide, the second polynucleotide may have a completely or partially identical nucleotide sequence to the first polynucleotide, facing or away from the complementary orientation of the first polynucleotide. Thus, the first and second polynucleotides may be perfect repeats of each other. On the other hand, the second polynucleotide may be an imperfect repeat of the first polynucleotide, i.e., the second polynucleotide may have a sequence that is not completely identical or is partially identical to the first polynucleotide, i.e., the second polynucleotide is an imperfect repeat. The second polynucleotide may also be oriented as a direct repeat or placed in the reverse complementary orientation of the first polynucleotide.

[0029] Each of the polynucleotides described herein, for example, a desired polynucleotide or a first or second polynucleotide, for example, may be identical to at least a portion of the target sequence, or have sequence identity to at least a portion of the target sequence. When a desired polynucleotide contains a sequence that is homologous to a fragment of the target sequence, i.e. has sequence identity to at least part of the target sequence, it may be desirable that the nucleotide sequence of that fragment be specific for the target gene and / or that a partially perfect or imperfect target sequence. that is present in the desired polynucleotide was of sufficient length to confer the target specificity. Accordingly, a portion of a desired polynucleotide that has sequence identity to a portion of a target sequence may contain a distinctive domain, binding site, or nucleotide sequence typically conserved by isoforms or homologues of the target sequence. It is therefore possible to design a desired polynucleotide that is optimal for targeting a target nucleic acid within a cell.

[0030] The desired polynucleotide may contain a sequence, preferably from 4 to 5000 nucleotides, more preferably from 50 to 1000 nucleotides, and most preferably between 150 and 500 nucleotides, which have sequence identity with the DNA or RNA sequence of the target nucleic acid. The desired polynucleotide may share sequence identities for at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47.48, 49, 50, 60, 70, 80, 90,

100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500 or more than 500, or any integer within this range, contiguous nucleotides that are 100% identical in sequence with the target sequence sequence, or the desired polynucleotide contains a sequence that is about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88% , 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71 %, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%,

49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%,

36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 8%, 27%, 26%, 25%, 24%,

23%, 22%, 21%, 20%, 19%, 18 %, 17%, 16%, 15%, 14%, 13%, 12%, 11%,

10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% sequence identity to the nucleotide sequence of the target sequence. In other words, the desired polynucleotide may be homologous or share homology with the full-length sequence of the target sequence or a fragment thereof.

[0031] Thus, the present invention provides isolated nucleic acid molecules containing a polynucleotide that shares homology with a target sequence and thus can hybridize under stringent or moderate hybridization conditions to a portion of the target sequence described herein. By a polynucleotide that hybridizes to a polynucleotide portion is meant a polynucleotide (DNA or RNA) that hybridizes to at least about 15 nucleotides, more preferably at least about 20 nucleotides, even more preferably at least about 30 nucleotides, and even more preferably more than 30 nucleotides of the reference polynucleotide. For the purposes of the present invention, two sequences which share homology, i.e. a desired polynucleotide with a target sequence, can hybridize when forming a double-stranded complex in a hybridization solution composed of 6X SSC, 0.5% SDS, 5X Denhardt's solution and 100 µg of non-specific carrier DNA. See Ausubel et al., Section 2.9, supplement 27 (1994). Such a sequence can hybridize under conditions of moderate stringency, defined as a temperature of 60 ° C in a hybridization solution consisting of 6X SSC, 0.5% SDS, 5X Denhardt's solution and 100 µg of nonspecific carrier DNA. For high stringency hybridization, the temperature rises to 68 ° C. Following a moderate stringency hybridization reaction, the nucleotides are washed in a 2X SSC plus 0.05% SDS solution five times at room temperature followed by further washes in 0.1X SSC plus 0.1% SDS at 60 ° C for 1 hour. Under high stringency conditions, the wash temperature is increased to a temperature that is typically around 68 ° C. Hybridized nucleotides can be those detected using a 1 ng radiolabeled probe having a specific radioactivity of 10,000 cpm / ng, where the hybridized nucleotides are clearly visible upon exposure to x-rays at -70 ° C for a period not exceeding 72 hours.

[0032] The construct may contain an expression cassette that produces a nucleic acid that reduces the expression level of a target gene that is correctly expressed by cells containing the construct by 99%, 98%, 97%, 96%, 95%, 94%, 93 %, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,

84%, 83%, 82%, 81%, 80%, 79%; 78%, 77%, 76%, 75%, 74%, 73%, 72%,

71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%,

58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, %, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%,

32%, 31%, 30%, 29%, 28%, 27% <> 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16 %, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% relative to a cell that does not contain a construct.

[0033] Each polynucleotide, be it a desired polynucleotide, a first polynucleotide, and a second polynucleotide, may have a percentage sequence identity to the target sequence. As explained herein, the target sequence may or may not be a partial or full length sequence of a gene, a regulatory element such as a promoter and terminator, an intron, exon, untranslated region, or any sequence upstream or downstream of the target sequence genomic. Accordingly, the polynucleotide may contain a sequence that is identical in length to the target sequence. On the other hand, the polynucleotide may contain a sequence that is partially identical to the target sequence. Hence; the desired polynucleotide may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68% , 67%, 66%, 65%, 64%, 63%, 62%, 61% or 60% nucleotide sequence identity to the target sequence.

[0034] The desired polynucleotide may contain a sequence that is derived from the target promoter. The target promoter may be naturally present in the genome of the cell, then the target promoter is endogenous to the genome of the cell, or may be introduced into the genome by transformation. A polynucleotide-derived promoter may be functionally active and contain a TATA box or a sequence similar to a TATA box, but no transcription initiation or any transcribed sequences prior to the initiation of transcription. Alternatively, a polynucleotide-derived promoter may be functionally inactive due to, for example, the lack of a TATA sequence. Such a derived promoter may only be part of the target promoter.

[0035] The desired polynucleotide may contain a sequence that is specific for an intron that is endogenous to the genome of the cell.

[0036] The desired polynucleotide may contain a sequence that is specific for a terminator that is endogenous to the genome of the cell.

[0037] The construct may contain two identical promoters that are functionally active in the target tissue. The construct may contain two different promoters, each of which is functionally active in the target tissue.

[0038] The construct may also contain one or more additional polynucleotides between box A and box B. For example, in the 5 'to 3' direction, the construct may include (i) a first promoter, (ii) a desired polynucleotide, (iii) an additional polynucleotide linker, for example an intron, (iv) an inverse complementary copy of the desired polynucleotide, and (v) a second promoter , wherein the first and second promoters are operably linked to the desired polynucleotide and the complementary copy, respectively, and are arranged to effect convergent transcription.

[0039] The additional linker polynucleotide may be of any length. That is, the linker polynucleotide may be an intron that is 2, 3, 4, 5, 6, 7, 8, 9, 0, 1.12, 3, 14, 5, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500 or more than 500 nucleotides or any integer within this range. If the linker polynucleotide between the two desired polynucleotides is long enough, transcription will never go from one promoter to the other. This means that whatever the cause, transcription may stop when the transcriptional mechanism is on a linker that does not contain a functionally active terminator element. Accordingly, the resulting transcript may include the full length sequence of the first desired polynucleotide and a partial intron sequence, but not a portion of the second desired polynucleotide. Thus, it is possible to design a construct of the invention with a linker polynucleotide that prevents transcription from passing from one desired polynucleotide to another. In such a situation, if one of the desired polynucleotides is oriented as an inverse complementary copy of the other, then preventing the transcription of the full read will avoid the synthesis of an RNA transcript that is intrinsically complementary.

[0040] Accordingly, depending on (i) the convergent pattern of promoters and desired polynucleotides, (ii) the number of copies of the desired polynucleotide, (iii) the lack of a construct terminator region, and (iv) the complementarity and length of the resulting transcripts, different populations can be generated from the construct. RNA molecules.

[0041] Thus, a single construct may produce (i) a single-stranded sense RNA transcript, (ii) a single-stranded antisense RNA transcript, (iii) a hairpin duplex formed by a single-stranded RNA transcript that hybridizes to itself, or (iv) an RNA duplex formed by from two separate RNA transcripts that hybridize to each other. A single construct may be designed to produce only sense or only antisense RNA transcripts from each convergent promoter.

[0042] The present invention also describes a method of reducing the expression of a gene, usually capable of expression in a plant cell, by stably introducing any of the constructs described herein into the genome of the cell.

[0043] Any type of cell, from any species, may be stably or transiently transformed with the construct. Thus, the construct can be transformed into bacterial cells, viral cells, fungal cells, algae cells, worm cells, plant cells, insect cells, reptile cells, bird cells, fish cells, or mammalian cells. The target sequence may be located in the nucleus or genome of one of these cell types. The target sequence may be placed in a gene in the genome of the cell. Hence, the target sequence may be located in at least one regulatory element of a gene, gene exon, gene intron, 5 'untranslated region of a gene, or 3' untranslated region of a gene. The regulatory element of a gene may be at least one promoter or enhancer of the gene.

[0044] Alternatively, the target sequence may be placed on an RNA transcript which is present in one of the listed cell types and which may or may not normally be produced by the cells. This means that an RNA transcript that contains the target sequence can be produced from a source that is foreign to the host cell. For example, an RNA transcript that contains the target sequence may be of viral origin but present in a plant cell.

[0045] The present invention also contemplates the in vitro, ex vivo, ex planta and in vivo display and integration of the desired construct into the genome of the cell or isolated nucleic acid preparations.

[0046] The constructs, for example, can be inserted into an Agrobacterium transformation-derived plasmid that contains the necessary T-DNA border elements for transforming plant cells. Accordingly, the plant cell culture can be transformed with such a transformation construct, successfully transformed cells grown in a desired transgenic plant express convergent promoter / polynucleotide cassettes.

[0047] Promoters can be constitutive or inducible or combinations thereof. Strong promoters, for example, can be isolated from viruses such as tungro rice rod virus (RTBV), maize virus, cassava vein virus, mirabilis virus, peanut kaulimovirus, leprosy mosaic virus, and chlorella virus. Other promoters may be cloned from bacterial species, e.g., the promoters of the octopine synthase and nopaline synthase gene. Various inducible promoters exist, but typically the inducible promoter may be a temperature-sensitive promoter, a chemically inducible promoter, or a temporary promoter. In particular, the inducible promoter may be the hsp17.7 G4 Ha promoter, the wheat wcs120 promoter, the Rab 16A gene promoter, the alpha-amylase gene promoter, the pin2 gene promoter or the carboxylase promoter. [0048] The present invention also describes a construct comprising an expression cassette that comprises (i) a first promoter operably linked to the first polynucleotide and (ii) a second promoter operably linked to a second polynucleotide, wherein (a) neither the first nor the second polynucleotide is operably linked to a terminator, (b) at least a portion of the second polynucleotide has a substantially identical nucleotide sequence to at least a portion of the sequence of the first polynucleotide. but is located in a cassette, at a different position to the first polynucleotide, and (c) transcription initiated by the first promoter is towards the second promoter and transcription initiated by the second promoter is towards the first promoter.

[0049] At least a portion of a second polynucleotide may be oriented as an inverse complementary copy of at least a portion of the first polynucleotide.

[0050] A transcription terminating sequence that is not operably linked to any polynucleotide may be a sequence at the 3 'end of a gene that is involved in 3' terminal formation and polyadenylation of the gene transcript.

[0051] The sequence that is involved in 3-terminal formation and polyadenylation may be a terminator.

[0052] The expression cassette must not contain (i) the nos gene terminator, (ii) the 3 'untranslated T-DNA 7 gene sequence, (iii) the 3' untranslated gene sequence of the Cauliflower mosaic virus major inclusion body inclusion body gene sequence, (iv ) 3 'untranslated sequence of the pea ribulose gene of the small subunit of 1,5-bisphosphate carboxylase, (v) 3' untranslated sequence of the potato 3-ubiquitin gene, or (vi) the 3 'untranslated sequence of the Potato Proteinase Inhibitor II gene, (vii) 3' untranslated sequence of the opine genes, (viii) 3 'untranslated sequence of endogenous genes.

[0053] The first polynucleotide may comprise a sequence partially identical to the target gene or at least one regulatory element that is associated with the target gene, target gene exon, target gene intron, the 5 'untranslated region of the target gene, or the 3' untranslated region of the gene target

The first polynucleotide may contain a sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87% identical. %, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,

62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54% 53%, 52%, 51%, 50%,

49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37 %, 36%, 35%, 34%, 3%, 32%, 31%, 30%, 29%, 8%, 27%, 26%, 25%, 24%,

23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,

10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% with target sequence nucleotide sequence

The target gene may be the COMT gene involved in lignin biosynthesis, the CCOMT gene involved in lignin biosynthesis, any other gene involved in lignin biosynthesis, the R1 gene involved in starch phosphorylation, the phosphorylase gene involved in the phosphorylation of starch, the PPO gene involved in the oxidation of polyphenolate , a polygalacturonase gene involved in pectin breakdown, a gene involved in the production of allergens, a gene involved in the biosynthesis of fatty acids such as FAD2.

[0056] In the present invention, (a) the regulatory element of the target gene may be a promoter or enhancer element associated with the target gene, or (b) the first polynucleotide may comprise a sequence partially identical to the intron sequence of the target gene, the intron comprising the sequence of SEQ ID NO. : 44

[0057] The target gene may be located in the genome of a cell. Thus, the cell may be a cell of bacteria, viruses, fungi, yeasts, plants, reptiles, birds, fish, and mammals.

[0058] The target sequence may be located in the DNA sequence which encodes the RNA transcript.

[0059] The first and second promoters may be functional in the plant.

[0060] The expression cassette may be positioned between the border sequences of the transfer plasmid DNA that is suitable for bacterial-mediated plant transformation.

[0061] The bacterium may be from the genus Agrobacterium, Rhizobium or Phyllobacterium. The bacteria may be Agrobacterium tumefaciens, Rhizobium trifolii, Rhizobium leguminosarum, Phyllobacterium myrsinacearum, Sinorhizobium meliloti and Mesorhizobium loti.

[0062] The construct may also include a linker polynucleotide sandwiched between the first and second polynucleotides. The linker polynucleotide can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 , 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500 or more than 500 nucleotides.

[0063] The first promoter may be an almost constitutive promoter, a tissue specific promoter or an inducible promoter, and the second promoter is an almost constitutive promoter, a tissue specific promoter or an inducible promoter.

[0064] A constitutive strong promoter may be selected from the group consisting of the potato ubiquitin-7 promoter, the potato ubiquitin-3 promoter, the tomato ubiquitin promoter, the alfalfa petE promoter, the alfalfa Pal promoter, the rapeseed napin promoter, the maize ubiquitin promoter, the rice ubiquitin promoter. the sugarcane ubiquitin promoter, the rice actin promoter, the rubisco small subunit promoter and the rubisco activation promoter.

[0065] The tissue-specific promoter may be a granule-bound starch synthase promoter or a glucose ADP pyrophosphorylase gene promoter.

[0066] The inducible promoter may be a temperature sensitive promoter, a chemically inducible promoter, or a temporary promoter.

[0067] The inducible promoter is selected from the group consisting of the Ha hsp17.7 G4 promoter, the wheat wcs120 promoter, the Rab 16A gene promoter, the α-amylase gene promoter, the pin2 gene promoter and the carboxylase promoter.

[0068] The present invention also describes a transformation plasmid comprising an expression cassette that includes in the 5 'to 3' direction (1) a first promoter that is operably linked to (2) a first desired polynucleotide that is adjacent to (3) at least one an optional linker polynucleotide, in which the 3'-end of one of the linker polynucleotides adjoins (4) the other of the desired polynucleotide that is operably linked to (5) a second promoter. wherein no desired polynucleotide in the expression cassette is operably linked to any known transcription terminator.

[0069] At least a portion of a first desired polynucleotide may be oriented in an antisense orientation, and at least a portion of a second desired polynucleotide may be oriented as inversely complementary to the first desired polynucleotide.

[0070] At least a portion of a first desired polynucleotide may be oriented in the sense orientation, and at least a portion of a second desired polynucleotide may be oriented as inversely complementary to the first desired polynucleotide.

[0071] At least a portion of the first desired polynucleotide may be a promoter sequence.

[0072] The promoter sequence may be derived from a promoter selected from the group consisting of: (1) R1 starch-related gene promoter (2), polyphenol oxidase gene promoter, (3) fatty acid desaturase gene 12 promoter, (4) microsomal omega-6 fatty acid desaturase gene promoter, (5) stearoyl protein delta9 desaturase gene promoter -acyl-cotton carrier (6), promoter of the omega-6 oleoyl-phosphatidylcholine desaturase gene (7), promoter of the Medicago truncatula caffeic acid gene / 5-hydroxyferulic acid 3/5-O-methyltransferase (COMT), (8), Medicago sativa caffeic acid gene promoter (alfalfa) / 5-hydroxyferulic acid3 / 5-O-methyltransferase (COMT), (9) Medicago truncatula kawoyl CoA gene promoter 3-O-methyltransferase (CCOMT), (10) Medicago sativa gene promoter (alfalfa) Kavoyl CoA 3-O-methyltransferase (CCOMT), (11) Apple Mai d 1 main allergen gene promoter, (12) Ara h 2 peanut main allergen gene promoter Gen (13), Gly m Bd 30 K soybean main allergen gene promoter and (14) a polygalacturonase gene promoter.

[0073] In the present disclosure, (i) at least one of the first and second promoters may be a GBSS promoter and (ii) the first desired polynucleotide may be a sequence from a polyphenol oxidase gene.

[0074] The first and second promoters may be a GBSS promoter.

[0075] The first promoter may be a GBSS promoter and the second promoter may be an AGP promoter.

[0076] The present invention also describes a method of reducing the expression of a gene normally expressible in a plant cell, comprising exposing the plant cell to any construct described herein, wherein the construct is maintained in a bacterial strain, the desired polynucleotide having a sequence partially identical to a target sequence in the genome of the plant cell.

The bacterial strain may be selected from the group consisting of Agrobacterium tumefaciens, Rhizobium trifolii, Rhizobium leguminosarum, Phyllobacterium myrsinacearum, Sinorhizobium meliloti and Mesorhizobium loti.

[0078] The present invention describes a construct comprising an expression cassette that includes in the 5 'to 3' direction (i) a first promoter, (ii) the first polynucleotide comprises a sequence partially identical to at least a portion of the target gene promoter sequence, (iii) the second a polynucleotide comprising a sequence partially identical to the inversely complementary sequence of at least a portion of the promoter of the target gene, and (iv) a second promoter, wherein the first promoter is operably linked to the 5'-end of the first polynucleotide and the second promoter is operably linked to the 3'-end of the second polynucleotide The present invention also describes a construct comprising an expression cassette that extends in the 5 'to 3' direction (i) the first promoter, (ii) the first polynucleotide comprises a sequence partially identical to at least a portion of the target gene promoter sequence, (iii) a second polynucleotide comprising the sequence, partially identical to the inversely complementary sequence of at least a portion of the promoter of the target gene, (iv) a terminator, wherein the first promoter is operably linked to the 5'-end of the first polynucleotide and the second polynucleotide is operably linked to the terminator.

[0080] The present invention also describes a plant transformation plasmid comprising the sequence shown in SEQ ID NO: 40 or 42.

[0081] The present invention also describes a method of reducing low temperature tuber sweetness comprising expressing any construct described herein in a tuber cell wherein (a) the first polynucleotide comprises the sequence of a portion of the R1 gene (b) the second polynucleotide is inversely complementary to the first polynucleotide. of the polynucleotide (c), one or both of the first and second promoters are GBSS or AGP, and (d) expression of the construct in a cell reduces the transcription and / or translation of the R1 gene in the tuber cell genome, reducing tuber sweetness induced by low temperature. The first polynucleotide may comprise the sequence shown in SEQ ID NO: 23 or 24. The tuber may be a potato. The first polynucleotide may contain two copies of the sequence of SEQ ID NO: 23 or 24.

[0082] The present invention also describes a method of increasing tolerance to darkening of tubers, comprising expressing any construct described herein in a tuber cell, wherein (a) the first polynucleotide comprises the sequence of a portion of a polyphenol oxidase gene, (b) the second polynucleotide is inversely complementary to the first polynucleotide. polynucleotide, (c) one or both of the first and second promoters are GBSS or AGP, and (d) expressing the construct in the cell reduces the transcription and / or translation of the polyphenol oxidase gene in the tuber cell genome and thereby increases the tolerance of the tuber to darkening. In one embodiment, the first polynucleotide comprises the sequence of SEQ ID NO: 26 or 27. The tuber may be a potato. The first polynucleotide may contain two copies of SEQ ID NO: 26 or 27.

[0083] The present invention also describes a method of increasing the level of oleic acid in an oil plant comprising expressing any construct described herein in an oilseed cell, wherein (a) the first polynucleotide comprises the sequence of a portion of the Fad2 gene (b) the second polynucleotide is inversely complementary. to the first polynucleotide (c) one or both of the first and second promoters are the napin gene, the Fad2 gene or the promoters of the stearoyl-ACP desaturase gene, and (d) expression of the construct in a cell reduces the transcription and / or translation of the Fad2 gene in an oilseed cell and thus increases the oil content of the seed. The first polynucleotide may include the sequence set forth in SEQ ID NO: 28 The napin gene promoter sequence may include the sequence set forth in SEQ ID NO: 30

[0084] The stearoyl-ACP desaturase gene promoter sequence may include the sequence shown in SEQ ID NO: 31.

[0085] The promoter sequence of the Fad2 gene may include the sequence shown in SEQ ID NO: 32

[0086] The oil plant may be a plant of the genus Brassica, canola, soybean, cotton or sunflower.

[0087] The present invention also describes a method of reducing the lignin content of a plant comprising expressing any construct described herein in a plant cell, wherein (a) the first polynucleotide comprises the sequence of a portion of the caffeic acid / 5-hydroxyferulic acid 3/5-O- gene portion. methyltransferase (COMT), (b) the second polynucleotide is inversely complementary to the first polynucleotide (c) one or both of the first and second promoters is the pet E gene promoter or the PAL gene promoter and (d) expression of the construct in the cell reduces the transcription and / or translation of the COMT gene in a plant cell; and thus reduces the lignin content of the plant. The cells may be in the plant's vascular system. In a preferred embodiment, the plant is alfalfa. The first polynucleotide may comprise the sequence shown in SEQ ID NO: 33 or 37.

[0088] The present invention describes a method of reducing pectin degradation in a plant fruit comprising expressing any construct described herein in a plant cell, wherein (a) the first polynucleotide comprises the sequence of a portion of a polygalacturonase gene, (b) the second polynucleotide is inversely complementary to the first polynucleotide , (c) both promoters are fruit-specific promoters, and (d) expressing the construct in a fruit cell reduces the transcription and / or translation of the polygalacturonase gene in plant cells, thereby reducing pectin degradation in the fruit. The first polynucleotide may comprise the sequence shown in SEQ ID NO: 39

[0089] The present invention describes a method of reducing the allergenicity of a plant-produced food comprising expressing any construct described herein in a plant cell, wherein (a) the first polynucleotide comprises the sequence of a portion of an allergen-encoding gene, (b) the second polynucleotide is inversely complementary to the first polynucleotide. polynucleotide; and (c) expression of the construct reduces the transcription and / or translation of the allergen. and thus reduces the allergenicity of food produced by plants.

[0090] In the present invention, (a), the plant may be an apple (b) the food may be an apple, (c) the first polynucleotide may contain a sequence from the promoter of the Mai dI gene; and (d) expressing the construct in the apple plant can reduce the transcription and / or translation of Mai d I in the apple.

[0091] In the present invention, (a), the plant may be a peanut; (b) the food may be a peanut, (c) the first polynucleotide may contain a sequence from the Ara h 2 gene promoter; and (d) expressing the construct in a groundnut plant can reduce the transcription and / or translation of Ara h 2 in peanuts.

[0092] In the present invention, (a), the plant may be a peanut; (b) the food may be a peanut, (c) the first polynucleotide may contain a sequence from the Ara h 2 gene promoter; and (d) expressing the construct in a groundnut plant can reduce the transcription and / or translation of Ara h 2 in peanuts.

[0093] The present invention describes a method for reducing the expression of multiple genes in a plant, comprising expressing in a plant cell a construct comprising the sequence shown in SEQ ID NO: 40, which down-regulates the expression of the polyphenol oxidase gene, L phosphorylase, and the R1 gene in a plant cell.

[0094] The present invention describes a method for reducing the expression of multiple genes in a plant, comprising expressing in a plant cell a construct comprising the sequence shown in SEQ ID NO: 42 that down-regulates the expression of the polyphenol oxidase gene, L phosphorylase, and the R1 gene in a plant cell.

[0095] The present invention describes a construct comprising a desired promoter which is operably linked to (i) a first promoter at its 5'-end and (ii) a second promoter at its 3'-end, the desired promoter having a sequence partially identical to the target promoter sequence in the genome of interest.

[0096] The present invention describes a construct comprising two tapered copies of a desired promoter which are separated by a polynucleotide, the desired promoter sequence partially identical to a target promoter in the desired genome of interest. The polynucleotide that separates the tapered-oriented promoters may be an intron.

[0097] The present invention relates to a construct comprising two desired promoters which are operably linked to a promoter and a terminator, the desired promoters having sequences partially identical to the target promoter of the genome of interest. Two desired promoters may have identical sequence to each other for at least a portion of their length, and one desired promoter may be oriented as inversely complementary to the other.

[0098] The present invention relates to a construct comprising two desired promoters which are operably linked to a promoter and a terminator, the desired promoters having sequences partially identical to the target promoter of the genome of interest. Two desired promoters may have identical sequence to each other for at least a portion of their length, and one desired promoter may be oriented as inversely complementary to the other.

[0099] The product disclosure relates to a construct which comprises four direct repeats of a polynucleotide of interest which are preceded by an antisense DNA fragment of the polynucleotide of interest. Such a construct is depicted as pSIM1111.

[0100] The present invention also provides a method for reducing the level of expression of an endogenous gene in an alfalfa plant comprising introducing a cassette into an alfalfa cell, the cassette having two alfalfa-specific promoters aligned in a convergent orientation, wherein the activity of the promoters in the cassette reduces the expression level of the endogenous gene alfalfa, which is operably linked in the alfalfa genome to a promoter which has partially identical sequence, with at least part of one of the cassette promoters. The sequence of at least one of the promoters is shown in SEQ ID NO: 54 or SEQ ID NO: 55

[0101] The present invention also provides a method for reducing COMT gene expression, comprising expressing a COMT gene fragment or a COMT promoter fragment in a cell that contains the COMT gene in its genome.

[0102] The present invention also features a method for reducing the expression of a Comt gene or a Ccomt gene, comprising expressing any construct described herein in a cell that contains the Comt gene or the Ccomt gene in its genome, the first polynucleotide comprising the sequence of the Comt gene or the promoter of the Comt gene. or the Ccomt gene, or the promoter of the Ccomt gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 shows a schematic diagram of the T-DNA of binary vectors that (a) constitute the negative control (pSIM714) and (b) contain constructs that are typical silencing constructs of pSIM374, pSIM718 and PSIM755. B is the border sequence of the transfer DNA; T is a terminator sequence; hptII is a resistance gene which confers resistance to hygromycin in a plant; P1 is a promoter sequence, and in this example, a promoter that is identical to the promoter driving the functionally active beta-glucuronidase (gus) gene in the transgenic gus plants; P2 is a promoter sequence which is also functionally active but different from P1; gus-S is a fragment of the gus gene; gus-A is the inverse complementary fragment of the gus gene; I mean intron. With regard to gus-S and gus-A, the solid thick arrows represent (part of) RNA transcripts which are partly identical to the part of the transcript produced by expressing the gus gene; the dashed thick arrows represent (part of) RNA transcripts which are partly identical to part of the inverse complement gus gene transcript; thin lines indicate portions of a transcript that are homologous or inversely complementary to another sequence, such as an intron, of the construct. The open left arrow (pointing to the gus-A element in the cassette) shows that the gus-A element is oriented in the expression cassette as inversely complementary to the gus-S, the arrow points to the right. Hence, the P1 and P2 promoters are arranged such that transcription from each is convergent, i.e. P1 transcribes in the P2 direction and vice versa.

[0104] Figure 2 shows T-DNA diagrams of binary vectors containing constructs that resemble traditional suppression constructs except that the terminator, pSIM728, PSIM140, and pSTM758 are missing. With regard to gus-S and gus-A, the solid thick arrows represent a portion of RNA transcripts that are partially identical to the portion of a transcript produced by expressing the gus gene; the dashed thick arrows indicate parts of RNA transcripts which are partly identical to part of the inverse complement transcript of the gus gene; thin lines indicate portions of a transcript that are homologous or inversely complementary to another sequence, such as an intron, of the construct.

[0105] Figure 3 shows diagrams of T-DNA containing the terminator-free colliding transcription (TFCT) constructs. In particular, it illustrates the T-DNA of pSIM715, PSIM717, pSIM756 and pSIM771. The meaning of these specific elements as well as the fixed and dashed arrows is the same as described in the legend of Fig. 1. In pSIM717, intron transcription starting with both P1 and P2 results in transcripts containing 5'-sequences identical to part of the gus gene transcript and 3'-sequences which are inversely complementary to the gus gene transcript. These transcripts can fold to form partially double-stranded RNA. Depending on the ability of the P1 transcription complex to function continuously, an RNA transcript, initiated from the P1 promoter, can transcribe sequences downstream of the gus-S sequence to which it is operably linked. Accordingly, when reading the top or sense strand of pSIM717 in the 5 'to 3' direction, the P1 transcript may contain the sequence of the middle intron (I) as well as the sequence of the inverse complement gus-S element. The upper strand sequence of the inverse-complementary gus-S element is antisense gus-S.

[0106] Figure 4 shows T-DNA diagrams containing the terminator-free colliding transcription (TFCT) constructs. In particular, it illustrates the T-DNAs of pSIM754, pSIM773, and pSIM767. The meaning of these specific elements as well as the fixed and dashed arrows is the same as described in the legend of Fig. 1. Pin indicates the part of the P1 promoter that is upstream of the TATA sequence. This sequence does not act as a promoter.

[0107] Figure 5 shows diagrams of T-DNA containing the terminator-free colliding transcription (TFCT) constructs. In particular, it shows T-DNA from pSIM782. The meaning of these specific elements as well as the fixed and dashed arrows is the same as described in the legend of Fig. 1. gusl denotes the intron of the gus gene. [0108] Figure 6 shows diagrams of T-DNA containing the terminator-free colliding transcription (TFCT) constructs. In particular, it illustrates the T-DNAs of pSIM765, pSIM922F, pSIM922G, pSIM774, and pSIM775. The meaning of these specific elements as well as the fixed and dashed arrows is the same as described in the legend of Figure 1. PPO means a fragment of the tobacco PPO gene.

[0109] Figure 7 shows an ethidium bromide stained agarose gel containing RT-PCR products. + = positive plasmid control; - = negative control; M = marker; T1 = P1 promoter transcript; T2 = P2 promoter transcript.

[0110] Figure 8 shows the autoradiograms of the RNA gel spots. The probe used for hybridization was from the gus gene.

[0111] Figure 9 shows sequence analysis of various promoter fragments and identifies an 89 bp sequence that can be methylated during promoter based silencing.

[0112] Figure 10 shows plasmid maps. G: fragment of the gus gene; H: expression cassette for the hptII gene; LB: left border area; RB: right border area; T: terminator; P1: P1 promoter; P1n: non-functional P1 promoter without TATA box; P2: P2 promoter; P3: P3 promoter; GB:

GBSS promoter; PP: fragment of the PPG gene; PT = fragment of the tobacco PPG gene. The direction of transcription is indicated by a small black solid arrow.

Detailed description of the preferred embodiments

[0113] The construct of the present invention can be used to effectively reduce or prevent transcription or translation of a target nucleic acid by causing convergent transcription of a desired polynucleotide. Accordingly, one object of the present invention is to provide constructs that produce nucleic acid molecules that prevent or reduce the expression of a gene or gene product, for example an RNA transcript or protein.

[0114] One particular feature of such a construct is that, unlike conventional silencing constructs, there is no terminator embedded therein operably linked to the 3'-end of the desired polynucleotide. As is well known, a terminator is a nucleotide sequence, usually located at the 3'-end of a gene, that is involved in the cleavage of an RNA transcript that is transcribed from the gene and in the polyadenylation of that transcript. Typically, the terminator is located downstream of the gene's stop codon.

[0115] The terminators used to build conventional silencing cassettes that are excluded from the constructs of the invention are derived from these 3 'regions in certain genes, and often also contain even more downstream, non-transcribed DNA sequences. Choosing which terminator to use is simply a matter of convenience. Accordingly, terminators and termination regions from endogenous previously characterized genes have been used in conventional silencing constructs. One of the more commonly used terminators, for example, is the terminator of the Agrobacterium nopaline synthase (nos) gene, which contains both 3 'untranslated sequences and additional further DNA. Other terminators include:

[0116] 3 'untranslated sequences of the T-DNA gene 7 (Genbank accession number V00090).

[0117] 3 'untranslated protein sequences of the major inclusion body protein gene of cauliflower mosaic virus.

[0118] 3 'untranslated sequences of the pea ribulose 1,5-bisphosphate small subunit carboxylase gene (GenBank accession number M21375).

[0119] 3 'untranslated sequences of the potato 3 ubiquitin gene (Genbank accession number Z11669).

[0120] 3 'untranslated sequences of the potato proteinase II inhibitor gene (Genbank accession number 00889094).

[0121] The 3 'untranslated sequences of the opin genes.

[0122] 3 'untranslated sequences of endogenous genes; that is, genes that are normally expressed by the genome of an organism.

[0123] In the present disclosure, however, none of these terminators, and even no functional terminator, is directly operably linked to the desired polynucleotide of the construct of the invention. Also, the desired polynucleotide is not directly operably linked to a terminator that is preceded by a self-cleaving ribozyme coding sequence.

[0124] Another characteristic of the construct of the present invention is that it causes the convergent transcription of one or more copies of a polynucleotide, whether or not directly operably linked to a terminator, through two opposing promoters. Due to the lack of a termination signal, the length of the RNA molecules in the pool that are transcribed from the first and second promoters may vary.

[0125] Occasionally, for example, a transcription machine may continue to transcribe past the last nucleotide that marks the end of the desired polynucleotide sequence. Thus, in this particular arrangement, transcription termination may occur either by the weak and unintended action of further sequences which, for example, facilitate the formation of a hairpin formation or by the action of unintentional transcription terminators located in the plant DNA flanking the transfer DNA integration site.

[0126] The terminator-less convergent transcription construct (TFCT) of the invention may thus include a first promoter operably linked to a first polynucleotide and a second promoter operably linked to a second polynucleotide, wherein (1) the first and second polynucleotides have at least partial sequence identity with each other and target sequence, and (2) the first promoter is oriented so that that the direction of the transcription initiated by this promoter is towards the second promoter and vice versa, (3) the construct produces molecules

RNA that is substantially different in size, some transcripts are RNA equivalents of at least a portion of the polynucleotide, and other equivalents that include at least a portion of both the polynucleotide and its inverse complement.

[0127] The desired polynucleotide may be linked to the promoter in two different orientations. In one direction, for example sense, at least the 5 'portion of the resulting RNA transcript will share sequence identity with at least a portion of at least one target transcript. An example of such a system is shown in figure 3 as pSIM717. In the opposite direction indicated as antisense, at least the 5 'portion of the predicted transcript will be identical or homologous to at least the inverse complement portion of at least one target transcript. An example of such a system is shown in figure 3 as pSIM756.

[0128] As used herein, sequence identity or identity in the context of two nucleic acid sequences or polypeptides includes reference to the residues in the two sequences that are the same when aligned with maximum consensus in a specified region. When percent sequence identity is used with respect to proteins, it is considered that residue positions that are not identical often differ by conservative amino acid substitutions, in which the amino acid residues are substituted with other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and therefore they do not change the functional properties of the molecule. Where sequences differ by conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitutions. Sequences that differ by such conservative substitutions are said to have sequence similarity or similarity. The means of adjusting are well known to those skilled in the art. Typically, this involves assigning a conservative substitution a score that corresponds to a partial rather than a complete mismatch, thus increasing the percent sequence identity. Thus, for example, when an identical amino acid is scored 1, and a non-conservative substitution is scored zero, conservative substitutions are scored between zero and 1. A conservative substitution score is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. (Intelligenetics, Mountain View, California, USA).

[0129] As used herein, percent sequence identity is a value determined by comparing two optimally aligned sequences in a comparative window, wherein the portion of the polynucleotide sequence in the comparative window may include additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) to optimally align the two sequences. The percentage is calculated by determining the number of positions where the nucleic acid base or amino acid residue is identical in both sequences, resulting in the number of matched positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying by 100 to obtain percent sequence identity.

[0130] Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be made using the homology algorithm of Smith and Waterman, Adv. Appl. Match. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by searching for similarity by the method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); by computer-based implementation of these algorithms, including but not limited to: CLUSTAL w. pragramien PC / Gene Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA; the CLUSTAL, this program has been described in detail by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 55-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994).

[0131] The family of BLAST programs that can be used to search a database for similarity include: BLASTN for querying nucleotide sequences against a database of nucleotide sequences; BLASTX for the study of nucleotide sequences from the protein sequence database; BLASTP for protein sequence research from a protein sequence database; TBLASTN for studying protein sequences from the nucleotide sequence database; and TBLASTX for studying nucleotide sequences against a database of nucleotide sequences. See Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al, Eds., Greene Publishing and Wileynterscience, New York (1995); Altschul et al, J. Mol. Biol., 215: 403-410 (1990); and Altschul et aL, Nucleic Acids Res. 25:33 89-3402 (1997).

[0132] The BLAST analysis program is publicly available, for example, from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm first performs the identification of pairs of high-scoring sequences (HSPs) by identifying short words of length W in the query sequence that either match, some meet a positive threshold score of T when aligned with a word of the same length in sequence from the database. T is referred to as the neighborhood word score threshold. These first neighborhood word hits act as seeds to initiate searches for longer HSPs containing them The word hits then extend in both directions along each sequence until the cumulative alignment score can be increased. Cumulative scores are computed using, for nucleotide sequences, the parameters M (reward for a pair of matching residues; always> O) and N (penalty for mismatched residues; always <o). For amino acid sequences, a scoring matrix is ​​used to calculate a cumulative score. The extension of the word hits in each direction is stopped when: the cumulative alignment score falls off the value X from its maximum achieved value; the cumulative alignment score goes to zero or below due to the accumulation of one or more negative residue alignments or after either sequence has been reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default parameters: a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default parameters: a word length (W) of 3, an expectation (E) of 10, and BLOSUM62 scoring matrices (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci, USA 89: 10915 ).

[0133] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the least sum probability (P (N)), which indicates the probability that a match between two nucleotide or amino acid sequences could occur randomly.

[0134] BLAST searches assume that proteins can be modeled as random sequences. However, many true proteins include regions with non-random sequences, which may be homopolymeric tracts, short-term repeats, or one or more amino acid enriched regions. Such low complexity regions may be matched for unrelated proteins, even though the other regions of the protein are completely different. Numerous low complexity filter programs can be used to reduce low complexity matches, for example, SEG low complexity filters (Wooten and Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chem. 17: 191-201 (1993)) may be used singly or in combination.

[0135] Multiple sequence alignments can be performed by the CLUSTAL method (Higgins Sharp (Higgins and Sharp (1989) CABIOS 5: 151: 153) with default parameters (Gap Penalty = 10, Gap Length Penalty = 10.) Default Alignment Parameters. in pairs using the CLUSTAL method are as follows: KTUPLE 1, Gap penalty = 3, WINDOW = 5 and recorded diagonals = 5.

[0136] Any or all of the DNA elements and sequences described herein may be native to one or more plant genomes. Accordingly, all DNA elements and sequences that are selected for the final transfer cassette may be endogenous or native to the genome of the plant to be transformed. For example, all sequences may be derived from the potato genome. Alternatively, one or more of the DNA elements or sequences may be non-native to a plant genome which is not the same species as the plant to be transformed, but which functions at any event in the host plant cell. Such plants include potato, tomato, and alfalfa. The present invention also encompasses the use of one or more genetic elements from a plant that is sexually compatible with the plant species to be transformed.

[0137] In view of the development of a native approach to generating genetically modified plants, as disclosed in Rommens et al. in WO2003 / 063980, US-2003-0221213, US-2004-0107455, and

WO2005 / 004585, concerns are publicly expressed, Rommens et al. describe the identification and isolation of genetic elements from plants that can be used to transform plants through bacteria .. Thus Rommens describes that plant-derived transfer DNA (P-DNA), for example, can be isolated from the plant genome and used in place of Agrobacterium TDNA for the genetic modification of plants.

[0138] The term plant includes, but is not limited to, angiosperms and gymnosperms such as potatoes, tomatoes, tobacco, avocado, alfalfa, lettuce, carrots, strawberries, sugar beet, cassava, sweet potato, soybeans, peas, beans, cucumber. , grapes, brassica, maize, turfgrass, wheat, rice, barley, sorghum, oats, oak, eucalyptus, walnut and palm. Thus, the plant can be a monocotyledonous or dicotyledonous plant. The plant and plant material also include plant cells, seed, plant progeny, sexually or asexually produced propagule, and descendants of any of them, such as cut pieces or seed. Plant material includes suspension cultures of cells, callus, embryos, meristematic regions, callus tissues, leaves, roots, shoots, gametophytes, sporophytes, pollen, germinating cuttings, seeds, and microspores. Plants may be at various stages of maturity and may be grown in liquid or solid culture, or in soil or suitable media in pots, greenhouses or fields. Expression of the introduced leaders, trailers or gene sequences in plants can be transient or permanent.

[0139] Thus, any of these plants and plant products may undergo transformation. Plant transformation as used herein is a process by which DNA becomes stably integrated into the genome of a plant cell. Stably relates to the permanent or transient storage and / or expression of a polynucleotide in and by the genome of a cell. Thus, a stably incorporated polynucleotide is one that is attached within the transformed genome of the cell and can be amplified and multiplied by successive daughter cells of the cell or resulting transformed plant. Transformation can occur under natural or artificial conditions using various methods known in the art. See, for example, METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Bernard R. Glick and John E. Thompson (ed.), CRC Press, Inc., London (1993); Chilton, Scientific American, 248) (6), pp. 36-45, 1983; Bevan, Nuci. Acids. Res., 12, pp. 8711-8721, 1984; and Van Montague et al., Proc R Soc Lond B Biol Sci., 210 (1180), pp. 351-65, 1980. Plants can also be transformed using improved transformation and precision breeding techniques. See, for example, Rommens et al. in WO2003 / 069980, US-2003-0221213, US-2004-010745, WO2005 / 004585, US-2004-0003434, US-2005-0034188, WO2005 / 002394, and WO2003 / 079765.

[0140] One or more traits of a tuber plant must be modified using the transformation sequence and elements described herein. The tuber denotes a thickened, usually underground, storage organ that holds the heels and scales of the bulbs and tubers. Roots and shoots grow from growth buds, called eyes, on the surface of the tuber. Some tubers, such as caladiums, shrink as the plant grows and form new tubers from the eyes. Others, such as tuberous begonias, grow because they store nutrients during the growing period and develop new growth buds at the same time. Tubers can be wrinkled and hard or slightly fleshy. They can be round, flat, oddly shaped, or lumpy. Examples of tubers include, but are not limited to, the following: yamy ahipa, celery, arracacha, arrow, arrowroot, baddo, bitter cassava, brazilian arrowroot, cassava, chinese artichoke, water Chinese chestnut, coconut, malanga, taro, collocation, alocasia, sunflower, goo nut, Japanese artichoke, Japanese potatoes, Jerusalem artichoke , ball, lotus, water lily, edible cassava, mexican potato, mexican yam, yamy, potato, sago, taro satooimo, seegoo, sweet cassava, sweet potato, cheapr, tannia, tannier, tapioca root, water lily root, yam bean, yam and yautia. Examples of potatoes include, but are not limited to, russet potatoes, white round potatoes, white long potatoes, red round potatoes, yellow flesh potatoes, and blue and purple potatoes.

[0141] Tubers can be classified into microtubers, minitubers, near-mature tubers and mature tubers. Microtubers are those tubers that are grown in tissue culture medium and have small dimensions. The term small size means about 0.1 cm 1 cm. A minituber is a tuber that is larger than a microtuber and is grown in soil. The almost mature tubers come from a plant that is beginning to age and is about 9 weeks old in a greenhouse. Ripe tubers come from a plant that has already grown old. Ripe tubers are, for example, tubers that are about 12 weeks or more. [0142] Therefore, the plant-derived transfer DNA (P-DNA) border sequence of the present invention is not identical in nucleotide sequence to any known bacterial T-DNA border sequence, but functions essentially for the same purpose. That is, P-DNA can be used to transfer and integrate one polynucleotide into another. P-DNA can be inserted into a tumor inducing plasmid, such as the Agrobacterum Ti plasmid in place of traditional T-DNA, and maintained in a bacterial strain, such as conventional transformation plasmids. The P-DNA can be manipulated to contain a desired polynucleotide that is designed to be integrated into the plant genome by bacterial mediated transformation of plants. See Rommens et al. in W02003 / 069980, US2003-0221213, US-2004-0107455, and WO2005 / 004585.

[0143] Thus, the P-DNA border sequence differs by 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20 or more nucleotides from a known T-DNA border sequence from Agrobacterium species, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0144] The P-DNA border sequence is greater than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87% , 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70 %, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%,

56%, 55%, 54% 53%, 52%, 51% or 50% similar to the nucleotide sequence of the T-DNA border sequence from Agrobacterium.

[0145] Methods for the identification and isolation of transfer DNA from plants, particularly potatoes and wheat, have been developed that use the consensus border sequence described in US-2004-0107455.

Plant-derived DNAs of the present invention, such as any sequences, cleavage sites, regions, or elements described herein, are functional if they facilitate the transfer and integration of a polynucleotide to which they are linked to another nucleic acid molecule, for example a plant chromosome. , with a transformation frequency of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88 %, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75 %, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50 %, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30 %, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 15%, or about 5%; or at least about 1%.

[0147] Each of these transformation sequences and elements may be modified or mutated to alter transformation efficiency. Other polynucleotide sequences may be added to the transformation sequence of the present invention. For example, it can be modified to have multiple 5 'and 3' cloning sites or additional restriction sites. The sequence of the cleavage site as disclosed herein, for example, may be modified to increase the likelihood that the companion vector DNA backbone fails to integrate into the plant genome.

[0148] Any desired polynucleotide may be inserted between any cleavage or border sequences described herein. For example, a desired polynucleotide may be a wild-type or modified gene that is derived from plants, or it may be a gene from a non-plant genome. For example, for transforming a potato plant, the expression cassette can be made such that it comprises a potato-specific promoter that is operably linked to the desired potato gene or fragment thereof and a potato-specific terminator. The expression cassette may contain additional potato genetic elements such as a signal peptide sequence fused in frame to the 5 'end of the gene and a potato transcription enhancer. The present invention is not limited to such an arrangement, and a transformation cassette may be constructed such that the desired polynucleotide operably linked to the promoter is not operably linked to the terminator sequence.

[0149] In addition to elements of plant origin, such elements can be identified, for example, for fungi and mammals. See, for example, SEQ ID NOs: 173-182. Several of these species have already been shown to be useful in Agrobacterium mediated transformation. See Kunik et al, Proc Natl Acad Sci USA 98: 1871-1876, 2001 And Casas, Flores et al, Methods Mol Biol 267 ,: 315-325, 2004.

[0150] If a transformation-related sequence or element, such as those described above, is identified and isolated from a plant, and if that sequence or element is subsequently used to transform a plant of the same species, the sequence or element may be described as native to the plant genome .

[0151] Thus, a native genetic element refers to a nucleic acid that occurs naturally in, comes from, or belongs to the genome of the plant to be transformed. Likewise, the term endogenous can also be used to denote a particular nucleic acid, such as DNA or RNA, or a protein as native to a plant. Endogenous means an element that comes from the body. Thus, any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA or cDNA isolated from the genome of the plant or plant species to be transformed or isolated from the plant or species that is sexually compatible or capable of reproducing with the plant species to be transformed. to be transformed is native to a plant species. In other words, a native genetic element represents all genetic material that is available to plant breeders for the improvement of plants through classical plant breeding.

Any variants of a native nucleic acid are also considered native according to the present invention. Accordingly, native nucleic acid may also be isolated from a plant or a sexually compatible species thereof and modified or mutated such that the resulting variant is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77% , 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or 60 % a similar nucleotide sequence as the unmodified native nucleic acid isolated from the plant. The native variant nucleic acid may also have less than about 60%, less than about 55%, or less than about 50% nucleotide sequence similarity.

[0152] Native nucleic acid isolated from a plant may also encode a variant of a naturally occurring protein product transcribed and translated from that nucleic acid. Thus, a native nucleic acid can encode a protein that is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88% , 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71 %, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60% amino acid sequence similarity to the unmodified native protein expressed in the plant from which the acid was isolated nucleic.

[0153] In a terminator-free construct that includes two copies of a desired polynucleotide, one desired polynucleotide may be oriented such that its sequence is inversely complementary to the other. The scheme of pSIM717 in Figure 3 shows such a setup. That is, the upstream or sense strand of the construct will include, in the 5 'to 3' direction: (1) a fragment of the target gene, and (2) a sequence inversely complementary to a fragment of the target gene. In this arrangement, a second promoter which is operably linked to this inverse complement of the desired polynucleotide will most likely produce an RNA transcript that has at least partially identical sequence to a transcript made from another desired polynucleotide.

[0154] The desired polynucleotide and its inversely complementary sequence may be separated by a DNA linker sequence, such as an intron, which is of any length. For example, it may be desirable to reduce the possibility of transcribing the inverse complement of a desired polynucleotide with the opposite promoter by inserting a long intron or other DNA sequence between the 3 'end of the desired polynucleotide and the 5' ends of its inverse complement sequence. For example, in the case of pSIM₇and₇ (figure 3) the size of intron (I) can be increased such that it becomes unlikely that the P1 transcription complex will reach the inverse complement sequence of gus-S before it is disrupted or deleted. Accordingly, about 50, 100, 250, 500, 2,000 or more than 2,000 nucleotides can be inserted between the sense and antisense copies of the desired polynucleotide.

[0155] A desired polynucleotide of the present invention, for example, the first and second polynucleotides as described herein, may have a sequence partially identical to all or at least a portion of the sequence of a structural gene or regulatory element. For example, the first polynucleotide may have a sequence partially identical to the coding or non-coding sequence of the target gene or a promoter fragment of the target gene. In one embodiment, the polynucleotide in question has a sequence of 100%, 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%. %, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66 %, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49 %, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24 %, about 23%, about 22%, about 21%, about 20%, about 15% or about 5% or at least about 1% identical to the gene sequence of a target or target regulatory element, for example a target promoter.

[0156] For simplicity, the term desired polynucleotide also includes other terms as used herein such as a first polynucleotide and a second polynucleotide, or any polynucleotide that is used in a construct of the present invention to reduce expression of a target gene or sequence. Hence, the desired polynucleotide may be a first or a second polynucleotide or both.

[0157] In a simplified form, the construct of the present invention does not contain two copies of the polynucleotide but only one copy. Suitably, the polynucleotide is operably linked to promoters at both its 5 'and 3' ends. In this particular arrangement, RNA transcripts will be produced containing sequences from each strand of the DNA duplex. An example of such a system is shown in figure 3 as pSIM772.

[0158] Terminator-free cassettes may exist as an extrachromosomal DNA molecule in the cell, or they may be integrated by any of a variety of mechanisms into the nucleus, chromosome, or other endogenous nucleic acid of the cell. If the terminatorless cassette is stably integrated into the genome of the cell, then it will be possible to generate a cell line, cell culture, biological tissue, plant or organism that contains the cassette in successive generations of the cell or organism.

[0159] Expression of this construct in a plant reduces or prevents expression of a gene (s) that have partially identical sequence or are inversely complementary to at least a portion of the desired polynucleotide. The present invention is not bound by any particular theory or mechanism, but it is recognized that transcripts may directly or indirectly affect the activity of regulatory sequences, such as the promoter, which is typically associated with the expression of a target gene in a cell; or transcripts may negatively affect the accumulation of a transcript that is endogenously produced in the target cell. Accordingly, transcript accumulation and / or translation of the transcript may be altered by the action of the transcript produced by the expression cassette of the present invention.

[0160] The plant according to the invention may be a monocotyledonous plant, for example alfalfa, canola, wheat, grass, corn, rice, oats, barley, sorghum, orchid, iris, lily, onion, banana, sugarcane and palm. Alternatively, the plant may be a dicotyledonous plant, for example, potato, tobacco, tomato, avocado, pepper, sugar beet, broccoli, cassava, sweet potato, cotton, poinsettia, vegetables, alfalfa, soybeans, peas, beans, cucumber, grapevine, cabbage, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy and cactus.

[0161] The performance of the RNA molecules which is produced by the terminator-free expression cassette of the present invention can be assessed by directly or indirectly measuring the level of the target nucleic acid or protein in the cell or in the environment in which the expression cassette is present. Thus, the effect of the expression cassette according to the invention to down-regulate, suppress, reduce or prevent or eliminate the expression of the target gene can be identified by reducing the amount of RNA transcript that is produced by the target gene or by reducing the amount of the target gene protein product. or both.

[0162] A desired polynucleotide lacking a terminator for a construct described herein may have a sequence identical or partially identical to the sequence of various regions of DNA, such as: (1) at least part of the target transcript coding sequence, (2) at least part of the intron of the gene encoding the target transcript (3) for at least part of the promoter of the gene encoding the target transcript (4), part of the terminator of the gene encoding the target transcript, where the polynucleotide is not is the terminator of (5) the 3 'untranslated region of the gene and (6) the 5' untranslated region of the gene. One or more nucleotides in one of these regions may be mutated, changed, or substituted to increase sequence identity to the target sequence, or to otherwise increase or enhance silencing of the target sequence.

[0163] The site of the location of the target sequence may thus be, inter alia: (i) the genome of the cell; (li) at least one RNA transcript, typically produced in a cell; or (iii) a plasmid, construct, vector, or other DNA or RNA carrier. The cell that contains the genome or that produces the RNA transcript can be a cell of bacteria, viruses, fungi, yeasts, insects, snail, worms, reptiles, birds, fish, or mammals.

[0164] Thus, the target nucleic acid may be one that is normally transcribed into RNA from the nucleus of the cell, which is then translated into an encoding polypeptide. Alternatively, the target nucleic acid may not be actually expressed in the particular cell or cell type. For example, the target nucleic acid can be a genomic DNA sequence found in the nucleus, chromosome, or other genetic material, such as a mitochondrial DNA sequence. Such a target nucleic acid may be, inter alia, a regulatory region, a non-translated region of a gene, or a non-coding sequence.

[0165] Alternatively, the target nucleic acid may be foreign to the host cell, present or expressed by an organism other than the host organism. For example, the target nucleic acid can be a DNA or RNA molecule endogenous or expressed by invasive parasites, viruses, or bacteria.

[0166] Moreover, the target nucleic acid may be a DNA or RNA molecule. present or expressed by a diseased cell. For example, the diseased cell may be cancer cells that express an RNA molecule that is not normally expressed in non-cancerous cells.

[0167] In plants, the desired polynucleotide may have a sequence partially identical to the target nucleic acid sequence which is responsible for the given trait of the plants. For example, a desired polynucleotide may produce a transcript that targets and reduces expression of a target polyphenol oxidase gene in a plant, thereby altering one or more darkening-related traits or phenotypes. Similarly, a desired polynucleotide may produce a transcript that targets and reduces expression of a starch-bound R1 target nucleic acid or a phosphorylase target nucleic acid in a plant, thereby altering one or more cold-induced sweetness characteristics or phenotypes.

[0168] The expression cassette in a construct of the present invention may be flanked by one or more transfer DNA (T-DNA) border sequences. Each of the expression cassettes described herein, for example, can be inserted into the T-DNA of an Agrobacterium-derived plasmid, such as the Ti plasmid from A. tumefaciens.

[0169] The border sequence may include a sequence that is similar to a traditional Agrobacterium T-DNA border sequence, but is in fact a sequence that is derived from a plant but that may facilitate transfer and integration of one nucleic acid into another. For example, such plant-derived transfer DNA (T-DNA) border sequences can be isolated from potato (SEQ ID NO: 44), tomato (SEQ ID NO: -46), pepper (SEQ ID NO: 47), alfalfa (SEQ ID NO: 47) 48), barley (SEQ ID NO: 49) and rice (SEQ ID NO: 50) as shown in the sequence table elsewhere in this application.

[0170] Accordingly, any expression cassette described herein can be inserted into a transfer DNA that is limited by such P-DNA border sequences that are capable of integrating the cassette into another nucleic acid, such as a plant genome or chromosome. . [0171] Accordingly, an Agrobacterium plasmid that contains an expression cassette according to the invention that does not contain a DNA region involved in the formation of a 3'-end polyadenylation of an RNA transcript may be stably integrated into the plant genome through Agrobacterium-mediated transformation. Accordingly, the progeny of this transformed plant will continue to express the expression cassette associated transcripts.

[0172] Promoters that are used to initiate transcription of a desired polynucleotide can be constitutive, tissue-preferred, or inducible promoters or permutations thereof. Strong promoters, for example, include potato ubiquitin-7 and ubiquitin-3 and the maize, rice and sugar cane ubiquitin promoters. They also include the rice actin promoter, various rubisco small subunit promoters, rubisco activation promoters, and rice actin promoters. Good tissue-preferred promoters expressed primarily in potato tubers include the promoters of pellet-associated starch synthase and the ADP glucose pyrophosphorylase genes. Various inducible promoters exist, but typically the inducible promoter may be a temperature-sensitive promoter, a chemically inducible promoter, or a temporary promoter. In particular, the inducible promoter may be the hsp17.7 G4 Ha promoter, the wheat wcs120 promoter, the Rab 16A gene promoter, the alpha-amylase gene promoter, the pin2 gene promoter or the carboxylase promoter.

[0173] Accordingly, to facilitate identification of a plant that has been transformed using a terminator-free expression cassette, it may be desirable to include a selectable or screenable marker sequence in the region delimited by the garage DNA sequences of the transfer sequence. The inclusion of the marker is a standard Agrobacterium-mediated transformation procedure used to allow rapid identification of successfully transformed plant material. In the expression cassettes shown in Figures 1-4, for example, the marker is hygromycin phosphotransferase (hptII) which confers hygromycin resistance to plants expressing the marker. Thus, in such cassettes, the terminator or region of DNA that is involved in 3'-terminal formation and polyadenylation of the RNA transcript is operably linked to the hptII gene sequence. Other selectable and screening markers can be used in place of hptll.

EXAMPLES

Example 1

Conventional silencing constructs

[0174] The efficacy of various silencing constructs was tested by interacting with the beta-glucuronidase (gus) reporter gene operably linked to the strong constitutive promoter of the leprosy mosaic virus, designated herein as P1 (SEQ ID NO: 1). The test setup is sharp as the gus protein is very stable. Thus, only a relatively large reduction in gus transcript levels results in a phenotypically detectable reduction in gus protein levels. Most silencing constructs contain at least one copy of the same 304 bp gus gene fragment (SEQ ID NO: 2) operably linked in sense or antisense orientation to a strong constitutive promoter followed, in some cases, by the Agrobacterium nopaline synthase gene terminator . The silencing constructs were inserted next to the expression cassette of the hygromycin phosphotransferase (hptII) selectable marker gene between the T-DNA borders of the transformation vectors.

The obtained vectors were used to remodel a tobacco plant that had previously been transformed with T-DNA containing the gus gene expression cassette (see also Figures 1 and 2).

[0175] To investigate the role of the element terminator in conventional silencing constructs, the following transformation vectors were generated: [0176] pSIM714: negative control vector pSIM714 that did not contain the silencing construct.

[0177] pSIM718: vector pSTM718, which contains a sense fragment of the gus gene operably linked to a terminator of the nopaline synthase gene (SEQ ID NO: 3) which is the strategies described in e.g. US Patents 5,283,184 and 5,231,020. This vector contains the gus gene fragment operably linked in sense orientation to a promoter followed by a terminator.

[0178] pSIM 140: Vector pSIM140, which is identical to pSIM718 except that the silencing construct does not include a terminator.

[0179] pSIM755: Vector pSIM755 which contains a terminator-containing antisense construct that represents the strategies described in e.g. US Patents 5,107,065 and 5,759,829. This vector contains the gus gene fragment operably linked in sense orientation to a promoter followed by a terminator.

[0180] pSIM758: Vector pSIM758, which is identical to pSIM755, except that the silencing construct does not contain a terminator.

[0181] pSIM374: Vector pSIM374, which contains a terminator-containing construct, including both the sense and antisense fragment of the gus gene, and represents the strategies described in, e.g., WO 99 / 53050A1. This vector contains two copies of the gus gene fragment, one in the sense orientation and the other in the antisense orientation, separated from each other by the intron shown in SEQ ID NO: 4, inserted between the promoter and the terminator.

[0182] pSIM728 and 777: Vector pSIM728, which is identical to pSIM374 except that the silencing construct does not contain a terminator. The vector pSIM777 is identical to pSIM728 except that the P2 promoter is on the other side of the expression cassette.

[0183] Binary vectors containing the various constructs are introduced into Agrobacterium. A 10-fold dilution of the overnight culture of the resulting strains was grown for five to six hours, precipitated for 15 minutes at 2800 rpm, washed with MS liquid medium (Phytotechnology, KS) enriched with sucrose (3%, pH 5.7) and resuspended in this medium alone to 0.2 OD / 600nm. The suspension was then used to infect leaf explants of a transgenic in vitro grown Nicotiana tabacum (tobacco) plant expressing the gus gene. Infected tobacco explants were incubated for 2 days in co-culture medium (1/10 MS salt, 3% sucrose, pH 5.7) containing 6 g / L agar at 25 ° C in a Percival incubator (16/8 hours light) and then transferred for M401 / agar medium (Phyto Technology) containing timentin (150 mg / l) and kanamycin (20 mg / l). The resulting shoots were transferred to a hormone-free rooting medium and three leaves of each obtained plant were stained for gus expression.

[0184] Table 1 shows that all plants retransformed with pSIM714 showed the same level of gus expression as the original gus, confirming that the reporter gene expression is not adversely affected by retransformation, single cell proliferation and regeneration.

[0185] Table 1 also shows that the constructs representing the three different methods of conventional silencing with different efficiencies induce gus gene silencing. pSIM374 is the most effective, which is in line with the literature on the subject. About half of the plants that were reconverted with these constructs showed some reduction in gus activity levels. Two other constructs supported a reduction in gus activity by only about 6% in the retransformed plants.

[0186] Importantly, Table 1 also shows that removal of the terminator dramatically reduces the effectiveness of the silencing constructs. For example, pSIM374 is more than six times more effective than its non-terminator derivative, pSIM728. Deprived of the terminator, pSIM758 shows almost no activity.

[0187] It can be concluded that the terminator plays an important role in optimizing the activity of conventional silencing constructs.

Example 2

Efficient gene silencing with terminatorless constructs containing at least two copies of the target gene fragment that results in convergent transcription

[0188] To investigate the effect of convergent transcription on gene silencing, the following transformation vectors were generated (see also Figure 3) [0189] pSIM715: The vector pSIM715 contains a construct that contains a first segment consisting of a fragment of the gus gene operably linked to the promoter (P1) and a second segment in the opposite direction, which consists of the same gus gene fragment operably linked to the 35S constitutive promoter of cauliflower mosaic virus, denoted as P2 and is shown in SEQ ID NO: 5, the first and second segments are separated by two different introns.

[0190] pSIM717: The vector pSIM717 is identical to pSIM715 except that the two construct segments are separated by a single intron.

[0191] pSIM789: The vector pSIM789 is identical to pSIM717 except that the P2 promoter is replaced with the P1 promoter.

[0192] pSIM771: The vector pSIM771 is identical to pSIM717 except that the P1 promoter is replaced by the potato ubiquitin 7 promoter which is shown in SEQ ID NO: 6 and referred to herein as P3.

[0193] pSIM770: The vector pSIM770 is identical to pSIM717 except that the P2 promoter that directs expression of the selectable marker gene is replaced with P1 and the P1 promoter of the silencing construct is replaced with P2.

[0194] pSIM772: The vector pSIM772 contains the gus gene fragment inserted between two oppositely oriented promoters: P2 and P3. [0195] pSIM756: The vector pSIM756 is identical to pSIM717 except that the gus gene fragments are oriented in the opposite orientation complementary to the promoter to which they are directly linked.

[0196] pSIM779: The vector pSIM779 is an example of tandem repeats of gus gene fragments inserted between two convergent promoters.

Q7

[0197] pSIM787: The vector pSIM787 is similar to pSIM779 but contains four target gene fragment repeats inserted between the convergent promoters.

[0198] pSIM1111 is identical to pSIM779 except that the four repeats precede the antisense DNA fragment of the gus gene which differs from SEQ ID NO: 2) and is shown as SEQ ID NO: 7

[0199] The gus tests performed on the re-transformed gus plants show that all the terminator-free constructs tested, containing two segments each containing a different promoter driving the gus gene fragment, in the reverse complement orientation, pSIM715, 717 and 756, 771 are more effective in gus gene silencing than pSIM374, the construct that represents the best conventional approach. Moreover, pSIM789 also causes efficient gene silencing in many double transform antennas.

[0200] The experiment also shows that the use of a single fragment of the gus gene (pSIM779) is ineffective. This result suggests that the convergent transcription of at least two copies of the desired polynucleotide is important for effective silencing.

[0201] The study also showed that the double repeat construct (pSIM780) triggers gene silencing. However, this arrangement was not as effective as the pSIM756 inverted repeat system (Table 1). Moreover, the four direct repeats (pSIM787) are more effective than the two repeats (Table 1).

[0202] To study the molecular basis of terminator-free silencing, RNA was isolated from three plants that had been re-transformed with pSIM717 and three additional plants transformed with pSIM715. In each case, one plant represented ineffective silencing, while two other plants showed near-complete silencing of the gus gene.

[0203] To test the production of transcripts from the two different promoters used in pS1M715 and pSIM717, polymerase chain reverse transcription reactions (RT-PCR reactions) were performed. The first primer used in these experiments (PG, shown in SEQ ID NO: 8) is sequence specific fragment of the gus gene and hybridizes the transcripts obtained from both strands. The second primer was designed to hybridize to single strand intron sequences (pIF shown in SEQ ID NO: 9, hybridizes to a linker region sequence derived from GBSS-intron transcripts produced by the P1 promoter and PIR shown in SEQ ID NO: 10, hybridizes to transcripts produced by the P2 promoter). Interestingly, these studies showed that the construct of non-silencing plants 717-7 and 717-13 contained only transcripts obtained from one of the two strands, T1 or T2 (Figure 7). In contrast, silenced plants 715-19, 715-38, 717-55, and 717-36 produced transcripts from both strands (Figure 7). Thus, effective silencing is only achieved if both promoters of the construct are functionally active simultaneously.

[0204] Hybridization in consecutive blots of RNA gels with radiolabeled probes derived from the gus gene showed that effective silencing at 715-38, 715-55, 717-12, 717-36, and 717-19 correlated with a sharp decrease in RNA gus accumulation ( Fig. 8). In addition, the transcripts produced by the silencing construct in fully silenced plants generally varied in size from 0.2 kbp to about 1.0 kbp (Figure 8). Although the RTPCR showed the presence of additional large transcripts that contain not only the polynucleotide but also further promoter sequences, the presence of these transcripts was difficult to detect in RNA gel blots. For example, hybridization with a probe derived from the P1 promoter required seven days of exposure before very faint streaks could be observed.

Example 3

Mute in potato tubers

[0205] To test silencing in tubers, various vectors were developed with terminator-free vectors. These vectors contained the neomycin phosphotransferase (NPTII) expression cassette as a selectable marker system (see also figure 6). The promoters used for gene silencing in potato tubers were selected from the group consisting of: (1) the strong potato ubiquitin 7 promoter, (2) the strong promoter of the tuber specific granule bound starch synthase (GBSS) gene (SEQ ID NO: 12), and (3) ), potato tuber and runner-specific strong promoter of the glucose ADP pyrophosphorylase (AGP) gene (SEQ ID NO: 13). See Figure 4 for DNA transfer maps.

[0206] pSIM764: The vector pSIM764 contains a tuber silencing construct that includes a first segment consisting of an appended element. trailer) 154 bp of the potato tuber-expressed PPG gene (SEQ ID NO: 14), operably linked to the GBSS promoter and a second segment in the opposite direction, which consists of the same fragment of the linked element operably linked to the GBSS promoter, the first and the second segment being are separated by an intron from the potato ubiquitin 7 gene shown in SEQ ID NO: 15

[0207] pSIM765: The vector pSIM765 is identical to pSIM764 except that the PPO gene fragments are oriented in the opposite direction.

[0208] pSIM217 is the control plasmid and contains two copies of the PPO gene inserted as an inverted repeat between the GBSS promoter and the ubiquitin terminator.

[0209] Ten-fold dilutions of overnight cultures were grown for 5-6 hours, precipitated for 15 minutes at 2800 rpm, washed with MS liquid medium (Phytotechnology) supplemented with sucrose (3%, pH 5.7) and resuspended in the same medium to 0.2 OD / 600nm. The suspended cells were mixed and used to infect 0.4-0.6 mm internode segments of a cultivar Ranger Russet. Infected stems were incubated for two days in co-culture medium (1/10 MS salt, 3% sucrose, pH 5.7) containing 6 g / L agar at 22 ° C in a Percival incubator (16 hours light), then transferred to callus induction medium (CIM, MS medium supplemented with 3% sucrose, 2.5 mg / L zeatin riboside, 0.1 mg / L naphthylacetic acid and 6 g / L agar) containing timentin (150 mg / L) and kanamycin (100 mg / L). After one month of cultivation in CIM, the explants were transferred to shoot induction medium (SIM, MS medium supplemented with 3% sucrose, 2.5 mg / L zeatin riboside, 0.3 mg / L GA3 gibberellic acid and 6 g / L agar) containing timentin and kanamycin (150 and 100 mg / L, respectively) until shoots are formed. Shoots emerging at the end of the regeneration period were transferred to MS medium with 3% sucrose, 6 g / L agar and timentin (150 mg / L). The transgenic plants were transferred to the ground and placed in an incubator (11 hours light, 25 ° C). After three weeks, at least 3 minitubers / line were tested for PPO activity. For this purpose, 1 g of potato tubers were pulverized in liquid nitrogen, added to 5 ml of a solution of 50 mM MOPS (3- (Nmorpholin) propane-sulfonic acid) buffer (pH 6.5) containing 50 mM pyrocatechin and incubated at room temperature by swirling through about 1 hour. The solid fraction was precipitated and the supernatant transferred to another tube for PPO activity determination. For this purpose, 1 g of potato tubers were pulverized in liquid nitrogen, the powder was added to 5 ml of a solution of 50 mM MOPS (3- (N-morpholino) propane-sulfonic acid) (pH 6.5) containing 50 mM pyrocatechin and incubated at a temperature of by swirling for about 1 hour. The solid fraction was precipitated and the supernatant transferred to another tube to determine PPO activity by measuring the change in optical density 410 over time. Experience has shown that pSIM764 and 765 trigger effective silencing in potato tubers (Table 2). A comparison with the data presented in WO 2003/069980 shows that the method of the present invention can be more effective than conventional terminator-based gene silencing, such as by pSIM217, PPO control.

Example 4

Multi-gene silencing in tobacco

[0210] To study the effect of the positioning of the gene fragments within the silencing construct, two constructs were generated. To this end, two copies of the gus gene fragment of pSIM771 were replaced by two copies of the gus gene fragment fused to a fragment of the tobacco polyphenol oxidase (PPO) gene (SEQ ID NO: 16) (also see Figure 6):

[0211] pSIM774: The vector pSIM774 contains a silencing construct with gus gene fragments directly fused to the promoters and adjacent PPO gene fragments fused to the central intron.

[0212] pSIM775: The vector pSIM775 contains a silencing construct with gus gene fragments directly fused to the promoters and adjacent PPO gene fragments fused to the central intron.

[0213] Retransformation of gus plants with these vectors is expected to trigger silencing as efficiently as pSIM771.

Example 5

Silencing multiple genes in a potato

[0214] Multiple gene silencing is accomplished by simultaneously acting on three unwanted potato tuber genes.

[0215] Plasmid pSIM1121 (Russet Boise II) contains the completely native transfer DNA shown in SEQ ID NO: 17, containing a silencing construct containing two copies of a DNA segment, separated by the potato ubiquitin 7 gene intron and placed as an inverted repeat between two convergent GBSS promoters at where the DNA segment contains (i) a fragment of an attached element expressed in the tuber of the wild potato polyphenol oxidase gene Solanum yerrucosum relative

Schltdl. TRHRG193, Accession No. 498062 (see LISDA, ARS, National Genetic Resources Program. Germplasm Resources Information Network (GRIN). [Online database] National Germplasm Resources Laboratory, Beltsville, Maryland. Available: http://www.ars-grin .gov2 / cgibin / npgs / html / acchtml.pl 1392998, September 12, 2005) (SEQ ID NO: 18) (ii) a fragment of the leader of the L phosphorylase gene (SEQ ID NO: 19), and (iii) a fragment of the leader of the R1 gene, (SEQ ID NO: 20).

[0216] The use of this plasmid produces transformed potato plants that contain only native DNA and exhibit the following new characteristics: (1) breakage tolerance due to silencing of the PPG gene expressed in the tuber, (2) reduction of cold-induced glucose accumulation due to silencing of the phosphorylase and R1 genes.

Example 6

Highly effective influence on the promoter

[0217] The following transformation vectors were developed to demonstrate that target promoter sequences can be used to silence target gene expression (see also Fig. 4)

[0218] pSIM773: The vector pSIM773 contains a construct comprising a first segment containing a P3 promoter linked to P1 and a second segment which is oriented in the opposite direction and which consists of P2 linked to P1. The first and second segments are separated by an intron. Thus, the construct contains four functionally active promoters. The two promoters in the middle are identical, constitute the target promoter, and are in a convergent orientation. The two outer promoters are different from each other and are in convergent orientation.

All four promoters contain the TATA box and follow a base pair upstream of the transcription start site.

[0219] pSIM1101: The vector pSIM1101 is identical to pSIM773 except that the P3 promoter has been replaced by the nos terminator.

[0220] pSIM788: The vector pSIM788 is similar to pSIM773 with the difference that the two central P1 promoters of the target gus gene only contain sequences upstream of the TATA box (SEQ ID NO: 21), thus constituting non-functional promoters.

[0221] pSTM1120: The vector pSIM1120 is similar to pSIM773 with the difference that the two central promoters of the target gene do not have a TATA box and are not oriented in a convergent but divergent direction.

[0222] pSIM1112: The vector pS1M1112 contains one non-functional P1 promoter inserted between the convergent P2 and P3 promoters.

[0223] pSTM1113: The vector pSTM1113 contains two convergent P1 promoters separated by an intron.

[0224] pSTM754: The control vector pSIM754 contains the P1 promoter driving the expression of the P2 promoter and vice versa.

[0225] Retransformation of gus plants with the pSIM773 vector resulted in 35 hygromycin resistant plants. PCR analysis confirmed the presence of pSIM773 transfer DNA. Surprisingly, gus staining showed a very effective, complete silencing of the gus gene (Table 1). Twenty plants (57%) showed no detectable gus expression. Thus, the action of the promoter using the pSIM773 strategy is highly desirable.

[0226] Similar results were obtained with target promoters of divergent orientation inserted between two convergent targeting promoters, 77% of plants that were retransformed with pSIM1120 showed complete silencing of the gus gene (Table 1).

[0227] Table 1 shows that gene silencing was also achieved by using one target promoter inserted between two convergent driver promoters (pSiM1112). However, this method may be less effective than methods that use two copies of the promoter arranged as an inverted repeat. [0228] Moreover, the efficacy of pSIM1113 shows that targeting promoters are not always necessary. It is possible to effectively silence a gene by simply using two convergent target promoters (Table 1).

[0229] Many (44%) of the plants, re-transformed with pSIM1101, showed complete gene silencing (Table 1). This result shows that promoter-based silencing does not require convergent transcription.

[0230] It has often been found that conventional silencing methods do not provide stable gene silencing across generations. In contrast, the four promoter constructs represented by pSIM773 resulted in complete silencing which is completely conserved upon transmission of the silencing cassette to the next generation. Increased stability was demonstrated by maturing double transformed tobacco plants and then determining the level of gus expression in the T1 progeny. This study showed that 100% of the progeny that had been obtained from the pSIM773 plant and contained both the gus gene and the silencing cassette had full gus gene silencing (Table 3). In contrast, none of the T1 plants harboring the gus gene and the pSIM374 silencing cassette showed complete gus gene silencing (Table 3). An intermediate phenotype was found by analyzing the progeny of plants carrying the gus gene and the pSIM717 silencing cassette (Table 3).

Example 7

Sequence requirements for running on promoto

[0231] The above experiments have shown that promoter sequences can be used to effectively trigger gene silencing. However, it should not be understood that any promoter fragment of the target gene may be used for this purpose.

[0232] To test the requirements for promoter-based silencing sequences, two vectors were created that contain two copies of only part of the P1 promoter, inserted as an inverted repeat between the driver promoters.

[0233] PSIM1118: The vector pSIM1118 contains two copies of the starting 300 bp fragment of the promoter shown in SEQ ID NO: 11

[0234] pSIM1119: The vector pSIM1119 contains two copies of the central 300 bp region of the P1 promoter shown in SEQ ID NO: 51

[0235] Retransformation of the gus plants with two different constructs resulted in 34 and 20 plants, respectively, which were analyzed histochemically. Interestingly, none of the plants tested showed reduced expression of gus, which indicates that the promoter fragments used did not induce effective gene silencing (Table 1).

[0236] Figure 9 shows the sequence analysis of various promoter fragments. The fragment that facilitates efficient gene silencing is present in pSIM773, 788, 1101 and 1120, but not in pSIM1118 and 1119.

Example 8

Reduced cold-induced sweetness in tubers of potato plants containing silencing constructs involving two copies of the promoter fragment of the R1 gene

[0237] The promoter sequence of the potato starch related R1 gene including the leader and the start codon is shown in SEQ ID NO: 22. Two copies of the short (342 bp) fragment of the R1 promoter (SEQ ID NO: 23) were inserted as an inverted repeat between or two convergent oriented promoters of the GBSS promoter (in plasmid pSIM1038) or the GBSS promoter and the AGP promoter in convergent orientation (in plasmid pSIM1043). The obtained binary vectors were used to generate transformed potato plants. These plants were able to develop tubers that were stored for about a month or more at 4 ° C. Analysis of the glucose content of chilled tubers showed that transformed plants accumulate less glucose than untransformed control plants. The reduced accumulation of glucose will result in less staining when frying the fries, thus reducing the blanching time and preserving the potato's original flavor to a greater extent. In addition, gene silencing through the R1 promoter will reduce starch phosphorylation and thus reduce the environmental problems associated with the generation of potato starch-containing wastewater. Other benefits of transformed tubers include: (1) the resulting fries contain less of the toxic acrylamide that forms from the reaction between glucose and asparagine, and (2) the resulting fries are more brittle, as judged by a professional tasting panel, due to their slightly altered starch structure.

[0238] Similar results can be obtained by using the shorter (151 bp) part of the R1 promoter, shown as SEQ ID NO. 24. The PSIM1056 binary vector contains two copies of this fragment inserted as an inverted repeat between two convergently oriented GBSS promoters; pSIM1062 contains fragments inserted between the convergent oriented GBSS and AGP promoters. This vector was used to generate transformed plants that exhibited reduced cold-induced glucose accumulation and all the benefits associated with this trait.

Example 9

Increased darkening tolerance in tubers of potato plants containing a silencing construct comprising two copies of the polyphenol oxidase gene promoter fragment

[0239] The promoter sequence of the tuber-expressed potato polyphenol oxidase gene is shown in SEQ ID NO: 25. Two copies (200 bp) of the PPG promoter (SEQ ID NO: 26) were inserted as inverted repeats between the convergent GBSS and AGP promoters. A binary vector containing this silencing construct, designated pSIM1046, was used to generate twenty-five transformed potato plants. The plants were allowed to develop tubers and tested for polyphenol oxidase activity. Such analysis shows that the target PPO gene expression level is reduced compared to the levels in untransformed control plants.

[0240] In a similar manner, the plasmid pSIM1045, containing two copies of the 460-bp PPO promoter fragment (SEQ ID NO: 27) inserted between the convergent GBSS and AGP promoters, can be used to lower the expression of the PPO gene.

[0241] Similar strategies can be applied to other plant species to reduce darkening. For example, the promoter of the leaf-expressed lettuce PPO gene can be used to reduce darkening of lettuce leaves, the promoter of the fruit-expressed apple PPO gene can be used to reduce the darkening of the apple fruit, and the promoter of the seed-expressed wheat PPO gene can be used to reduce darkening wheat grains. In all these and other instances, the promoter can be isolated simply by designing primers that hybridize to the known PPO gene sequences and performing known DNA isolation methods such as reverse PCR.

Example 10

Improved oil content in rapeseed containing a silencing construct that includes two copies of the Fad2 gene promoter fragment

[0242] The promoter sequence of the brassica Fad2 gene including the leader and the start codon is shown in SEQ ID NO: 28. Two copies of the fragment of this promoter containing no transcribed sequences, such as the 441-bp fragment shown in SEQ ID 29, can be inserted inverted a repetition between two convergent oriented promoters which are expressed in cruciferous seeds. Examples of driving promoters are the napin gene promoter (1.7S seed storage protein gene) shown in SEQ ID NO: 30 or the stearoyl-ACP desaturase gene promoter (SEQ ID NO: 31).

[0243] The silencing cassette can be placed within the DNA sequence of a transfer binary vector that can be used to transform plants of the genus Brassica. Some of the resulting plants will produce seeds that contain increased amounts of oleic acid.

[0244] Other promoters that can be used in silencing constructs to improve the oil content of oilseeds such as rapeseed, soybean, cotton, sunflower, include promoters of other genes in the fatty acid biosynthetic pathway. For example, a 12 target fatty acid desaturase promoter or omega-6 microsomal desaturase gene (FAD12) (e.g., Genbank accession no. AF243045 for rapeseed and AB188250 for soybean), such as the soybean FAD12 promoter shown in SEQ ID NO: 32, it can be used to increase the concentration of oleic acid in crops such as rapeseed and soybeans.

[0245] Furthermore, the promoters of the stearoyl acyl carrier protein delta 9 desaturase and oleyl phosphatidylcholine omega 6-desaturase genes can be used to increase the stearic acid and oleic acid poisomes in cotton plants, respectively. This promoter can be identified by guiding methods such as PCR using the reverse known sequence of the target genes (Liu et aL, Plant Physiol 129: 1732-43,2002). Two copies of the freshly isolated promoter can be used in strategies similar to those shown for pSIM773, wherein the seed specific targeting promoters can represent both foreign DNA and native DNA.

Example 11

Reduced lignin content in the vascular system of alfalfa plants containing a silencing construct containing two copies of the COMT gene promoter fragments

[0246] The promoter of the caffeic acid / 5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene, Medicago sativa (alfalfa), containing the leader is shown in SEQ ID NO: 33.

[0247] Two copies of the 448-bp promoter fragment containing no transcribed sequences (SEQ ID NO: 34) were inserted as an inverted repeat between the two convergent oriented guide promoters. The first drive promoter is the petE gene promoter shown in SEQ ID NO: 35; the second promoter is the Pal gene promoter shown in SEQ ID NO: 36. A binary vector containing this silencing construct, designated pSIM1117, was used to generate transformed alfalfa plants. Stem tissue of these plants was examined and it was shown to contain reduced amounts of lignin.

[0248] Lignin reduced content may be determined according to the following protocol: (I) cut stem fragments and place them on a watch glass, (ii) immerse the cut stems in a 1% potassium permanganate solution for 5 min at room temperature, (iii) remove the potassium permanganate solution with a disposable pipette and wash the samples twice with water to remove excess potassium permanganate, (iv) add 6% HCI (v / v) and wait for the section to change color from black or dark brown to light brown, (v) add additional HCl if necessary, to facilitate dark color removal, (vi) remove HCl and wash samples twice with water, (vii), add a few drops of 15% sodium bicarbonate solution (sometimes may not go into solution completely), dark red or reddish purple color will develop for hardwood (higher S-unit content) and brown color for softwood (higher G-unit content).

[0249] Nineteen transformed alfalfa lines were tested for lignin reduction, six plants were found to accumulate reduced amounts of lignin S-units.

[0250] Instead of the promoter of the COMT gene, it is also possible to use the promoter of the caffeoyl CoA 3-O-methyltransferase (CCOMT) gene. The sequence of this promoter, together with the attached leader is shown in SEQ ID NO: 37. In order to lower the lignin content, a fragment of SEQ ID NO: 29 which does not contain the transcribed sequences as shown in SEQ ID NO: 38.

[0251] Similarly, the lignin content of trees can be reduced by using promoters of genes involved in lignin biosynthesis. It is also possible to use SEQ ID NO: 59 and reduce the lignin content of maize using the promoter-based silencing methods described above.

Example 12

Extended shelf life of tomato plant fruit containing a silencing construct comprising two copies of the polygalacturonase gene promoter fragment

[0252] A polygalacturonase target gene promoter such as the tomato promoter shown in SEQ ID NO: 39 can be used to reduce pectin degradation, leading to slower cell wall degradation, retard softening, improve viscosity properties, and increase tomato durability by inserting two copies of the fragment promoter as an inverted repeat between the fruit-specific convergent driving promoters.

Example 13

Reduced allergenicity of plant foods containing a silencing construct consisting of two copies of the promoter fragment of genes encoding allergens

[0253] The promoter of the mai d 1 apple major allergen gene can be isolated by using reverse PCR methods with a known gene sequence (Gilissen et aL, J Allergy Clin Immunol 115: 364-9, 2005), this promoter can then be used to develop variants apples that contain lower levels of the allergen.

[0254] Likewise, the Ara h 2 major peanut allergen promoter (Dodo et al., Curr Allergy Asthma Rep 5, 67-73, 2005) can be isolated by reverse PCR methods and used to develop peanut varieties that contain lower allergen levels.

[0255] Furthermore, the major soybean allergen promoter Gly m Bd 30 K (Herman et aL, Plant Physiol 132, 36-43, 2003) can be isolated by reverse PCR methods and used to develop peanut varieties that contain lower levels of the allergen .

Example 14

Multi-gene silencing based on the combination of gene fragments and promoter fragments

[0256] The plasmid pSIM870 (Russet Boise III) contains all native transfer DNA shown in SEQ ID NO: 40 includes: (1) a first silencing cassette containing two copies of a DNA segment situated as an inverted repeat between two convergent GBSS promoters, the DNA segment comprising (i) a fragment of an attached element of the tuber expressed Solanum verrucosum polyphenol oxidase gene, (ii) a fragment of the leader of the L-phosphorylase gene and (iii) a fragment of a fused element of the phosphorylase L gene (SEQ ID NO: 41) and (2) a second silencing cassette containing two copies of the R1 promoter placed as an inverted repeat between the AGP and GBSS driver promoters, respectively.

[0257] Plasmid pSIM899 (Russet Boise IV) contains all-native transfer DNA shown in SEQ ID NO: 42 containing a first silencing cassette containing two copies of a DNA segment positioned as an inverted repeat between two convergent GBSS promotions, the DNA segment comprising (and ) a fragment of an attachment element expressed in a tuber of a Solanum verrucosum polyphenol oxidase gene, and (ii) a leader fragment of a phosphorylase L gene; and a second silencing cassette containing four copies of the R1 gene leader operably linked to the AGP promoter, followed by an inverted repeat containing the sense and antisense fragment of the R1 gene.

[0258] Transformation of potato with any of these three plasmids produces plants which, compared to non-transformed plants, exhibit the following properties: (1) decreased expression of the polyphenol oxidase gene expressed in the tuber and, consequently, (i) increased tuber polyphenol content, which can be determined from xx, and (ii) increased tolerance to darkening of the tubers, which can be determined from xx, and (2) significantly reduced expression of the phosphorylase gene and the R1 gene, and hence, (i) reduced phosphorylation of starch and, consequently, reduced phosphate content in the waste water containing potato starch, and (ii) reduced conversion of starch to glucose during cold storage as determined by the glucose oxidase / peroxidase reagent (Megazyme, Ireland) resulting in (a) less caramelization and hence reduced color during frying which allows storage at elevated temperatures and / or shorter blanching times (b) reduction of acrylamide formation, and (c) increased crispness of fries. Example 15

Action on introns

[0259] The polynucleotide used to generate the TFCT construct may contain a gene intron that produces the target transcript. The concept of intron directed silencing can be represented by the intron of the gus gene expressed in transgenic tobacco.

[0260] The following transformation vector was developed to demonstrate that target intron sequences can be used to silence target gene expression (see also Fig. 5).

[0261] The vector pSIM782, which contains a construct that contains a first segment composed of the gus gene intron operably linked to the promoter (P1) and a second segment in the opposite direction, which consists of the same gus gene intron operably linked to a second constitutive promoter (P2 ), with the first and second segments separated by an intron.

[0262] An example of an intron that can be used for gene silencing is the intron of the starch-bound R1 gene Solanum vernei SEQ ID NO: 44. Silencing the R1 gene reduces the degree of cold-induced sweetness of tubers during storage.

Example 16

Action on a terminator

[0263] The polynucleotide used to generate the TFCT construct may contain sequences downstream of the transcribed target gene sequence. This concept can be demonstrated by the sequence downstream of the gus gene that is expressed in transgenic tobacco.

Example 17

Reduced lignin content in the vascular system of alfalfa plants containing a silencing construct comprising two copies of the COMT gene fragment

[0264] The binary vector designated pSTM856 was assembled containing the expression cassette consisting of the two COMT gene fragments shown in SEQ ID NOS: 52 and 53, positioned as an inverted repeat between the two convergent alfalfa promoters shown in SEQ ID NOS: 54 and 55 therein. the way that the promoters are operably linked to the first antisense fragment and then to the sense fragment. The expression cassette is inserted between the alfalfa-derived sequences that act as Agrobacterium border replacers and are shown in SEQ ID NOs: 56 and 57. The complete transfer DNA shown in SEQ ID NO: 58 is inserted into a plasmid that carries the expression cassette into the gene Agrobacterium IPT in the backbone.

[0265] Transformations were performed as described by Weeks and Rommens in patent application US20050034188A1. The two transformed plants were tested for lignin content and in both there was no apparent accumulation of S.

TABELS

[0266]

Table 1. Efficacy of conventional and non-terminator quench constructs.

<td>Construct for the second transformation</td><td>Tobacco plants tested</td><td colspan="4">expression of gus</td>

<td></td><td></td><td> 50-700%</td><td> 70-50%</td><td> 7-70%</td><td> 0%</td>

<td>lack</td><td> 3</td><td> 3 (100%)</td><td> 0</td><td> 0</td><td> 0</td>

<td>PSIM714</td><td> 8</td><td> 8 (100%)</td><td> 0</td><td> 0</td><td> 0</td>

<td>PSIM374</td><td> 36</td><td> 13 (36%)</td><td> 11 (31%)</td><td> 9 (25%)</td><td> 3 (8%)</td>

<td>PSIM718</td><td> 35</td><td> 33 (95%)</td><td> 1 (3%)</td><td> 1 (3%)</td><td> 0</td>

<td>PSIM728</td><td> 23</td><td> 15 (65%)</td><td> 5 (22%)</td><td> 3 (13%)</td><td> 0</td>

<td>PSIM715</td><td> 37</td><td> 10 (27%)</td><td> 11 (30%)</td><td> 15 (41%)</td><td> 1 (3%)</td>

<td>PSIM717</td><td> 35</td><td> 11 (31%)</td><td> 3 (9%)</td><td> 19 (54%)</td><td> 2 (6%)</td>

<td>PSIM754</td><td> 38</td><td> 38 (100%)</td><td> 0</td><td> 0</td><td> 0</td>

<td>PSIM755</td><td> 36</td><td> 35 (97%)</td><td> 0</td><td> 0</td><td> 1 (3%)</td>

<td>pSIM756</td><td> 37</td><td> 18 (49%)</td><td> 12 (32%)</td><td> 5 (14%)</td><td> 2 (5%)</td>

<td>PSIM758</td><td> 29</td><td> 29 (100%)</td><td> 0</td><td> 0</td><td> 0</td>

<td>PSM770</td><td> 38</td><td> 35 (92%)</td><td> 3 (8%)</td><td> 0</td><td> 0</td>

<td>PSIM771</td><td> 35</td><td> 20 (57%)</td><td> 3 (9%)</td><td> 9 (26%)</td><td> 3 (9%)</td>

<td>PSM772</td><td> 35</td><td> 34 (97%)</td><td> 0</td><td> 1 (3%)</td><td> 0</td>

<td>PSIM773</td><td> 35</td><td> 15 (43%)</td><td> 0</td><td> 0</td><td> 20 (57%)</td>

<td>PSIM774</td><td> 35</td><td> 31 (89%)</td><td> 2 (6%)</td><td> 2 (6%)</td><td> 1 (3%)</td>

<td>PSIM775</td><td> 36</td><td> 22 (61%)</td><td> 6 (17%)</td><td> 7 (19%)</td><td> 1 (3%)</td>

<td>PSIM777</td><td> 36</td><td> 33 (92%)</td><td> 1 (3%)</td><td> 1 (3%)</td><td> 1 (3%)</td>

<td>PSIM778</td><td> 36</td><td> 32 (89%)</td><td> 2 (6%)</td><td> 2 (6%)</td><td> 0</td>

<td>PSIM779</td><td> 36</td><td> 33 (92%)</td><td> 1 (3%)</td><td> 2 (6%)</td><td> 0</td>

<td>PSIM782</td><td> 35</td><td> 34 (97%)</td><td> 0</td><td> 1 (3%)</td><td> 0</td>

<td>PSIM787</td><td> 32</td><td> 20 (63%)</td><td> 7 (22%)</td><td> 3 (9%)</td><td> 2 (6%)</td>

<td>PSIM788</td><td> 35</td><td> 14 (40%)</td><td> 0</td><td> 0</td><td> 21 (60%)</td>

<td>PSIM789</td><td> 35</td><td> 19 (54%)</td><td> 4 (11%)</td><td> 6 (17%)</td><td> 6 (17%)</td>

<td>Construct for the second transformation</td><td>Tobacco plants tested</td><td colspan="4">expression of gus</td>

<td></td><td></td><td> 50-700%</td><td> 70-50%</td><td> 7-70%</td><td> 0%</td>

<td>PSIM1101</td><td> 34</td><td> 14 (41%)</td><td> 0</td><td> 5 (15%)</td><td> 15 (44%)</td>

<td>PSIM1111</td><td> 36</td><td> 21 (58%)</td><td> 9 (25%)</td><td> 6 (17%)</td><td> 0</td>

<td>pSIM1112</td><td> 36</td><td> 33 (92%)</td><td> 1 (3%)</td><td> 0</td><td> 2 (6%)</td>

<td>pSIM1113</td><td> 34</td><td> 24 (71%)</td><td> 2 (6%)</td><td> 3 (9%)</td><td> 5 (15%)</td>

<td>PSIM1118</td><td> 34</td><td> 34 (100%)</td><td> 0</td><td> 0</td><td> 0</td>

<td>PSIM1119</td><td> 20</td><td> 20 (100%)</td><td> 0</td><td> 0</td><td> 0</td>

<td>pSIM1120</td><td> 35</td><td> 8 (23%)</td><td> 0</td><td> 0</td><td> 27 (77%)</td>

Table 2. PPO activity in potato minitubers at t = untransformed wild-type plants; '401' = transformed plants harboring transfer DNA containing only the expression cassette npt11 gene of the selectable marker, OD = OD260, SE = standard error.

<td>Control sample</td><td>rep-1 (OD)</td><td>rep-2 (OD)</td><td>rep-3 (OD)</td><td>% TUE</td><td>SE</td>

<td>Tue-1</td><td> 0,127</td><td> 0,121</td><td> 0,137</td><td> 87</td><td> 2,6</td>

<td>Tue-2</td><td> 0,129</td><td> 0,141</td><td> 0,125</td><td> 89</td><td> 2,7</td>

<td>Tue-3</td><td> 0,138</td><td> 0,146</td><td> 0,123</td><td> 92</td><td> 3,7</td>

<td>Tue-4</td><td> 0,134</td><td> 0,157</td><td> 0,159</td><td> 101</td><td> 4,4</td>

<td>Tue-5</td><td> 0,152</td><td> 0,173</td><td> 0,169</td><td> 111</td><td> 3,6</td>

<td>Tue-6</td><td> 0,153</td><td> 0,152</td><td> 0,151</td><td> 103</td><td> 0,3</td>

<td>Tue-7</td><td> 0,173</td><td> 0,158</td><td> 0,167</td><td> 112</td><td> 2,4</td>

<td>Tue-8</td><td> 0,149</td><td> 0,165</td><td> 0,152</td><td> 105</td><td> 2,7</td>

<td>Control sample</td><td>rep-1 (OD)</td><td>rep-2 (OD)</td><td>rep-3 (OD)</td><td>% TUE</td><td>SE</td>

<td></td><td></td><td></td><td></td><td></td><td></td>

<td> 401-1</td><td> 0,138</td><td> 0,155</td><td> 0,174</td><td> 105</td><td> 5,7</td>

<td> 401-2</td><td> 0,182</td><td> 0,193</td><td> 0,163</td><td> 121</td><td> 4,8</td>

<td> 401-3</td><td> 0,139</td><td> 0,145</td><td> 0,152</td><td> 98</td><td> 2,1</td>

<td>pSIM784</td><td>rep-1 (OD)</td><td>rep-2 (OD)</td><td>rep-3 (OD)</td><td>% TUE</td><td>SE</td>

<td> 1</td><td> 0,051</td><td> 0,055</td><td> 0,060</td><td> 37</td><td> 1,4</td>

<td> 2</td><td> 0,071</td><td> 0,072</td><td> 0,068</td><td> 48</td><td> 0,7</td>

<td> 3</td><td> 0,063</td><td> 0,070</td><td> 0,075</td><td> 47</td><td> 1,9</td>

<td> 4</td><td> 0,035</td><td> 0,032</td><td> 0,030</td><td> 22</td><td> 0,8</td>

<td> 5</td><td> 0,045</td><td> 0,031</td><td> 0,030</td><td> 24</td><td> 2,7</td>

<td> 6</td><td> 0,053</td><td> 0,056</td><td> 0,056</td><td> 37</td><td> 0,6</td>

<td> 7</td><td> 0,079</td><td> 0,108</td><td> 0,117</td><td> 68</td><td> 6,3</td>

<td> 8</td><td> 0,035</td><td> 0,042</td><td> 0,041</td><td> 27</td><td> 1,2</td>

<td> 9</td><td> 0,039</td><td> 0,042</td><td> 0,043</td><td> 28</td><td> 0,7</td>

<td> 10</td><td> 0,081</td><td> 0,073</td><td> 0,077</td><td> 52</td><td> 1,3</td>

<td> 11</td><td> 0,059</td><td> 0,061</td><td> 0,052</td><td> 39</td><td> 1,5</td>

<td> 12</td><td> 0,056</td><td> 0,046</td><td> 0,053</td><td> 35</td><td> 1,5</td>

<td> 13</td><td> 0,036</td><td> 0,039</td><td> 0,032</td><td> 24</td><td> 1,1</td>

<td>Control sample</td><td>rep-1 (OD)</td><td>rep-2 (OD)</td><td>rep-3 (OD)</td><td>% TUE</td><td>SE</td>

<td> 14</td><td> 0,052</td><td> 0,068</td><td> 0,062</td><td> 41</td><td> 2,6</td>

<td> 15</td><td> 0,037</td><td> 0,033</td><td> 0,034</td><td> 23</td><td> 0,7</td>

<td> 16</td><td> 0,066</td><td> 0,057</td><td> 0,066</td><td> 43</td><td> 1,7</td>

<td> 17</td><td> 0,063</td><td> 0,061</td><td> 0,057</td><td> 41</td><td> 1,0</td>

<td> 18</td><td> 0,063</td><td> 0,041</td><td> 0,047</td><td> 34</td><td> 3,8</td>

<td> 19</td><td> 0,045</td><td> 0,049</td><td> 0,041</td><td> 30</td><td> 1,3</td>

<td> 20</td><td> 0,061</td><td> 0,051</td><td> 0,048</td><td> 38</td><td> 2,2</td>

<td> 21</td><td> 0,043</td><td> 0,039</td><td> 0,039</td><td> 27</td><td> 0,7</td>

<td> 22</td><td> 0,111</td><td> 0,102</td><td> 0,112</td><td> 73</td><td> 1,8</td>

<td> 23</td><td> 0,058</td><td> 0,049</td><td> 0,057</td><td> 37</td><td> 1,6</td>

<td> 24</td><td> 0,043</td><td> 0,041</td><td> 0,042</td><td> 28</td><td> 0,3</td>

<td> 25</td><td> 0,041</td><td> 0,040</td><td> 0,045</td><td> 28</td><td> 0,8</td>

<td> 28</td><td> 0,044</td><td> 0,042</td><td> 0,042</td><td> 29</td><td> 0,4</td>

<td>pSIM765</td><td>rep-1 (OD)</td><td>rep-2 (OD)</td><td>rep-3 (OD)</td><td>% TUE</td><td>SE</td>

<td> 1</td><td> 0,044</td><td> 0,035</td><td> 0,039</td><td> 27</td><td> 1,4</td>

<td> 2</td><td> 0,041</td><td> 0,048</td><td> 0,055</td><td> 32</td><td> 2,2</td>

<td> 3</td><td> 0,064</td><td> 0,060</td><td> 0,058</td><td> 41</td><td> 1,0</td>

<td> 5</td><td> 0,122</td><td> 0,118</td><td> 0,102</td><td> 77</td><td> 3,4</td>

<td>Control sample</td><td>rep-1 (OD)</td><td>rep-2 (OD)</td><td>rep-3 (OD)</td><td>% TUE</td><td>SE</td>

<td> 10</td><td> 0,042</td><td> 0,066</td><td> 0,059</td><td> 38</td><td> 3,9</td>

<td> 14</td><td> 0,087</td><td> 0,103</td><td> 0,111</td><td> 68</td><td> 3,9</td>

<td> 15</td><td> 0,045</td><td> 0,049</td><td> 0,059</td><td> 34</td><td> 2,3</td>

<td> 16</td><td> 0,033</td><td> 0,042</td><td> 0,035</td><td> 25</td><td> 1,5</td>

<td> 19</td><td> 0,033</td><td> 0,048</td><td> 0,045</td><td> 28</td><td> 2,5</td>

<td> 20</td><td> 0,043</td><td> 0,040</td><td> 0,052</td><td> 30</td><td> 2,0</td>

<td> 21</td><td> 0,044</td><td> 0,035</td><td> 0,033</td><td> 25</td><td> 1,9</td>

<td> 24</td><td> 0,046</td><td> 0,049</td><td> 0,047</td><td> 32</td><td> 0,5</td>

<td> 28</td><td> 0,046</td><td> 0,048</td><td> 0,033</td><td> 29</td><td> 2,6</td>

<td> 29</td><td> 0,071</td><td> 0,082</td><td> 0,078</td><td> 52</td><td> 1,8</td>

<td> 30</td><td> 0,051</td><td> 0,059</td><td> 0,056</td><td> 37</td><td> 1,3</td>

<td> 32</td><td> 0,105</td><td> 0,134</td><td> 0,129</td><td> 83</td><td> 4,9</td>

<td> 34</td><td> 0,045</td><td> 0,047</td><td> 0,038</td><td> 29</td><td> 1,5</td>

<td> 35</td><td> 0,143</td><td> 0,168</td><td> 0,171</td><td> 109</td><td> 4,9</td>

<td> 36</td><td> 0,115</td><td> 0,128</td><td> 0,097</td><td> 77</td><td> 5,0</td>

<td> 37</td><td> 0,057</td><td> 0,049</td><td> 0,040</td><td> 33</td><td> 2,7</td>

<td> 38</td><td> 0,082</td><td> 0,067</td><td> 0,063</td><td> 43</td><td> 0,8</td>

<td> 39</td><td> 0,048</td><td> 0,055</td><td> 0,045</td><td> 33</td><td> 1,8</td>

<td>Control sample</td><td>rep-1 (OD)</td><td>rep-2 (OD)</td><td>rep-3 (OD)</td><td>% TUE</td><td>SE</td>

<td> 40</td><td> 0,040</td><td> 0,036</td><td> 0,036</td><td> 25</td><td> 0,7</td>

<td> 41</td><td> 0,083</td><td> 0,069</td><td> 0,072</td><td> 50</td><td> 2,3</td>

Table 3

<td>Line parental</td><td>PCR positive for both the gus gene and the silencing construct</td><td>Partially muted</td><td>Completely muted</td>

<td> 374-18</td><td> 25/50 (50%)</td><td> 24/25 (96%)</td><td> 0</td>

<td> 717-54</td><td> 35/50 (70%)</td><td> 28/35 (80%)</td><td> 3/35 (9%)</td>

<td> 773-4</td><td> 23/50 (46%)</td><td> 0</td><td> 23/23 (100%)</td>

Sequences

[0267]

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 1</td><td>EMV promoter (P1)</td><td>MSTA0aaGCWTCca ^ T6eGSTcmcĄAcaaG6TAC0Aecaawa0i CTiTATTCAAMIGGTATCaCCAAAACCAAGMGGAACTCCCAtCCSCAAA GGmGWGGASGAMrCTCSSTCCWGGCTCAACAAGGTCAGGGTACA GAGTOiCGAĄAGCaSIAGCCMaaGCTACAGGAGMCAATGAAGAATCTTC AMcAMGTAAACTACiGTTCCAGCACASGCATCAttGGTCAGTĄAGTITCA gaamagacaiccaccgaagacttamgttagtgggcatctmgaaagtaa rCTTGTCAACAiCGAGCAGCTGGCTTGTGGGGACCAGAOAAfiAAAGGAATG GTGCAGAATIGIEAGGCGaACCTACCAAAAGCATOTTTGdCIiCTATTGCAA AGATAAAGCAGASTCCIOTAGTACAACTGGGGAACAAaArAACGTGGflm GAGCTGICCI'GACAgCCCACTCACTAA5; GCGTATGACGAACGCAGTOACGA CCACAASAGAAlrCCCTCIATATAAGAAGGCAMCATICCCAilTGAAGGA rCATCAGATACTCAACCAAS</td>

<td> 2</td><td>a 304 bp fragment of the gus gene</td><td>caacgcgsaaactggaccćgaogcgtgcgatcacctgcgtcaatgtaaigt yCTGCGACGCSCACAOCGASACGATCAGCGATCiCTSTGAttGlGCIGIGCC TGAACCGITATTaCGGATĘGWGTCCAAAGCGGCGATITGGAAACGGOAG AGBAGGTAGTGGAAAAAGAACTTCSGGCCTGGGAGGAGAAACTGCAICAGC CGATTAiCATCACCGĄATACGGCGTGGATACGOTAGCCGGGClEGCACTCAA TGTACACCGACATGTGSAGTGAAGAGTATCAGTGTGCASGGCTGGAm</td>

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 3</td><td>pSIM718 terminator</td><td>CGTTCAAACATńGGCMTAAAGTTTCMAAGATIGAMCGTGTTGCCGGT CTTGCGATGATTATCAiMAATTTCTGTTGAATSACGTIAAGCAiGIAATA ATTAACATSTAATSCATGACGTWTTATGAGATGGGTTCSSTATGATTAGA GECPCGCMmiACATTSAATACGCGATAGAAAACAAAATATASCGCGCA AACTAGGATAAAUTATCGCGCGCGGTGTCATCTATGTTAOTAGATCGGG</td>

<td> 4</td><td>intron pSIM374</td><td>STGGTAAGIETTTACTCATGTCCTCCAATTATTTCTGASTTCMGCATGMa; CGCTAGATIC®A®TAIGAATCGTGTTATGG3GTATAAAC6TTGMTCATAT CTęAJCTpATPJAM.CTGASeiSyGATICJjai &amp; TGCP.TAC.TGAAtSTGACCal¹ACSGTAATCGGSGATAAATOTGAMGCtSCOTCTICTiCTTCTTClICTUA. GAAMĆAMTTCTGIMTGTTMTGITCATCTGTAGC ™</td>

<td> 5</td><td>35S promoter from Cauliflower mosaic virus (P2)</td><td>JAGClWATGGAGTCAAAGAWraAmiaGSA ^ AAAGAGTGGGGAAGAGISCATACAGAGtCCTCieTACGAęiCAATGACAAGAA gaaaatcttcgtoaacatggiggagcaggacacacttgtgtactccaaaaa TATCAAAGATACAGtfCyCAGAAGACCAAAGGGCAATTGASACTTTTCAACA AAgGGTAAIASCCGGAAACC1'CGTCG0AITCCATIGCCCAGC5? ATCTGTCA CTTSATTGlfGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCGAICA MGCGATAAAGGAAAGGGCATCGTTGAAGATGCCTOSGCCSACAGTGGiCC CAAAGArGGACCCCCACCCACGAGGAGGATCGTGGAAAĄAGAASACGTICC AACCACGTCTTCAAAGCAAOTGGAMGATGTGATATCTGCAOTGAGGTAAS G0ATGACQCACAATCCCACTATCCTTęGCAAGACCCTTCGTOTA7ATAAGS AAGMcAWCAWGGAGAGAACACGGGGGAPiC</td>

<td> 6</td><td>the promoter of the potato ubiquitin-7 gene</td><td>TaGAGCAĆAraGATTGAGTMSATAiCGCAATATAGTAATAATAAitAATATa 'TGTTATAAAGCAAGAGGTPAATTTSSETKTASIATACCAACGTCACTAaAT TATATTTGATAATGIAAAACAATTCAATTTTAaTSAAATATCATOTAATAA ACTATTTTraSAACCAAATCACTAAATTmaOAATAAAAAAAAGSCATTA AGAAGACaTAAAATAAAISTGAGTAAAAAGAGTGAAGWCGACTGACmTI' tttttitatcataagaaaataaatsattaactttaacctaataaaacacta ATATAATMCATGGAATCTAATACTrACCTCTTAGAAATAAGAAAAAGTGr STCTMEAGACOCTGMTTTACATTAAATATOTCCMTCAAATTTAAATAA CAAATATCAATATGAGGTCAATAACAATATCAAAArAAlATGAAAaAAGAG ClUrfACATAATATAAGAWJAAGATISMSTSCGMWCZUlGGTAGTATT ATATCCTaATCTGCUAATATTTAAACTCTTATAM ^ AAGGTCATGTKCATG ATAAACTTGAAATGCGCTATATTAGAGCATATTAAAATATACTATGAWITAMTACTATTAAAATATACTATGAWITA</td>

<td></td><td></td><td>'TJTTCTGGGTiTSiCTAMGGAGCGTGIAAGTGTCGACCTCATTCTCGTAA • rfTTCCCCACCACATAAAAATTAAAAAGGAAAGGTAGCTCTTGCGTGMGT TTIGGTACACTACACCTCATTATTACACGTGTCCTCATATAATTGGTTAAa CCTATGAGGCGGTTraGTaiAGAGTCGGCCATGGGATCmAAAATGAAGa tttctgcacctcatomtttcatcttctatctgatttctattataatwtc® CTCAATiGCCTJSCAAATTICTCTTTAAGGTTAGAAATCJITCTCTATTMTG gtttttgtctgthacattctcgaattagctaatcaggtggtgttatagcc CTTAA</td>

<td> 7</td><td>antisense fragment of the gus gene</td><td>'CCęmccaGGMGCCAGAGGTGCGGATTCACCACMGCAMGICCCGCWA GiGCGMGTCCAGTTGCAACCACCTGTTGATGOGOATCACGCAGTTCAACG CTGACATCACCATTGGCCACCAGOTGCGTAAGTGACTGATCAGGACTGATCAGCGACGAT</td>

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 8</td><td>PG starter</td><td>CWCGmCTC (&amp; CCC®CGCGTC</td>

<td> 9</td><td>starter plE</td><td></td>

<td> 10</td><td>PIR starter</td><td>TGGAGGAGATGAGTAAAAGTTACCACG</td>

<td> 11</td><td>5 'fragment of 300 bp from the P1 promoter</td><td>A3H5tAGCAGCAMCCAGAl! TGGGITCAA5? CfflACAAGeSACHRGCpA5A! FCA CTIWICAWTGGW.TCGCWiĄBAGCP.AGMGGM.CTCCGAGCpTCnM gqittstaaggaagwtctcagtccwgcctcaacm.ggicagggtaca GAGTCTCCMACCATTAGCCAaAAeCMAGGASATCAATGAAGAATCTTC AMCABAGTĄAACTACTGTICCAGęAęATGCATCATGGTCAGTAAGTTTCA GWAMACATCCACCGAAGAOT®WGnWrGGGCATCTTTGA</td>

<td rowspan="2"> 12</td><td rowspan="2">Potato GBSS gene promoter</td><td></td>

<td>Gaat ^ AMiÓMeJÓMTĆiraAAIACSAaAiUULTGCAACAAAASWGTAfiTGG AGGGACCAGTACCAGTACAiTAGATAWASCMSTATTAOTATAASAAOT TTIAATTAACACGAGACATAGGAAiGTCAAGTGGTAGCGGTAGĆAGGGAGT TGGTTCAGimmGATACTAGGAGACAGAACCGGAGGGGCCOATTGCAA GGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATAG SGTCGAIGAGCATJTCCarATAATACAGTGTCCACAGUffGCCraCCGCTAA GGGATAGCOACCCGCTAiTCTCSTGACACGTGTCaCTGAAACCTGCTAGAA ATAAGGCAGGCACCJCCICATJOTCACACTCACICACIOAOACAGCKCAAC ĄAGIGGTAAęTTTTACTCAUCTCCTCCAATTATTTOIGAłTSOATGOATGT lUCCCSACATTCTATTAiGAATCGTGSTATGG ^ GTATAA &amp; CGTiGTTTCM ATCTCArCTCATCTATTCTGArTTTGAiTCTCTTGCCTACTGAATTTGACG CEAOrGIAATCGGTGATAAATGTGAAaGCGTGTGATAAATGTGTCT0CGTGTGiTCSTTTAGTCCTCTCSTTAGTCCTGCSTTAGTGTCTCTCSTTAGTCCTCTCSTTAGTTCTGC</td>

<td> 13</td><td>the promoter of the potato AGP gene</td><td>, cggcagigtgccagggctgtcggcagauggAcataaatggćacaccgcscg 'ecrCGTGGAAAGAGTATGGTCAGSTEOA-STGMAAGTATTTACSCGTATSC GGffGTTTACATCAAGITAATATGTTCAaACACATGTGATASCATACATCCA TIAGWAASfATAĄATGCCAACTTTTTACTTGAATCGGCGAATAAATTTAC TTACGTCCAATOTTTAGTTTłGTGTGTCaAACATAICATGCACtfATTTGAi TtóGAMAMTAMCGATGTGTAMMGAMACCAAWlGAaW ^^ GACGGGAnGATGTTCTGTGAAATaMTGGMATTGGACGGACGAMAM • łflTGATCGTOCA ^ TOAAGCAiAGCAACATGGGJGmAGICATCATCAISA TGTTATAATTAIMICaTGAAACISGATAcACCAACl ^ MTTGGGAAAGT gacagcatagtataaactataatatcaatictggcaattmgaatiattcc AAATCTCTCTTGTCATlTCAraiCCTCCOTATGTCTGCAAGłACCAAmA TTTAAGlB, CAAaAAATCJJGAT5AAA &lt;lAATmmrCTCAĆTAAmTOA CAmAATCATCAACGGTTCATACACGTGTGTCACTCSSiTUTTAWCTCT CAAGCGCATGTGMCATACCAATTATTTAAilTACAAAAAATCTTGAWTAAA camioagtttctoagtaatmtcacamtaatcatcmc tm ^ ^</td>

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td></td><td></td><td>And ATCCGTCACTCrrmilMiCłCTCAAGCGCMGTGATCMACCAATTAT TrAAATACAMĄAATCTiGAtłĄAACAATTCMTTTClCACTAAttAATCAC ATTTAATCATOAACGOTITATAOACGĆCCSCCACTCTTTITTtAWCTCTO AAGCGTATGTGACOATATCSAACTClCGTGCAAAGAAGłGAAATGACGTTG ACTAATAAATAATCTTTTGAATACOTTGiTCAGTTTAATITATCTAAmG ATAA</td>

<td> 14</td><td>154 bp attachment element of the potato BPO gene</td><td>TIAGTCICJATTGAATC®GCTGAGATTACAC'J5! TGASGGA5! GATGCTCIGT TTTTGTTISCIIGrTCTSIilTTTCCTCiaTTGAAAlCAGCnTGMGCTT GATMCATTGAAGTTGTTATOCAAGAATAACAATCAGtTTGTATOCAAGAACAATCAGt</td>

<td> 15</td><td>the intron of the potato ubiquitin-7 gene</td><td>GMAGAAAICTTCTCtóramGGTTTSIGICTGaWTAGAMCSCGAMCTA GCIAASGAGGJGęTGTIASAGCCCTSAATTTTGAGTTTIMTECGGITGTT SIGATGjSAmGGCCTAAAAmCMOTTTMTOACGETGGm ^ AGgcęTAmWeGAGWSSCCCCGmmSGAKOMAWaCCOTAGTi TGAGMTTISTlWTSTCGASTCGATTCIfAAAGGTilTAAAATTAGAGrtiT TĄCAyjTGraTGAIGAAaAAGGCęriAAAtSTGAGraTJSCCGGMGaTTT GMGAAAAAGCCClAGAATMGTGEimTGGTCGGTSIGĄSICIGAAGGC CrAAAAiTTGAGTTTOrCCGGCiGETSa / GAfGAAAAAGCCCTAAATTrGAG TTTCTCcaGCIGffSfTGAiOMAAAGCCCiFAAMEffGAGirraffSCCCCGT GTTSTAGAmGia / IrGGlrTTAATffCTCGAATOAGCTAairCAGGGAGIGJSA AAAGCGOTAAAAJirGAGTTTT'JTTCGJ? TGTTCTGATTGItGITTTTAIGA ATTTGGAG</td>

<td> 16</td><td>fragment of the tobacco PPO gene</td><td>! rTXi! J'.ę! rĆ? ATOMTC5'.GCmGAmCACII^,and^,GATG9ATGATGCTPTGr TJTTGSfróCTIGT ^ CTGrTffyiTCpTĆJGTTGAAATCAGCTTTGTTSGTT GMSrcATTGMGMGTmTC.AAGAATAAATCAGTTACMWMGTTTGG G.</td>

<td> 17</td><td>Transfer DNA Russet Boise II</td><td>TGSCAGGATAiATACCGGTGTAAACGAAGTGTGTGTGGTTGATCCAAAATC TATCGTACCTTTAGAAAGUGIAGCTAIGAAGGATAGTCTCACMAiGAAGA ACTACCTATTOAGATSCTTGATCGTCAGGiaCGAAGGTTGAGAaAMTAGA agtcgcttcagitacggcttigtggaggagsaagggtaccgmccatgcat CTCAATCiTAATACTAAAAAASGCAACAAAATTCTAGSGGAGGGACCAGTA ęKA0TACA! PTAGATAtSArcma »mACTATAAT ^^ CGAGACATAGGAATGTCAAGifGGTAGCGGTAGGAGGGAGTCGGTICAGTIT WmGATACTAGGAGACAGAACCGGAGGGGCCaATTGCAAGGCCCAAGMG AAGTCCAGCCGMaATCAAGnBAGAGAGGGCCCATAATTCCAGACAGGCCCATAATTCCAGACAGCGATGTAGA ccgctattctcTtgacacgsgicacigaaacctgctacaaataaggcaggg ACGSCCPCATOCECACACTCACTCACTCAOAGAGOTCAACAAGTeGTAACI? ITTACTCAJCTCCTCCAATTATreGTGAITCCATGCAtrGmcCCTACAtr CMTATGAMCOTGTTATGGTGTATmCGTTGIPKifCATATCTO TCTATTGTGAOTSTGATTCICTTGCGPAC ^ ^ ^ GG GAATTTGACCejAGlfSTAATC GATAAAffGTGAATGCTTCCTCTrCTSCrSCTTOTSCTOAGAAASCAAT ęTCJGyTMGiiTKTGirCATCTGTAGCriGGTAGATTCCCCMSITGTAG ACCACACATCACGGA! E! GCCCCAAACATAAtrKGTAACIGATTTAIICWGAA TAACAACTrCAATGAAAICAAGCAACAAAGaTGA5TTCAACATGAAĄAAAC AGAACAAGAAAACGAAMCAGAGCA ^ GATCCATCAAAGTGSiATCTCAGCA GATTCAASAGAGACmCTCGAGGTGCTCTCTATGCAAAICTAGCTMTCG AATGAGAGTGATAAGAGaGTGAGGATIGTGAATTATTTTATACTAGTAGTTATACTAGGATGACTIGATACTAGGATTCTIGA</td>

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td></td><td></td><td>ACGNłAATCTCGTGCACEAGECCCCSGCACEClOETATAlTEGTCCATTIC OCACAmOCCTAACTTAATTACTffACCCACAGTaAAGCTTCMGAClEGIi aGGmGGMTCCCTGGrACAGCAAGmWCCCTAAGGAMTACECAIA ICCECCCACTGGCiMAETCACTCĄAGTECAGGTAGAAACGSCGATWTCTA GTGAAGSAACaAGGĄSATTAGCSAAOAŁGCGICGAGĄAATOCGATGAAGAS! GAAISCAGGAAGCAAAATGAAGATTGGAGCAGAGAG ^ ATGGGGATTGGAGA GiGGAAAGTGGEAGTGAAATAAGGEOCGaGGGCTAAAmCiATGAMmEG AACTCĄA ^ AGCIETTCATAATGAGCAAffATTATCTTTCĄTAGCGCTGACATTCGCGTGACATTCGCGTGA CEAiGMTACAAWGATTCTTGEAGCIGCAGACCITATITCACTACCACTT TCCACTCTCCAMCCCOMAClOTCTGCrCCAMCTlOATWrGCOTCGEG AATECAECTTCATCGAATrTCTCeACGCTECTECGCTAATSTGCEGGTTAC eecaotagaaascgacgtttgtagotgaacteoagtgasstaagcgagtgg GAGGAIAEGAGTAATSCCSEAGGGAAWOTGCTGTACCAGGGAIIFCCTA ACGTGMCAGTGTTGJWJCTEGAGTGTGGGEAMJIAMWAAGWAGGGAIE TGTGGGAAATGGACAilATATAAGAGAGTGCAGGGGAGTAGEGCAGGAGAM WCOTGCITWATEGATAAAEAAAAAAAGćGTGACAOTIAATTTCCACAAG AGGACGCAACACAACACACETAATECCTGEGIGTGAATCAATAATTGACTT CTCCAATCMCATOAMAAAArAMTCACAATCCTCAGTCTaiTATCAGW TCAlTCGAAAAAGCaAGAtStGCATAGAGAGCACCTCGAGTTAGIGTCTATT eAAKaiGCTeAGAWACACTTTaATeSATGAiraęCCIGTTTTCGTAGTGlTCTITTTCATGGlTCTITTTCATTGlTCTITTTCATGTTTTCGTAGTGlTCTTTTTCATGLTCTIT TGTWTOAAGAATAAATCAGTTACAATIAIGTTTGGGTCSAGAGTGAtGT GraGTCTACAAAAAGGGGAATCPACCAAGCSACAGA ^ GAACAAAAACAAAA CAGAAATTGATTECTGAGAAGAAGAAGAAGAAGaGGAAGCATTCACATCTA TCACCGATraCAGTAGGGTęAAACTCAGSAGIGCAAGAGAATCAAAĄTCAgA ATAGAIGAGATGAGATATGAAACAACGTTTATAOACCATAACACGATSGAI AATAGAATGTAGGGAMCATGCASGĄMTCAGAAA ^ AAilGGAGGAGAIGA GTAAAAGajrACCACreGiTGAGCSGTGTGAGSOAGTeAGTGTGAGAATGAG GAGGTGCGlGCCTWTTGTAGCAGGTilCAGTGACACGTGTCAAGASAAi AGCGGGTGĆCiPATCCOTTAGCGGAAGGCAAGTGTGGACACTGTATlMAGS GW ^ ^ GGTCA CGACflGWTATGSGCCCTCICimĆTTGATrCACGGCTS GACtfTCAACTIGGGCCrTGCAATGGGCCCCTOCGGWCTGTCTCCTAGTAI CTAAAAAAClOĄaCCAACTCCCłCCTACCGCTACGACTTGACATIGCSATG TCTCGTGTrAAOTARZWU? ATCATTATAGTAATAAMGATAATATCTAMGT ACTGGTACTGGIOCOTCCACTAGAATSITGTSGCASTTTTTAGTAJTAAGA KIGAGATGaAIGGTiĆGAeCICCSiCAACASGTTATAAACTaOACATATTC AGTTGGGMTAGGCTSSATAATGACTTGGAGTACGTTATGiCCCCCICAAG TCCOAGAATTATGTGGCCCaGTATCTTATAAGTECGACGCCGATGAATTCGACGCCEGATATCGACGCCEGATA</td>

<td> 18</td><td>fragment of the attached element expressed in the tuber of the Solanum verrucos PPO gene</td><td>CaCAAAGAEAA ^ EGTAACTGATraAIirCTEGMTAACMaTTCAATOAAAT CAAGCAACAAAGCEGATTTCMGATGAAAAĄACAGAACAAGAAMCGAAM CAGAGCAiCATCCATCAAAGTGTAATCEOAGGAGAEECAATAGAGAGTM</td>

<td> 19</td><td>leader fragment</td><td>GTGCTGEĆteGCAAATCIAGCWilECGAAEGAGAGTGAmGAGAGIGAG GATMTGAATTAWCTATTGAEGAAGATSGGAGAAGmGAATTAEEGAETęA CACAęAeGAATTMGTGTGESGTGTSGCGTdCTCTTaccGaAttegataaaaaa</td>

<td></td><td>phosphorylases</td><td>CCTGOACTCTCTTATATirGTCCAmGCCACMMCCCmairTTAAmc TTACCCACACTG</td>

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 20</td><td>fragment of the R1 leader</td><td>CMCAGTairGaGSTTiiGGIASTCCCTGGTACAGCAaGIiAITCGCTMlGGA ATTŁCICAfAycCiCGCACTGGCTmTTGACTCWGWICAGCTAGWCG TCGATITGTAGlGMSTMCGAGGAAATSAGGGMGAAGCGTCGAGiWkTT CGaTGAAGATGAATTCACGAAGCAMATGĄAGAWGGAGCAGAGAGmGG GOATTGGAGAGTGGWGTGGTAGWAASAAGGT</td>

<td> 21</td><td>non-functional P1 promoter without the TATA box of pSIM788</td><td>ATMAGCAGCATTCCAGAWGGSTTCAATCAACAAGGTACGAGCCATATCA CTTrAffSCAAAMGGTASCGCCAĄAACCAAGAAGGAACTCCCAICCTCAAA GsmGmGGAAGAATICICAGROTUiAgCCTCAAcaAGGJCASGGTACA GAGTCTCCAAACCaWAGCCAAAASOIACAGGAGATCaATGAAGAAlOTIC AATGAAAGTAAAGTAGTGTTCCAGCACATGCAtCATGGTICAGTAAGTiTCA ^ 0AAAAmCATCCACCGAAflACmWSmGKeG TGTFGTCAAOATCGAGOAGOIiGGCTTOTSGGGACCAGACAAAAAAGGJATG GTGOMSWIGmAGCCGCMCWCAWGCMcmcCcmATTGCAA A0ATAAAGCAGAKTCCTCTAGTACAAGiGGGGAACAACGACCAACGTACGACCAACGTACGACCAACGTACGACCA</td>

<td> 22</td><td>the promoter of the potato R1 gene</td><td>TEOAAAITiGAmeTGTCAiAirAAAOTGAGACATATAATyGiCGSCACAT GGIcaEGiAICCAAAcAAGGATAMWGATCATCTATMITOATATMGA mSTACGAmramcwmAM CAATAASTAACAAGmAAm ^ ^ TAAAaGTCArATAAAAaATJAATTGACTCTCAAAATTOIGIAAfJTACrATA AAITAAAATaAMAACAACSIAAGAAWtCAAAGTCATAAAAAATITGG! FG GCICiCTAAAATATAIłCAATGTCACATAAAAASmCAWAMAraCAGA AATiACGTAAAAGATACGACAAAmCAATAATTAACAACffTGAAATATTT ΛΑΒΑΪΑΟΑΡΑ ΛΓΑΛΙΙίΑΑ ^ ^ ^ ΪΑαΑΑΑΤΤ TeGGACAACGAAAIAAlATATACTATTAMTTAMATTATGTAAaAAAATA ACTaTAAATCATGATAATTAATAACTTAAAatairAWAAAAATCATAHAA AmTmATAATWTCAGGMSCAGCCGTAmCATAAATTAGGATAAA WTAW ™ 7WGSGCCGW.GCT? GCAaęG ^^ IGATAATCTrTTAATOTMTCIAClnAGCATGGGGTGCĆSTaiClAGMifA MTAATAAATATCGTTirCTAGJCTATCTCTTCTGATGC ^ AAATAAAGTCAG 5ΉΑϊϊΑΤΤ55®ΤΑΑΪΪΤΪΤΪΟΤΑΟΤΑβ (ΐΙ5! ΑΑΤαϊΑΑΑΑΪΤΟΪ®Αϊβ! Ε1! ΑΑΟ CAAATAAATIGAGAGAAATJAAdTCAGMAACCAGAGTirAAGAGTAAAGirA ctaitgcbagaaaatatcaaasgcaaaagaaąagatcatoaaagaaaatat CAAAGAAAAAGAAGAGGTffACĄATCAAaCiGCCASAAAACTCCAAAAATAA · aomtgaaatpggaaaaacasgcamcaaatsgcsctagitcacggsgccc acgg</td>

<td> 23</td><td>a fragment of 342 bp from the promoter of the R1 gene</td><td>AAAATTcmTGTTAACCAaATAAAraGAGACAMTIAATTCAGITĄACCA GAGTTAAGAGMAAGTACTASSGCAAGZUiWAICAAAGSOAAAAGAmG ATCATGAMGAAAAiAiCAAAGAAAAAGflAGAGGTTAOAATCAAAaSCCCA TĄaMCSGCAAAAATAMCAracASATTGGAAAAACATCGAATCAAATTGC 5CTACTTCACGGGGCCCACGCCG0CI3CATCTCAAAGimCCCACGTOACA TCCCAJAACAAWJCACCACCGTAACCCTJCTCAAAACTCSACACGTCACiC rTSWCTCmA! TSfACaATAAAAAATATAC0I? &lt;Young CC</td>

<td> 24</td><td>a 151 bp fragment of the promoter of the R1 gene</td><td>CATTCAMiTGCAiyWACATCCAATCAAA ^ IGGifGTACTTCACGgGGGCęA CGCCGGCTGCATGTCAAAGTTaCCACGyGACATCCCATAACAAATCACCA CCGTAACCCra / CSCAAMCTCGACACCTaACTGATTmCSOTAT</td>

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 25</td><td>promoter of the PPO gene expressed in the potato tuber</td><td>TAATATAACATACCATGGGIGGAGCTAGAAGTCIGAaiACAAAETTCGTCA AAAAIATA? ACAMTTaAGGTCAAGGATTCAGaTAamGęCTTTAAAAaO GaGTGATAAAATTCWGaEAAAWTCWACraCCCCGEGCETAACATTA csmmiAAAmATOTWTcTOGTAeAWutwAoiatm ^ TOTaTAGAGGrcSACrATAGATCBCCTTAGATTAAMACICCASTTTPAA TAWTTCCECAflAAKATTAiaCTTAATCaACCAGOIACCGGAGTCAGĄAA TATATTAAATGWiATA®TCTATCTATTAA3TTATGATCTACCTATTGATA ATTTGTĄATCTAGTCAAAATGATGGCĄĄAAĄAAATATAMATCTAGACTGA agtecteagicaatagcgtaaatgaaagąaaaaaaaaaaagcicaagaaga AACATGATATCTTTGlTGGTCTGATTCGSAAiłAAAAlWlACATAGTAACTT CAiAAMEAECaTATCGTTtGGACAGAGCGATGAAAAAAATATAITACTAG TAATAGTGASAITAGSPACGTGAGAOTASriOOrAsaTSaSGTmGATS¹!: GAlTTATmGGAAAATiATGTTTGAACGGCCATGCTTATCCATGCATTAT TAAWaATCAATATATTACTAAATGCTATTACFATAGGaiJGCTTATATGTTC IGTAATACEGMTATGATGTAEAACEAMACAaACAWAAATTCTOTAATA aatctajcaagagaaggctaagagattaacaaatactactattatccagag TAAGTTATSIISCTGTTTACTAGAGATCCITCCAAGAACAAAAACTTAATA ATJGTATGGCSGCTATACATAATTCCCGŁCflTACGGCTICCTGGWrAATT GATATGGAAGCCGCOSCTĄAAATTGAATAATilATACTGTffiSTACATATTAT ATAA</td>

<td> 26</td><td>a 200 bp fragment from the PPO gene promoter</td><td>AAGmirraiCWITACWAGATCCnOCMGWWWCraAW. T5GTATGGC! (GCTATACATAATCCCCCACCTACCGCITCCTGGAATAAT! C &lt;3 MATGGMGCGGCCTaTWMTGAB.mTTATACTGT5? T5'ACATATTATA TAAAGCAAGGTAIAGCCCAAlGAATMTCAMAGCAAAGCT</td>

<td> 27</td><td>a 460 bp fragment of the PPO gene promoter</td><td>CTAGWlWTGAGmAGmĆGTGA®CWm'OTATCMCTGTmG MTTGMTTATTAAGGĄWTTATGTTTCAACSGCCATGCTTATCCATGCA ttattaatgatcaatatattactaaatgctattactataggssggitatat GTiCTGrAATACTGWATGATGTATAACTAASACATACATJTAAATTCSCl 'AATAAATCTATCAACASAAGCCTAAGAGAMAACAAATACTACTATTATCC AGACTĄAGTfAT TMCTGTTTACSACAGATCGTTOCAAGAACAAAAACTT AATAATEGTATGGCTGGTATACATAACTCGCCACCTACCGGTTCCIGGAAT AAITGASMGGAaGCCGCCTGTAAAAWGAATAAT ^ ^ ATACIGiSSTACAWA raTATWKJMGGtAmGCCWWTWCATTCWiAGCTAGGAAT A</td>

<td> 28</td><td>Brassica Fad2 gene promoter</td><td>ASTGAGCSTGAAGGAACATTCGAGĆAGATAAACGAAGCGAGCGCAATGGTT AGAGAGCTGATTGGGAGGCTSAATSCCGOATCTAGGAGACCACCSGGTGGC GGTGGTSGCGGGGGSGGGCTTGGiMGĄAGGGAAACCAGATCCAGGAAGC AACTTCAAGACGAAGASGSGTGAGAGATTCTęSAAAGGAAGCTGTACATTT ggigatagatgscactttgcjcacggggaagcagagctacgcaggtcatga ATTGCGCaTASAGJTACTGGTGMACAAGICWCTOTCASISGSTGTGGTGA TTCCTAAlATCA ^ CTEGTCaTAOTlGTMrTAGTTOTCłJTCGraTmGAA AGTACMEGTTTAGTTTTCATTGTCAGEGl / AAGEETTCCCCATMGGiaM iTTTEAGĄATCTAGTTTGAATTEGAGATGGGGCAAGCTTGATGAAIGATEG GCAAAACAGEGGTrAGGATTlOEGEGCEGrcaCEACEIAATATTECATGET ITAECTAGTTTATTTTGGTCAGCAAGTrGATGTGTl'TCTGTGATGIGaGIG TGATTAaCAGCEEAGATTAaaTIGEGAGTATGCTAGAGEGTATAACTAATC GIEGTCGATeSTATAGaECTCaaATAATGETTGAEAGACTATAEAAGTAAA AAEaCAEGTJASTAATAGCCGTCGCEGATAGTAACAGCTGAAaAAATGAAA ϊεΑΑΑΕΟΑϊβεΤΑδθϊβΑϊθΑΜΤΓΑΑΆΟΑΆΤδΤΤΑΆΑΑΑΤΑΑΙ'θϊβΤΟΟ TTATAAGCGGTAATGCAaAaAAAAAGTCTAAaCATcamcATAAGAGAGA GCGAEAGCaaTAATAAAGTACTOAAAEEAATTAĆTAGECGGGAGTCGCTGC CEACTTSTGIACaACCaAAATTAATETATTATaAaAEATGACGAAaCTOCA AAGaACATCACACACACTCGGGGGTArECACGIGATClCAACCACWGTC iGCAGataawaSTTAAGTIETOTaCTCAOATGGGAGAAGAAGAAGCCHiAG</td>

100

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td></td><td></td><td>CACG</td>

<td> 29</td><td>5 fragment 441 bp from the Fad2 promoter</td><td>CCGGGIACCACTAACTTCTAGAGMCTACTSGTGAGTCGGCAAGGAeGTTT CCICATO5AAAGTAaaGACATCAAaTACCATAATCTCAATG0TAATiAAG GIAACeGATGAGWSCTATAACATAACCOAAACTASTCTTTGTGMACATTAG GATTGGGTAAACCAĄiATTiPAGATITTAAAAACAAAArACAMiAAGAAACG TGATaAACMMTAAZaGCAAMATAfGATCACGGCATCTOmCAGTTM CGGTAAATATAWAAGTGGiGTAAAiAiCAGATAtMGGAGTAGAAAĄAA AAAAAAAGMAAAAGAAAWflAAGaGAGGAMiAATGGAGGGGCCCACTT GTAAAAAAGiaAiGAAWmTGTCACTC ^^ ^ ffCAA TAAAĆGGCCTGCCiECrGCCCAATCGC</td>

<td> 30</td><td>the seed-specific napin gene promoter</td><td>WCSWCŚGAłCGGieAMGATTCCimfiAAGACIWGTTTCraATCT SSCTIC5GAGGCAAGW! RCASTS? ACCACTSAl? AT10TGGAC'nTCTGACT GCATccTCftirmawaTmMAmcaCTAiTOGCTGAM ^ TCTTffGAGGAAGtAAACAAKICAGAiGGCAGAAATGTATCRACCAATGCATA TATAGMASGTACCTGCTGTTGirCAAAACATCIATCGGAIGGTTCCATTTG CMTGTCATCCAAStCAGTGACTACrnATATmTCAaTCCyCTiTAtiAC TATTTiCAiGCGAGGTTGCCATGIACAWTAIATTSGTAAGGATSGACGCTA MGAGCGIUTJrCTTOAACTrTęTITAMTSAGACATGGGTATGAĄATGGT, TGI.TAGĄSXTG ($ AGGIIC ęiGW®lGW ^ ^ ^ YrajĄG aAGGA ^ GACClfĄęĆCATTĆm TĆAGAGTAAAATGIG! FACCmAAĆl! CilMMCGA3! TGAaATÓTA®CGAW CAAGATAAAATTĄĄACCAGCCiGCACPSGCAS CCACAITiGAAGTATTiTC AAACCGWCSGęTGCtATCCACCGGeiemCAAGACeGASTCCGRASTTS eAAGArT SGAOTCAAATTCCCAATSrTATATWACGGrGACTAAATGAAGie WAACWCTAaWECSOATIĄlMSCTCCCAATiWATTCCCWICGGCAC® ACCTCCAAAATimAGAGTC®GArcCCCTTTTAAACCAACTTAGTMACG ^ ^ TTTTTTIffTirAWTimG.WiTTAAGTT STACCTTGTTTTWAAflAGA ATCGtffCATAAGATGCCAWGCCAGAAOAiraASCTACACGSTACACATAGCA TGCAGCCgGGGAGWMTmoreCGCCACTWCACTCGmOMAGA CCTAAGAGCTWCEClOTCACAGCACACACATACAATCACATGCGTGOAliGG ATTA</td>

<td> 31</td><td>the seed specific Brassica napus promoter</td><td>CTBCAGGTACAAAGAGGAGCSCSACTTAGTTIATGACITTAIGCCCAGSGG AAGCOnGACAAGTAGCfOTACACCGSATGAGATCAAGAATATATeAAGTI CACĄAGAAAAATCTTAGTATISGIMAGSGTATCTTTCaaTGTAAAIGTGE STTTGCnOTCAAAAAAGAGCMIGAGAAAAATTAAAGAAGASAACTtGTC WAAaCTATimGg TęGGGraTTOGGGAGAAGSrCTGAAAAiMTGACA MTA ^ ^ ^^ GmiGOCOSCGATGCSaflWiAACATITTC AAGTiCCWTGJACAGTAiAfUCATifArACTAAAATAAAlCTTAAAATAAWi! TAATASIAWSITAAAWATATAATTAAAAAACHAraAIMGaTffAATAT mAmmwTWGJmAACTmiAciaaATacTmTCAGMAA CAATACAAiACATATATATAGAAATTATCTMTTlMAAASTATATWTTTA GATCTTGGATAATTiAATATTATAOTEJAWATATAATlAAGAAISmi mjAWiTAGCTATjmiATTCMGimAFACTimWATAiAAT ttaAaGsmTAAcmmcftAmTACwow ^ aiACATAAATTATCTataTOPIAAAATMAWTSKTC</td>

101

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td></td><td></td><td>TWmAGGATATAAACGATAJTAATCASTCTAAWITAACGTGAGAtMT GGAMCCMAAASTTACMCGCAAAIAMJAGTAIATATCWkGCTTATiFAGG GCyTMAMGGTtraGGTTrCTTWCGCMTAATTGT ^ TTJTTTMACTAirA astgtaaacgtgttaaacataaciaatcagkgttaaaacttggsiiattm ATTTTTCCAAOTTTTĄGATWWIGCATAAAGTSTTACCATMAAAAGAAGA ITAAAGCATAAAGAGATATCAUTTGGTATAATMTTATGCaAACGSAiAAT TrGGlT ^ ^ TASClTSWGCMACGCATAGACMO GGACTTGAAAaAAACG AGAAMAGGACACTTAACACAAACTCCCAAAATGTCACITAAAGCTATAG® TCTGTOACGGffCTCTCAATGGAAAATCGIGGTGaTATCAAIGAAMAACGT TGIGCtGAJ5AC5GGCAGAG0ACAAAACyATAGICTAAAAA.GGATTGAATGA AGCAAAAMłGCAAGAAĆGAMGGCACAACGAtfrCYGACATTTGAAYGATA TTAGAAATATTAGJTTCATTTGCCAAGiGGACACATCCACGTGGTAGAGGA GCCATGCOACTTGTCTCTTtCGTGGGiTCTCACGCCCGAaTTACATTTGAA AAfTADAAAĄTAAAAGAACATTTTGTATATGlASSGACATrETKAGCCTCG CMATACAWOTGiraTAmcmATTCACAACTAATC ^ ITIAGiTAACTAAGAGCAUęMTGTTMCCAIGAITCmTTliGMATTAA TTCMGATTifGAlATWTATTATTTEATMTTAmiTraTGGTTAAAAAC AGACTCmTATCWmTITMGAGATAGTTCTTAATOTTCTTAAlTAAA AGITAAGAAACaGJTCTęAACCAAATGTAMMCCATATTGTAAGAGCaaG GAMWWGATcmGmACSCACGAGTCMSlrCACCTMTCAaATCTA AAATATAGTAATTATAGCTMACCGAOASGTGATACTGCCAAAAATAATAA ATAATAIATAGACACAAGAAGSATaTGATAGTGAGGAATCTGGAGGGCATAATATCATAtAGAAAATATCATAtAGAA! SACACGATAGATGACGTCATTirTCCATTTGTTTrGlOAATWiGTTATTTA MTCęKTCTCTCTISCCTCCGŁGISSAGACTCTIOCCTCAAACCGCTGa STCTCRrAACCATATJCrCWTaTGTGGACCAGTAGGAGAACTCAGTAGAAGAACTCAGTAGAAGAACTCAGTAGAAGCA</td>

<td> 32</td><td>soybean Fad12 gene promoter</td><td>CAGAAAAAGGGAAIAGSJSTGGATAAAATAGAWTTAGGTCrCTCAATTĆGT -AGTCflAAATTAGTCTCAATCATTATGCTTTAAAAATGATGAWilGACACT ϊδβρΑβΑΤΑΑΟΑΑΤΑΪΪΪΟΤΤΑΑΑΟΤΪΙΘΑΤΤύΤΟΆΑβΤΪΝδϊΑΤΑΪΑΪβΑ MSAGraTGTTeGGAGAAGtWCICJWAAAATAAATSTTSfATAMTTAGA GATGAiPTCTCTaATTCTTATGAAGGłSASATACTAGAAAAAAAATGATTTT TATTMTTTATTTMTATiTATGAAAOirTAAGAATTAAGATACCAeGATGAG ggacaaaagtcattaatmttaaaaaaąaatacaagaatcaagattattat TTITAAAATATAAAAAAAaCTAACWSGAWATAAAGAAAlOCAaGGGAT ATAATACACACTCTATaCCAAATATTrGGTTAAACCCCCAGGGGCCCAATG STSCGiCTTTOCTCAACAGWAAATTGCTAATGATAMAKTTGTCIlOAA TTGGSJTGCfGTGGGTAGCAWCTGTGCAACTTGTGCAAGCATITGGSAAT TCAATTAAGAATATńTAATATACTTTAAAtTTAGTAGGAliGCAiAAAAAAC CCTGIGACITGiCTGACCAAGACTIGCCAAATPCTTEWATGAilGCATTACA AAAACCAGCCATTTGraTUTATKTMTGGAMlOmiCTSTCCAAfeTGAA GGCCTAACAGATAMTSGCATCTCTAATIJ / CCCCITGTTATTAGAGAAMA AGAAM ^ ^ ATAAGCTTTTGCiCTTGAGmiTGAAOAifATSM OACTęMSGG AGGTIGCTirTTAraTGGAAGACCAGAGGAGSJCAAAATAACAGTGTCGaę GFAAGTAAeUGCTCGACAMCTGGAATĄGTCTCTSATIGCGTAfTGTGCCA 'JCATSTrGASGCCTSGTtlGGCETGaATCACCATTOAAAGAAATmiMGA TGGTTAAAATGGWATACCTTTfGTCTTCATTATSACECGJUiTUACAilSAG AMG</td>

102

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 33</td><td>the promoter of the alfalfa COMT gene</td><td>MATGAAAGAGAGTTAASGAWGAMTGAAAOTGGTAAAAAACAGCWTAIT MAAAACAlCrTATTOAAMCMCmTWTAWAAMaWTTAmiA jrCaWACATGTO? T®ATAAGTTRTWMTGWATAAGCIGM^:TTGW AAGCTGmTTAAAATAAGGTGCTTMCATAaAAiaAGriGTIWSGTTAA ZUłSAAOTSeTmiSCAmARGCKeiSWBWAAGCTSOTWSmTĄB. ATAAGTCGEWTGAAmGCIGSITOTffyTTAAATAAGMGSTTTTyTMAT wcraimewAAfmerammmGGiGtTWGmAwwe CTGTTTrGaAtAAGTTSTTITGAATAAGTEGTMyGAATAAGCTGTTTTTI mAwiwitttMmcAwaAiaafiCTOTwmAia TGfATAĄATAAGcTTITTASAATMeCTATTCAAAmSTIGTTTmTGS AMGATCCAAaAAAGAGTffGAAGJGGITSCMSAAAATAAMTAAĄĄiiGT ^^ ^ CAAGTGGTIIGGTICGGIICĄaACGGTTCGGTTCGGTTCMGATGGitCSG TOATGGTTCWAACTOTAATAAmAACGGMOGGTTCGTCAMCATTA TAACGAITCSSTTAT5M9? GGTS! CGOTCGGTTCGGGCaGWCGGTICGeT TCATGGSWCTTTSTGCCCACCCCTASAGAAMTHAATGAATGGTGGSSGRE TArTCTTAAAASGATraGTITrCTAGAAIAftAGAGITAAfAAGGGGGirCĄŁ MGAGCAACOATCWGGTAWmcWTTTAGAGmATGCGGITm ASTTGGATATAACACidfTTGTTGACCAAAATGICTCTTATGAAiAAGACTG AAAGAAGSAAlAATTTAAAAAAAAAAAATCCaGCEGTTGCATrTTiirJWAA GMTAMCCGMGAAWATOTITGAAAMTGTETATAATGP.GAAGTTATT WGAGmmSTOCTIfWAAAAAmAATGmTmCAmiCT ACCCATAAAAaTACiICTGTSTTMTAAAGA ^ &amp; TCTCTAAAAAFCAAMICT AAACGSCAABAGTTTTJrAffAOAASłAGTiTAGGGTGtrTJCTATGAGGGTS TGAłAATATSyęrACGACTATATAJATTMTTWJJTAAGGAAATiCTACGA CTACJfTGIAGTIGGAATMGGGAATACGACTACimCTATGAAGAGCAGG ETACGGSAGACACAAAAGGIGACTCCIGCGaĄAAGCJTGJTCAACCCĄATA GyGACATATTAGGAAATGAAAAATACCCTAATGaciCCTSTTCAATAęJCA AGAAAAGTCCTCCMACCATĄTTGTCCaASTJTCTTTAAGAGCAGAGAAGA ACACATTIGraCACACCMCATGaTTITTG1? ATGGTT0J AAATGAAMGC3: JCTAGyTArCCAGCTCAACCCGJGACTAAGGTCTATTCAAilTGGTTAGAA ^ AGęiCAttw; wwGCiy &amp;! T; r;? And AęWATGAACTffGigGS.GTCAC.GTAĄGijsAATaAOACTATCTAAATTTGA ^^ ^^ GSACAGTACimcCSGTCACGGGGAGaMMlCACTCAAAATCAMTGm gagataąrttttgtatcataaaitaattaattwacaattacatcaatma KcmitmrrAAPcmiiWAłATGAauAAcwrcmt GAGCaAAAAcamamTAAWGCAT &lt;MAACG &lt;KIWACm &lt;^ TmATTCG (OTGAGTAC! RCAGTCaGmW! ACGAATTCTCSiAGTOŚGTCCACAWG CC ^ ^ ^ GGTGGJTGAGTGGGACCCAAitTG taatggasggcccacatacacckaactgaaccaaacaasetctcatmagt jCTmTAMaecwccACWGcam</td>

<td> 34</td><td>a fragment of 448 bp from the COMT promoter</td><td>1 CAaCAACASGAmTTTGTAlGCTiGTMATGAAAAGCTTCTAGTTATęCAG CICAAGCCfiTGACTWGTCWTCAAITTGCJraW ^ ^ TGftSGCATCAAT TATGATGęAAATTTMGTACTCATrACTCAAMCAAAĄACTATA GAACra ATGGirGięACGTAAGIGAATAAGACTAiaTAAWTTGAGrACTrCTCCTea CACGGGSAGAAAAACACTCAAAATCAAirWCATGCAACGGCAACACAriTC TGTITACAAITATATICGSTGAGTACTCAG ^ OAGSATAACCOAATTACCAC ATATGCAGGAAMOTCITAGTGGGTCOACAETGTGGTGGMGAGTGGGACC CWTGTAATGGA7G0CCCACATAGACCAAACrCAACCAM.CAAWrCTCA TMaGT ^ _ CTATATAATAGCAATCCACTWGgATCATTGAG</td>

103

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 35</td><td>Alfalfa Pet gene promoter</td><td>ATAGIGGACCAGTIAGGTAGGTGGAGAAAGAAASWJTAmAAATATATT TAlATGrTeiCAAATAACaCAAAAATCATAAAAGJTTAAGTTAGCAAGTGT ccacAMTmTTTGSAcwaGiATMaccTAcmaTGTTATMaircAiis AJJAAACAlSAGAGtAHAGAAATATGGATGATAAGAMAAGAGiAGIGATA TTTGAOAACM5I ^ t? GTTACAACATTTGAGAĄAAWJ! 3? GMGMCTCTaT HrTCATrGGTCAAAAACAATAGAGAGAGAGAGAGAAAAAGGAAGAGGGAGA AiAAAĄACATAAlGSGAGTATGAaAGAGAAAGTTGTACAAAAGITGTAaCA AAMGGTT0TACAAATaTCMTGAGGAAT®tfaACAAAAGCTACACAAATaA GGGilAATIGaffGTaAaTAAATAAGGATGACGCAT ^ AGAGAGATGTACCAT TAGAGAATTTTTGGCAAGTCATrAAAAAGAAAGAATAAAfSATTTTTAAAĄ srowitmoAGicATKraMmmT ^ GAGMTTGGATTAAAGiTGTATTAAlieATTAGAATSIGGTGrCAAAIWAA WTGACATTTGATCTiriCCTATATATSGCCGOATAGAGTCAiTTAACTOA TraWAWTSCATAGATOAAATAAGAGAAATAACGGTATATTAATCCCTC CAACAAAAAAAAAAAAAAAACGGlAmiTAC ^ AAAAAATCTAAGGaACGC AGGAGGAlAACATCęAASCCAACGAATęAOAAGĄATCCTGATGAGATMCC oactwaagcccacgcactctgtggcacatctacastatctaaatcacaoa STGTTCCACACASOTGAGCCAGACAAAAACCAADCCACAilOTTTA ^ CATCC ATTGTATAAAaAATCACACTMGTGAGTCTACACCTTGATTCCCTSfaAAAC ACATACAaAGAGAAGAaAGTAATTAATJAATTAAłCAlCTTGAGAGĄAGC 0</td>

<td> 36</td><td>Alfalfa Pal gene promoter</td><td>AGAGAGGAGGCAGTGTACACAGGGGCAGAGAGAGGTGAGffCGTCTTTęffGG TAGGGCTGGTGitTGGGGATAGTGGTTGGTTTGAGAGTCAGGTGGIGAGgAG GGCrGGCGATGGGGTTGATACGTSGTTTtGGTTGGATAGGSGGCTAGGAGA TGCTCOmaTGTGTTTGTTięAeGAGÓTTGSTTGAGTTAACAGAGAACAA ATMęlGTCSGIGGCTAATTTGTTATCIGTTGACTCGGAGCAGTGGGGGGA GGIGTTGAGGIGAAGCGTATGSSGGCAGAGGTGGTGGęAGAGGTGAAGCGT ATGGrGGCAGCTGAGGGAGGCAGTGSACACAGAGGTGeAGAGAGAGGAGAG agaagagagaacagagagaaaasggagaagagagaagagaagaoagagaag ACAAAWTTSGTGTGTGTGACCAAACCAAAAT ^ CITGGTCCTGGTCCACAC AAGASTJJICTaCCAACCĄAGGTACAAGAAłACCACGATCCiAAGAGiGCCAG GTIGCAACATCATAACCGIIOAATAGTAAGAGATAATCGAAGGGCCATAA⁴? TAAIOTTCAACAAACCCAarmTSCCIPCCTACitTITGCAACTTGłCCCTC ATCACCTACCAAACACACATAGGACACCAACACACAl? AATAATAiTATAA5 aamgtaaatatatgtagcctccaaattagbaa.gajacgtctatataaagc CTAAaTAaTTCOTTCACAAATCAGGAAASTGACAACTCSAATAiCTCASWTG TTTCCTAAlCA ^ WAGAATTTCaAMCSTATAAAAiTCTAGGTACCACCACA CAACAAATAAAGGiiACATfAATCAATACTATTAAGATGGATa</td>

<td> 37</td><td>the promoter of the alfalfa Ccomt gene</td><td>CJmATTMTGATTmTCAACCTmTWWWAGGMGGTGACCTTAT TTTGCĄAATAATCCATGCATGGAAATGCATCATCCTiMGAAAAlGGGATJ! AICiGAAiTCITAAGTTACGUGAWtSTAATACATTTCMTTTAGASAAA WAmiSAmiit ^ OACmGAWKTOAAAAAraitóC ^ AACAAMCAAATAAAATATACGACGAAATGAGTGTAAlSTAGSgJGSSiAAG CAiCGiGAAGCIIGGAGAGAAAGATCATAGtTTGATCTITGAAAACrAaAa TAWGAAAAGGGTGAAGATATCJAAACATCCAAACAAMTTiPAmTGATA GICGMTCZiAATmCMAATTIGTGAWiTATTTTGTŁWTGTTAAGir GGCAAAAAlATGTTAAWTTUJCAAATTACCASETGCAOATETTacTAATCTC AAATCACATTTĄaGGGATGTTGACTACtl ^ AGTTTTGTACAAAICilTACA ASTTTAAGA ^ raATAAAArGTGTrSGGGTAGATAAAAAGTGiGAGTATiGT TTATAAGAGATTGTGTtTlTCTTTTGTTTAAACTTATMAATńAATATATA stttattttattttaatgtoagattgtaagaattgattataagaitatgsc ATTCCCTCAAAAGAAAATSAGATGATGTCAiTTTCATAACMAmTaTM AAATACAGAaaATCCTCAARAATGAAAAAĆCTCAGTCAAAAAAfAAAAGAA AAACATCAA AGTGGACIGGCCCACAC ^ ^ CATTGCTTSGCTSSAGrATAAGA AAGiAGACCSCACCAACCACGAACCGGAaGCCAAGCGmCAACCAAACAT TACACCAATOTCCTTAACCATACCGSTmTCCCTCGCmTATAACCAT CTTCCTACCTCTIAECTAACCAAGCTCCAMCAACTCirCAACACATAUCA</td>

104

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td></td><td></td><td>GAMCAGAĄaAaGMGCMMCATMCMIiMTTMCR</td>

<td> 38</td><td>a 171 bp fragment from the Ccomt promoter</td><td>cwamaGjwkCTGGoccacĄiMsmiMOT TAGACCTCACCAACCaCGAAGCGGACGCCMCCGGTTOMCCĄAACATTAC AGCMTTMCCSTAACCATAGCSGTTTTTCCCWCCTIńiATAACGATCTT CCTAĆOTCMAICTAACC</td>

<td> 39</td><td>promoter of the tomato polygalacturonase gene</td><td>AAeĆrfCiffAAAAAGGCaAaTTGATTAATTSGAAGICAAAATAAStcaAilA (WMtfMTCOTAAACOACCITAAGAAACCATAOTT ^^ (MTATATATWTmcWGMAMATAAAAAmTIiaiW GaCTAAMXATXCiCAMGTAWCGAAAXGGTACMMCTAGCATCCGSCC AOCTASTGACTCCaAAATAAAATTATTATCCACCTTTGAGTOTflAMTlCGA CmcmTATAACAmmWTTAMCiAWMWACMMAAWilra CASGGaGTTCWTAraTMTATAAITSMMTATGMmCAmAtĄIUl ęcAACCAACFACOAACTCAT5? AATCATXAAAXaCCACGCWriCTACTAX CAĄaA®XGTCCTAAACAaTACTAMAęAAGACGAAAXTGXTCGAGTCC0AA ICGAAGCACcmCTAATJ? TAGGMGAGCCQCATATTlAGGAQGACACS?! IT CAATAGTATmSTTCMGCATGAAMSGAAATTTAAGATTAATGGTAMG MGTAGTACACCC0AAWTMTTCAT0Cęm2ITAAMA! L? AAWAXATAAA IATSTMGATTTGWTIAAMMTAAAACTTGAMATMEAMTSTAAAAA ĄATTATCTATTflAGXACCATęilCATAATłGAGACGAGGAATAMTAA0ATG TAGrWWMWA W ^ ^ ^ ĄAiijpftl STAG! ® ^ ^ TATGAATMGT ĄAATWĄAĆAaATATyjGAGCGCGATGWTTTAAaAa AJĄTrĄAATAAGWrTGAATTiAAAACCGTTAGATAAAIGGICAATTMGM CCCAAAAGiGGATSAGAP.GGGTATSTMOAGCCAATAGGGGraATGAGAAGG ATATmGMGCCAAWGTGAiGGAJGGAGGATAMiMGrATCAMTCi AATACTTTAAAGATATSmGGTCAlilSCCCTECTTTAGITTATAGACTA TAGU¹</td>

<td> 40</td><td>Transfer DNA pSIM870 (Russet Boise III)</td><td>XGGCAGGAmAIAaCGSTGTAAACGAAaXGTGTGTGGn¹GAXCCAAAATC SATGGTACGTTTAGĄAAGTGTAGCMGAAGGATAGTCTCACiTATGAAGA actaccsattgagattcstgatcgscaggtccgaaggttgagaaaaataga AGTCGCSWCAGTTACGGCITTGSGGAGGAGTAAGGGSACCGAACCATGCAI C2ęAATCMAATACXAAAAAATGCSACAAAATXCTAGXGGAQGGAGCAGTA aCAGTACAMAGATATTATaimATTACIMAATAATATMTAATTAACA CGAGACATAGGAĄTGXCĄaGTGGTAGCGGXAG0AGGGAGTTGGXTCAGXXT XiXAGATACTAGGAGACaGAACCGGAGGGGCCCMtrGCfiAGGaCGAAGTTG AAGSCCAGCCGTGAATCAACAAAGAGAGGGCCCAXAAXACTGXCGATGAGC ATTTGOCTATAATACAGTGTCCACAGTiGCCMCCGCTAAGGGATAGCCAC CęGCJATTCTCTTGAGACGTGTGACTGAAACCTGCTACAAATAAGGCAGGC ACCrcaKCAiTCTęAaAOECACrCACSCACACAGCTOAAGAAGtGGTAACif TCTACTCATCTCCTCCAilKTASMCTGArtfTCASGCATGTiTCCGrACACT CWTATGAATCGXGmi'GCmttXASACGTTGTXTCATATCTCATCTCA TCTATTCTGAmTGATTOTaiTGCCTACTGAATTTGACGCTAGIGTAATC GGTGATĄMTG GWGCTTCCTlWTCTS ^ / ^ Grj.O TCT ^ CTCAGĄAATCAAT TSCSaCJTTTGTTSTiGTICATCrGTAGCiTGGrAGATTCCCCmiSGSAG ACCACACATCACGGATGCCCCAAACATAATTĘffAAGTGATTTATEGTTGAA TaACAACTl^,CM.TGMATCAAGGMCAAAGai'GATXTCAACATGABAAAAC AGAACAAGAAAACGAAAACAGAGCATęATCCATCAAAGTGTAATCiCAGCA</td>

<td></td><td></td><td>ΘΑΪΪ</td>

105

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 41</td><td>fragment of an element of the attached phosphorylase gene</td><td>tWATCAIGTGATOAAGGTATATAATGiATOIGTMGAGGATGATGmMA CCACATAATAA.GAGA ^ G ^ GAGICSCMMTCTGCjy</td>

<td> 42</td><td>Transfer DNA pSIM870 (Russet Boise IV)</td><td>MGCAGGATATATACCGGTGTAAACGAAGTGrGTGTGGTTGATCCAAAATC TATCGTACCTITAGAAASTGTAGCTATGAAGSATĄGTCTCAOtiPAIGAAGA AOIACCTATTGAGMrcTTGAi! AeTCA0GTCCGAAGGxm®AAWmGA AGTCGaSJUOAGTTACGGCTIlGTGGAGGAGTAAGGGTAGCGAAGCATGCAT CTCAA5fGTTAAiACSAAAAAAIGCAACAAAA®iGTAGl'GGAGGGACCAGIA CaaerAOATTAGATACTATCTTmmCifATAATAAmjTEWTAACA CGAGACATAGGAATGICAAGTGGTAGCGGIAGGAGGGAGTTGGTTCAGTTT TiTAGATACPAGGAGAęAGAACCGGAGGGGCOCAMGCMGGCCCAAGTIG MGiOCAGCCGTGAAiTCAACaAAGAGAGGGGOCAiAATACTGTCGATGAGC AraiCCOTATAATACAGTG ^ CCACAGOTGCCMCCGaTAAGGaATAGCCAC CGGCTATTCTCTliGAGAaGTGTCACTGAAACCTGGIAaAAAIAAGGGAGGC ACCTCCTCATTCSOACACTCACSCAGTCACACAGCTCAACAAGTGGCtAACT reiACTCATCICCTCCSASIAiMaSeATTECAtfGCATGTTTCCCTACATS CWWGAAKOTGWTPGrCTATAAACGOTGmiCAmCTCAICICA TCTATICTGAMTTGASTCTCTTGCCTAeTGAATSSGACCCTACiGTAATa GGSGATAAATGTGAASGGMCęTCTTCTlaTTCiTCTTCTCAGAAATCAAT aKCTasmGłmMiiiwcmAecwGGTAeATSCC ^ ACCACACATCACGGATCCCCCAAACATMTTGIAACTGATTTATTCTTGAA TAACAAĆTTCAATGAAMCAAGCAAOAAAGCTGATTlęAACAiGAAMAAG AGAACAAGAAAACGAABACAGAGCASCAIGCATCAAAGTGTAAWCTCAGCA GATTCAATAGAGACiAACTCGAGGjGCICSCTATGCAAATCTAGCSTIICG AAlGAeAGTGATAAGAGAGTGAGSATTGiGAArTMSTiCATTGASGAAGAT IGGAGAAGTCAATTAU ^ GATICACACACASGAATTAAGMMTGTGraGC GTCCrCTTGWAAAiffAAATGTCAGCCITMTISATmTaAAlAAAAGC ACGMMTCTCCTGCACiACTCCGGrGOAGraOOTATATTTGSWAm CCACAAATCCCISACMAATTAGTTACCCACAGTCAAGCTTAAGCAGiłAAA IGAGAOSCgSCATGTGTTAmifGiaGSAATAAGAICATCCTCTEACACAT ACAiJIAiATACCMCATGACA ^ GATAAAAAGABAnACTAaiTACTGTGaCa: WSmiJPmaASJJcACaWCCCCCfOCCGOGGeBTAAAgJIFCASGAMm TGAACrCAATAGGITSTCATAA ^ ^ GAGCAATATTAICWWCTTCAGTAGOaA ATCCACAWGCTOrTATGeTCGMGńAATAGMTTGGCCGTGGAGTITCACO ASOTATGTTIĄCAATIOA TGWSGTAGCSSCAGGAGGGGGAAGSGAAiGAA mm &lt;mASG (WlAWAAGTACTT! FCttam! L! ^^ TATCamGAT0AA ATATAATGTATSTGTMGAGGATGATGTTAraACCACATMTAAGAGATGA AĆAGSGtCAiETTCIGGMAAGCMGAGtfOTGGGTAAGTAATOAAOTAGG GATKfOTGGGAAATGGACAAATAWGAGAGTGCAGGGGAGSAGTGCAGGA GATTTTCGTeCiMTAfTGaTAaATAASAAAAGGGTGACATTTAASITCCA</td>

106

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td></td><td></td><td>CAAGASGACGCMCACAACACACTTAATOCCTGTGTGTGAMCAATWWTG ACTXCSCCAASCMCATCAATAAAMAM! ECAC # TCG! FGAGTCTC! ITATC ACTCTCATTCGAAAAGCTASATTTGCATAGAGAGGACCirCGASCTAGTCTC TAMGAAICTGCTGAGAHACACTTTGAlOGATGASGCifCTGTCTTCGTTE TCirTGTTCIGTKaTWCATGiSGAAATCAGCIT ^ GTrGCTTGAWCATTG AAGtSSOTATTOiiASAATAAAWAGTEACAAWATBITTGGGTCJAGAGIS ATGtGIGGTCTACAAAAAGGGGAATCrACCAAGCTACAGATGAACAAAAAG AAAACAGAAATIGATMCTGAGAAGAAGAAGAAGAAGAGGAAGCATTCACA SIWCAGGGATTACAGTAGGGSCAAASTęAGTAGGCAAGAGAAiCAAAAT CAGAATASA GAGATGAGATATGAAACAACGTSIUATAGACCATAAOaCGAT TCATAATAGAATGiAGGGAAACATGCAJGAAATCAGAAATAA ^ ^ TGGAGGAG ATGAGrAAAAGTTACCACMOITGAGClTGTGTGAGT (? A (MGAGTGTGAGAA TGAGGAGGTGCCTGaCMATTTGIAGCAGGTTTCAGtfGACACGTGTCAAGA GMTAGCGGGTGGCTAICCCOTAGCGGAAGGCAACTGTGGACACIGTATTA JAGGGAAATGGTCASCGAGAĆTATTATCGGCCCTCTGTTaGiTGASTCACG GCIOGACSJCAACjfTGGGCCiSGCAAJGGGCCCCTCGGGTTOIGICWCTA GTATCSĄAAAAACTGMCCAACTCGCTCCTACCGCTACCACMGACATTCC tatgigtcjgigwaaitaaaatattattasagtaAtaaaagataatatcta AIGTACTGGTAOTGGTCCCICCAOiAGAATOTTGTiGCATmiTTAGffATI 'AAGATTGAGATGCATGGITCGAGGTOCCGCAGTGTGCCAGGGCTGIOGGCA gatggacataaasggcacagcgctcggctcgsggaaagagtatggtcagtt miTaATAAOTATmCTCGTAIiCGGTGTITAGATCAAGTIAATAtGiy CAAACACATGTGATATCATACATCCATTAGTTAAGTATAAATGCCAACTST TIACTtĆAMJCGCCGAAlWWWACWACGTaCAATATCTASKWGSGJ GTCAAACAmCAIGCACTAiraGATmGAATAAATAMCGATGTGKAAT STCAAĄACCAASiAGAAAAGAAGTATGACGGGASTGATGTTCTGTGAAAIC ACTGGTAAATTGGACGGACGATGAAATITGATęGTCCATTiMGCATAGCA Acaja / GGGTC TTAGlaATOATCATTATGITATAATTATTITCTIGAAACTT GATACACCAAGSMCAiiCGGGAAAGTGACAGCATAGIAPAMGTATAATAT OAATffCTGGCAATTICGAATEAITCCAAATCTCTSTSGTCATMCATOECG ^ a ^ ^ cęęrAT Tc.TGPAAG.W.CMiSJAiiTAAGTACAĄAAAATCTTGATTAA .'ACAATTTATra ę.TęACTAATAATĆACAT ^ ^ ^ taji CATCAACGGTTCATACA ĆGTĆTGTCACKCŚimTTAWCTCTCAAGCGCATGIGAKSWPACCAATOA TJTAAATACAAAAAATCIIGaSIAAACAAKCAGTTTCTCAGTAATAATCA CAWiEAATCATCAACGGTTfcATACACATCCGTCACTCiETTTTTTATTCTCT CAASCGCATGtGAICAraCCAATTATTSAAATACAAAAAATCTIGATSAAA CAATTCAMItCTOACTAATAAICACATMAATCATCAACGGTOTATACAC aTCCGCCACTCTTTTTTTATTCTCTCAAGGGTATGTGATCATAICTAACTG TCGTGCAAACAAGTGAAATGACGTTCACTAATAAA5ĄATCffI! TIGAATACT ITGTłCAGTajTAATWATtiSAATTTGAIAAGAATSSTTTSATfCATTęAATT tetattgtsttaaattaaaaataagwaaatatatcaaaatatcttttaat TTTATTn'TGAAAAATAACGTAGMCAAACAAa ^ TAAAAraGAGIAACTGT IWSCGAAAAATAATGATTCTAATAGiTATATTOTmiCAtCAiTAGATAI IllSraTAAGCTAAGTACAAAAGTCATATTTCAATCCCCAAAATAGCCTCA ArCACAAGAMTGęWTAAATCCCCAAAATACCOlfCAATCACAAGACGTGTG i7lCOAAlWrACOWm «G'CTCGTAMTTGCGACM ^ AAAGTTACCCOTGATATCAGTATmAAAACTAAAAATCTCAGCSGTAATT CAAGUGęAATCACACTCTACCACAOACTCTCSAGTAGAGAGATCAGWGAil? AAOARGCTTGTTAACGGATCCAAAAiffCTTATGTTAACGAAATAAATTGAG ACAAATIAAETCAGffiTAACCAGAGSSAAGAGirAAAGTACTATIGCAAGAAA atatcaaaggcaaaagaaaagascasgaaagaaaataicaaagaaaaagaa Gagg jjacaatcaaaotcccaiamacigcaaaaataaacattcamttgg AAAAACATCCAATCAAAilGCTCTACSTCAGGGGGCCCACGCCGGCTGGAT ^ ^^ ctcaaacittcccacgtgacaicccawaacaaatcaccaccgtaacccttc TCAAAACTCGACACCTGACTCWmcraATATTACWAAAAiŁA GTGICCCCGGGGGTJAAATTGATGATrTTMGAACTCAATAGCilifSTCATA ATGASCAATAS ^ ATCTWGTTCAGTAGCAAATCCAOATGGTCTTATGCTCG</td>

107

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td></td><td></td><td>CKGAAATAeTTMGGCCGTGGAGTOTCAęCATCTATGMTACAATTGATTC HTGTAGCTGCAGGGACACGifATAMTITTATTGTAAiATAGAGAAAAAGAG TGAGGTGflCGAGTTMGAGAAGGGTrACGGTIGGGGATTGTSmir¹CACGTGGGAAAGTTCGAGATGCAGCGGGCGTGGGCCCCGTGĄAGiAGAGGA ATTTGAITGGATGTmSGCAATTieAATGTSTATmTCIGWiTTASeG ΟΑΰϊϊΤβΑΪΤβΤΑΑΟΟΤΰϊϊεϊΤΪΚΓΟΤΤνΟΑΤΑΤϊ ^ ^ ΤΟΪϊΕΟΑΤβΑΤαΤΤ TICMTiGCCTSTGMATiTTCTTGCiUTAGTACTT ACICrW-CiClGG TTAACTOmiCAATETGTCIGAAaimWTGGTTAAaATĄASAAWIIETCI AGAGJGATGyGTSGSOPACAAAAAGGGGĄAICFACCAAGnTACAGATGAAC AAAAACAAAACAGAAATrGASITOTGAGAAGAAGAAGAAGAAeAGGAAGCA TTCACAJTrATCACCGATTAGAGTAGGGTCAAASICAGTAGGOAAGAGAAI¹CAAAAiCASAAyAGATGAGATGAGAmGAĄACAACGStCTATACACCATAĄ CACGAWCATAATAGMTOTAGGGAMCATGCMGi ^ TCAGWWITG GAGGAGATGAGTAAAAGITACĆACWGTTGAGCUGSGTGAGTGAGTGAGTG SGAOAATGAGGAGGIfGCCTGCaiTATaTGTAGCŁGGTTICAGTGACAcgtG JCAAGAGĄATAGCGSGTSGCJATCCGSSAGCGGAAGGCAACTGTGGACACT STAMATAGGGAAATGCICATCGACAGTATTATGGGGCCTCirCTTTGMGA IICACGGCTGGACTTCĄa.CTTSGGGCOiTSCAATGGGaCGCTCGGGMCSGT CTCCIAGTATęTAĄaAAACIGAACCnAGTCCGTCCTACCGCTACCACTOA CATICCTATGTCTCGIGTTMMAAMTATCAKTASAGSAATAAAAGATM αMCTMΪGTACTGΘTACTGGTCCCϊCCΆC AGAAT ¢: mGϊXGCATΪΪfl '¢ ^ ϊ AGTATTAAGATTGAGATGCATGGMCGAGCTCCTTGAACAT0TWAAACr CAęAfATTGAGSTeGGSATAGGCTTTATAATGAGWGGACSACGTTATGr CCCCCTCAAGTCCCAGAATTAIGTGCCCCCGTATGTTATAAGffCCCCiJOTG cgggcatcaatttagtgatcacgccagacatgcctctatacgtcggccagg ftTATŁTTTGTTGGTAATG</td>

<td> 43</td><td>intron R1</td><td>GTAAAMTCTAGTGATTATACTGTACATTTCGCaTAATTTAGGATCGTATT TGMATGTimCGCTTGA ^ TGAlTCGAGAACTTAAAGCTMMGATCTGA AATirCTSJ and / GGCATZiCSCGAGSIGńGATCGlGGTTAruATCAGlGTUA ^^ ^ ^ TTCGAI GAAMTSASGAAAATMGGPGTTGATMTCAGJATTTTGATGGr TTAArGiATATBAACAABCTTMTtrayTCAAATiCAAGGTCGTSTAACCM STWTACAGCMamCT0GAAM1Wm &lt;Mi &lt;^^^ TATTCGŁGAAAAAAACAAAAACGAAAAAAGSGGAAASGfiiG1! 'FCTGTA ^ Gr ACAAAAAGTGATTGATCAGCTmGGICACCSAOATACAT ^ TGATTAGTAC atacacgagtcatacgagtasatttccgATGTGTTAGAGATTGTATTAGATAGATGTATTAGA CACTETACTOTAAWAAGTCTWCTGTGAACTGACAAATACTGAęęAGGC ACACATTAaAACGTiCAnTGASTATCCGCGAAĆGATCCATTGCTTTTGTG TATTłGCITMGiSATIGACTGATTISGTATiGyAtmGGAG</td>

<td> 44</td><td>limit st02 of potatoes</td><td>CATTACCAACAAATATATCCTGGCC</td>

<td> 45</td><td>border of the tomato Le01</td><td>CATTACCAACAAATATATCCTGGCC</td>

<td> 46</td><td>border of the tomato</td><td>CTCTACCTCTGAATATATCCTGCGG</td>

<td> 47</td><td>border of pepper Ca01</td><td>CATTACCAACAAATATATCCTGGCC</td>

<td> 48</td><td>limit of Lucerne Ma01</td><td>GTATACCTCTGTATACATCCTGCCG</td>

<td> 49</td><td>Hv01 boundary for barley</td><td>ATATACCAAATGATACATCCTGCCC</td>

108

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 50</td><td>border Os01 rice</td><td>ACTTACTCAAGGTATATCCTGCCT</td>

<td> 51</td><td>the central 300 bp fragment of the P1 promoter</td><td>GCGTCAACAAGGTCAGGGTACAGAGTCTGCAAACCATTAGCCAAAAGCTAC GCATCATGGTCaGTAAGTIiCAGAiLAGGACATCCACCGAAGACSTAMGT TAGTGGGOATCSSSGAAAGTAASCiTGTCAACATCGAGCAGCTGGCTTGTG GGGACCAGACaAAAAAGGAATGGTGCAGAAWMGCAMTACAGAAWGCCTGTACGAAWGMCC</td>

<td> 52</td><td>fragment of the Comt gene</td><td>AATGCCCCCAICAAGGACTGCATCmfTAGGTGGTACCAGCmCCAtGAS CACTWTATCGIGATJCATGAGATJAAGAGCAGAMffGGATAGACCAiCSTC ATICITAACęaAATACTSAGCAACAGTAGCęAAacCAiAAAGSOTCTGAaC CITICCATCTTGTTGaGTAGGAACTGAACAAGTGAGGACATTGTAAcaAGG CMGAGACGCMCATOCGGTCCMCATAACTGSTGCATCAGGGMAGTTGT TCGTAGCTGAGMGCAAmCmAGGTGMATTTGAGGACCAGGSCGA</td>

<td> 53</td><td>fragment of the Comt gene</td><td>MTGCCCĆCAICAAGGACTGCATCmiAGGTGGtACCAGmTCĆATGAG CACTTTATCCTGATTCATGAGAiTAAGAGCAGAAATGGATACACCAKTTC ATTCTmCCĄMTACTTAGCAACAGTAGCCAAACCATAAAGICTCTGAAG CrarcCATCTMMGAG ^ ACGAACTGAAĆAAGTGAGeATATTGTAACAAGC caagagacgcaacatbcggtccsacataactgstgcatcagggttagttgi '.reGfAGCTaAGAJ.GCAAITTCAATAGGTGAAATTIGAGOACCAĆGTCCAGA ATTCAATCTCACAMAACCTCATCAATCACAACCMGGGTTCAACAGGTGA AACTCAAATMCACCMCCCACMATCAGATGMGAAGCAAACCTCTICGC CATGGAACTAGCAAGTGCTICAGTWCMCCCATGATTITGAAATCAGCTCT OTAACTTGAICTCTTAGAMTCATTGCTAAAGC</td>

<td> 54</td><td>alfalfa promoter</td><td>gggcccmagtggacgagttaggtaggtggagaaagaaactattaaaaaaa fflTATTTATATGraGTCĄAAIAACTCMAAAttCAIAAAAGTOIirAAGTTAGC AAGTGTGCACATTTTTATITGGACAAaAGirATICACCTACTACTGTTATĄA ATtaTTATTAAACATTAGAGmAGAAATATGGAitGATAAGAATAAGAGTA GTGATATTTrGACAACAAlSSMrTAęAACAT ^ TGAGAAAATITTGTIGTT CTCTCTSJSTęAęTGGICAAAAACAATAGAGĄGAGAGĄGAGAWlAAGGAAGA GTACCĄAĄATGGiiałGIACAaATATÓAMGAGGAAtTTGAGAAAAGCTACAC aaataagggitaattgctgtaaataaataaggatgacgcattagagagato lACCATMGAGAATTSTTGGCAAGTCATTAAAAAGAAAGAATAAATTAiiiT TTAWATEAAAAGTTGAGTCATI GA ^ ^ TAAACATGTGMTATTTAATGAAT TGAIGAGAGAGMGGMIAAAGSTGTATTMTGATTAGAMTTGGTGTCAA ATTTAAT TTGACATTTGAJCTTJjrCCMTATA TGCCCCATAGAGTCATTT ^ '^ SACTCATI JTATATTTaATAGArCAAAiAAGAGAAATAACGGTAaiAMAA TCCCTCCAŁCAAMAMUUWiiWOiACGGTATAmACTAAĄAWOTAAG CCAGGTAGGAGGATAilCATCCAATCCAAGCAATCACĄAGAATCCTGAIGAG ataacccaciitaagcccaggcacictgtggcacatćtacattatctaaat CACACAłfTGTTCCACACATCIGAGCCACACAAAAACCAATCCACATCKTTA TCATCCAifrCTATAAAAĄATCACŁCTIiGTGAGiCłACACTTTGAETCaCT TCHIAACACAVACĄAmmsA (^ TAA! RmTTAMTAAiCM GAAAGCC.</td>

109

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 55</td><td>alfalfa promoter</td><td>AGAGAGGAGGCAGTGTACACAGGGGCAGAGaGAGGTGAGTCGTCTTTCTGG TAGGGCTGGTGWGGGGATAGtGSTTGGTTIGAGAGTCAGGTGGTGAGGAG GGłTGGCGATSGGGTTGAiacGTiGSSTTGGTlGGATAGGTGGTTAGSAGA IGCTCGEmrGTOTTGmcBSGAGGTTGCTTGACTmCAGAGAACAA ĄTTIGTGTCSGTGGCTAAWTGWatCSGTSGAClOSGAGCAGTGGGGGGA GGTGTSGAGgTGAAGCGTATGGTGGCAGASGWGGTGGCAGAGGEGMGCGT ATGGTGGCAGCTGAGGGAGGCaOTGTACACAGAGGTGGAGAGAGAGGAGAG A &lt;i; AA (3ĄSAGAAGAGAGAGAAAATG0AGAAGAGB.QAAI3AGAAGAGAGAGAAG ACAAATtTTl? GTGTGiGTGACCAaAGCAAAAT3? CTTGGTCCTGGTCCACA0 MGATOTICTCCOAACGAAGGTACAAGAATACOACGATCCAAGaGTGCCAC GJTGCAACAtCATAACaGTSGAATAGTAASAGAiaATCGAaCGGCCATAAT</td>

<td></td><td></td><td>saatsttcaackaacccactstuiccicctacttttgcaacttgtccctg atoacctaccaaacacacatagcacaccaacacącataataatatwaat WW'm? MATGTASCCTCCAWTAGmGAWCTCTAWW \ GC GSAACi? AOTTCCTTCACAAaTOAGGAAATTCACAACTCTAATATTCATTTC TTTCCTMTCATTAGAACMCOAMGSTATAAAATTCTAGGTACCACCACA CAACAAATAAAGGaACMTAATCAATAaTATTAAGAT</td>

<td> 56</td><td>alfalfa DNA fragment that functions as an alternative to the left border of Agrobacterium</td><td>cggcaggatgtatacagaggtatacaattotatattacatttataittstg ΪΙΆΑϊϊΟΑΪΙβΑΑΤΙΤΤίαΟΪΪΤΤΑΤϊϊϊΤΤΑσϊϊϊφΑϊΑΑΈΟΑΑΟΙβΤΟΤ AAAGMmmGAAAAAIAmATAamATAmmJSTOSiWATG</td>

<td> 57</td><td>a fragment of alfalfa DNA that functions as an alternative to the right border of Agrobaotherium</td><td>CTAGATWGCGGGCmCGGGaTGCCCGCGGCCCmGGGGCTAGCGGTA ACGGGTACCC-GGCCCCf ^ CAGGMGTAtACAGAGGWAG</td>

110

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 58</td><td>complete transfer DNA pSIM856</td><td>CGGaaGaATGTMaCAGAGmTACAATTTTATATTACŁTmTATIKJrS TTAATTCATGGMTITTCACTMTATTiTTTACTTTGATAATC ^ ^^ erGTGT AAAGAAmmtmwTAWMAATTTATAeAAimmT gggcccatag5ggaccagtta0gta &lt;3gtggagaaagaaattatimaaaaa TATABTTATATGMGTCAAATAACTCAAWWGATAAAAGTTmGffTAGG MGTGTGCACAaimtfAmGGACAAMGTATTCACCTACTACTGTTATAA ATGATTATTAAACATTAGAGTAAAGAAATATGGATGATAAGAATAAGAGTA GTGATATTTTGACAACAATTITGITAaAACATTTGAGMAATTTTGiTGrT CTCiCITttGATTGGTCaAAAAOAAWAGAGAGABAGAGAGAAAMGGAAGA GGGAGAATAAAAACATAA ^ GTGAGWGAGAGAGAAAGTTGTACAAAAGTT OTACCAaAATGGMGTAGAAATATCATSGAGGAATTTGACAAAAGGTACAC AĄATAAGGGraAATTGCSGTAAATAAATAAGGiaTGACGCATTAGAGAGATG TACGAmGAGĄATTmGGCAAGTCAiiEAAAAAGAAAGAATAAATiATTT TSfAAAAMAAAAGTTGAGTCATTTGATTAAACAUGifGAWATTTAATBAAB TGATGAGAGAGSTGGATTAAAGSiEGJAlTAATSAtrTAGAATTTGGTGrCAA ^! And ATaKAAmGACaiSmTCTTTTCCTATATATBGCGCCATAGAGSCASa AACJ! CATTMTATATnGATA0Al? CAAATAAGAGAAATAAC0GTATATTAA łCCęrCCAAOAAAAAAAflAAAAAAAACGGTATASTTACTAAAAAATCTAAG CCAGG? AGGAGGATAACAiGCAATCCAACCAATCACAACAASCGIGATGAG ATAACCCACTrTAASCCCACGCACTCEGTGGGACATCiACAtTATCTAAAl? CaCACmcraCCACACAraWGCGACACWGmCOM.TCCACATCTTTA TCATCCAWCTATAAAAAATCACACTTWBAGTCTAaACSWIGATTCGCT 'E^,CAAACACATACAAAGAGAAGAGACTAAiTAATTAATl'AATCATCa'TGAGA GAAAGCCCTGCAGAATGCCCCGATCAAGGACTGCATCITTIAGGTGGSACC agctticcaisagcacmwcctgattcatgagattaagagcagaaatgg</td>

<td></td><td></td><td>ATACACCATCTTCATTCTTAACCAAATACTTAGCAACAGTAGCCAAACGAT AMGTCTCTGAACCTTTCCATCTTGTTGAGTACGAACTGAACAAGTGAGGA TATTGTAACAAGCCAAGAGACGCAACATTCGGTCCAACATAACTGGTGCAT CAGGGTTAGTTGTTGGTAGCTGAGAAGCAATTTCAATAGGTGAĄATTTGAG caccaggtccagctttagcaatgatttctaagagatcaagttcaagagctg atttcaaaatcatgggaagaactgaagcacttgctagttgcatggcgaaga ggtttgcttcttcatctgatatgtgggttggtgttatttgagtttcacctg TTGAAęcCATGGTTGTGATTGATGAGGTTTTTGTGAGATTGAATTCTGGAC CTGGTGCTCAAATTTCACCTATTGAAATTGCTTCTCAGCTACCAACAACTA ACCCTGATGCACCAGTTATGTTGGACCGAATGTTGCGTCTCTTGGCTTGTT ACAATATCCTCACTTGTTCAGTTCGTACTCAACAAGATGGAAAGGTTCAGA GACTTTATGGTTTGGCTACTGTTGCTAAGTATTTGGTTAAGAATGAAGATG gtgtatccatttctgctcttaatctcatgaatcaggataaagtgctcatgg ĄAAGCTGGIACCACCTAAAAGATGCAGTCCTTGATGGGGGCATTGGATCCA TCTTAATAGTATTGATTAATGTTCCTrTATTTGTTGTGTGGTGGTACCTAG AATTTTATAAGAATGG71AATTCTAATGATTAGGAAAGAAATGAATATTAGA GTTGTGAATTTCCIGATTTGTGAAGGAAGTAGTTAGGCTTTATATAGAGGT ttctttctaatttggaggctacatatatttacamtattataatattatta TGTGTGTTGGTGTGCTATGTGTGTTTGGTAGGTGATGAGGGACAAGTTGCA aaagtaggaggaaamagtgggtttgttgaaaattaattatggccgttcga ttatctcttactattgaacggttatgatgttgcaacgtggcactcttggat cgtggtattcttgtaccttggttgggagaaaatcttgtgtggaccaggacc aagaattttggtttggtcacacacacaaaaatttgtcttctctctcttctc TTCTCTCTTCTCCATTTTCTCTCTCTTCTCTCTTCTCTCTCCTCTCTCTCC ACCTCTGTGTACACTGCCTCCCTCAGCTGCCACCATACGCTTCACCTCTGC CACCACCTCTGCCACCATACGCTTCACCTCAACACCTCCCCCCACTGCTCC gagtcąacagataacaaattagccacagacacaaatttgttctctgttaac TCAAACAACCTCCTGAĄACAAACACAAAAAGGAGCATCTCCTAACCACCTA TCCAACCAAAACAACGTATCAĄCCaCATCGCCAACCGTCCTCACCACCTGA CTCTCĄAACCAACCACTATCCCCAACACCAGCCCTACCAGAAAGACGACTC ACCTCTCTCTGCCCCTGTGTACACTGCCTCCTCTCTCTAGATTATGCGGGC TAACGGGCTGCCCGCGGCCCTTTCGGGCTAGęCCTAACGGGTACCGGGCCC CGGCAGGATGTATACAGAGGTATAC</td>

111

<td>SEQ ID NO:</td><td>Name (if any)</td><td>Sequence</td>

<td> 59</td><td>COMT promoter from corn</td><td>TTCCACGGCAGCTGCCACCGTCGCTATCGCTGACCĄACCCGGCTGGTCGCC TCTGTGCTCCATCCATGCATGTTACAACTATGGAGATGCAGCCGAAACAAA CACTGGCTAGAAAGGCAGCCCAACGGGCCTACTGTCATTCGCTCCGGCATG CTACTGGTGGGCCCACTTGCACCGGCCGATGACCAGTTCATCATTTTTCTC GACGĄATTTGTGCACAGAATTTGCTĄAĄAATTCTTCGCACGTGGCAAAACC AGGGGGAAAATCGACAACTAGTCGGGGTTTTTTTAATTCCCTGATAGĄATA GTCCCTGCTAATCATCCATGĄAAACCAAACACGTACTCTACGTCACCGTCA TGGATGGAGCGAGTGAACTGATGATTTTTTCCCCATCCCGCACGCAACAGC ATGGGTGACAACAACCACICCCGCTGCGGTTGGGCGAGCACATCTCTACGC acttgacactcacgcaaacctaacgcatactagagtaatcatcgccaccaa ctatcggcgacagaaacgatgggccccgcttctcttaatcacggtgcttga ATTAGTGCGCGCATAGTAGTGAAAAAATAATA (? TGAAAAAATAGTGTAAATAATA</td>

112