Transgenic plants containing molecular decoys that alter protein content therein

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DESCRIPTION

Transgenic plants that contain protein molecular decoys inside.

Field of the invention

The present invention describes a process for the production of transgenic plants such as transgenic tobacco plants with altered protein content within, leading to altered phenotypes such as reduced nicotine levels, together with transgenic plants produced in this way and seeds. for such plants.

Background of the invention

Production of tobacco with decreased nicotine levels is of interest, given concerns regarding the addictive nature of nicotine. Additionally, tobacco plants with extremely low levels of nicotine production, or no nicotine production, are attractive as recipients of transgenes expressing products of commercial value such as pharmaceuticals, cosmetic components, or food additives. Various procedures have been designed to remove nicotine from tobacco. However, most of these procedures remove other ingredients from the tobacco in addition to nicotine, thereby adversely affecting the tobacco. Classic plant breeding techniques have produced tobacco plants with lower levels of nicotine (approximately 8%) than those found in wild-type tobacco plants. Tobacco and tobacco plants having even further reductions in nicotine content are desirable.

Nicotine is formed mainly in the roots of the tobacco plant and is subsequently transported to the leaves, where it is stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden, Hutchinson and Ross, Stroudsburg, Pa ( 1972)). Nicotine is produced by the condensation of two precursors, nicotinic acid and N-methylpyrrolinium, which arise from two different biosynthetic pathways (see Figure 1) (Bush and Saunders (1977) Proc. Am. Chem. Soc. Symp., New Orleans, pp. . 389-425; Hashimoto and Yamada (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 257-285; Walter and Dermer (1981) in: The Biochemistry of Plants: A Comprehensive Treatise, PK Stumpf and EE Conn, eds. Academia Press, pp. 317-395). The pyridine nucleotide cycle synthesizes nicotinic acid (Wagner et al. (1986) Plant 167, 226-232; Wagner and Wagner (1985) Plant 165, 532-537), while N-methylpyrrolinium cations are synthesized from ornithine or arginine by rot (Leete (1980) In: Encyclopedia of Plant Physiology, Secondary Plant Products, Vol. 8, EA Bell and BV Charlwood, eds, Springer-Verlag, pp. 65-91; Tiburcio and Galston (1986) Phytochemistry, 25, 107-110). Reciprocal grafting experiments have shown that nicotine is synthesized in the roots and transported through the xylem to the leaves and other organs of plants (Dawson (1941) Science, 94, 396-397).

Two regulatory loci (Nic1 and Nic2) regulate nicotine production. Legg et al. ((1969) J. Hered, 60, 213-217) incorporated genes from cultivars of pure Cuban low alkaloid content into Burley 21 cultivars. These researchers demonstrated that lines with low alkaloid content differed from cultivars conventional at two loci, Nicl (originally identified as A) and Nic2 (originally identified as B). These two loci are not linked and the gene action is semi-dominant and mainly additive (Legg et al. (1969) J. Hered, 60, 213-217). Collins et al. ((1974) Crop Sci., 14, 77-80) prepared tobacco double haploid breeding lines of these four alkaloid genotypes. The genotype of the conventional cultivated varieties is Nic1 / Nic1 Nic2 / Nic2 and that of the low nicotine lines is nic1 / nic1 nic2 / nic2. Nic1 / Nic1 nic2 / nic2 is a higher intermediate and nic1 / nic1 Nic2 / Nic2 is a lower intermediate (Legg and Collins (1971) Can. J. Genet. Cytol. 13, 287-291). These lines are similar in days to start flowering, number of leaves, leaf size, and plant height. Enzymatic analyzes of roots of single and double Nic mutants show that the activities of two enzymes, quinolinate phosphoriboxyl transferase (QPTase) and rot methyl transferase (PMTase), are directly proportional to the levels of nicotine biosynthesis (Saunders and Bush ( 1979) Plant Physiol 64: 236). Both Nic1 and Nic2 affect PMTase and QPTase activities in roots, and thus regulate nicotine synthesis (Leete (1983) In: Alkaloids: Chemical and Biological Perspectives, SW Pelletier, ed. John Wiley and Sons, pp. 85-152).

Hibi et al. ((1994) Plant Cell, 6, 723-735) isolated the cDNA encoding PMTase, PMT, and demonstrated that PMT transcription levels are regulated by Nic1 and Nic2. The QPTase cDNA and genomic clones (NtQPT1) have also been isolated and the transcription levels of NtQPT1 are also regulated by Nic1 and Nic2 (Song, W., Mendu, N., and Conkling, MA (1999) Plant Cell, in preparation). Thus, it appears that Nic genes regulate nicotine content by regulating the transcription levels of genes that encode the two rate-limiting enzymes, PMTase and QPTase. Furthermore, Nic1 and Nic2 have been shown to be positive regulators of NtQPT1 transcription and that promoter sequences upstream of the transcription start site contain the cis-acting sequences necessary for activation by the Nic gene product of NtQPT1 transcription. . Since QPTase and PMTase expression are coordinately regulated by Nic gene products, it is likely that Nic gene products also directly regulate PMT gene transcription.

One approach to reducing the level of a biological product, such as nicotine, is to reduce the amount of an enzyme required (ie, QPTase and PMTase) in the biosynthetic pathway leading to that product. Where the affected enzyme occurs naturally in a rate-limiting amount (relative to the other enzymes required in the

ES 2 267 809 T3 pathway), any reduction in the abundance of this enzyme will decrease the production of the final product. If the amount of the enzyme is normally not rate-limiting, its presence in a cell must be reduced to rate-limiting levels to decrease production of the pathway. Conversely, if the naturally produced amount of the enzyme is rate limiting, then any increase in enzyme activity will result in an increase in the end product of the biosynthetic pathway. Modification of nicotine levels in tobacco plants by antisense regulation of rot methyl transferase (PMTase) expression is proposed in US Patents 5,369,023 and 5,260,205 to Nakatani and Malik. Wahad and Malik PCT application WO 94/28142 describes DNA encoding PMT and the use of sense and antisense PMT constructs. Additionally, PCT application WO 98/56923 by Conkling et al. describes DNA encoding a plant quinolate phosphoribosyl transferase (QPRTase) enzyme, constructs comprising said DNA, and methods of altering QPRTase expression to increase or decrease nicotine production in plants. Despite previous efforts and successes, there remains a need for new approaches to reduce the production of gene products in plants (eg, nicotine).

Summary of the invention

A first aspect of the present invention is an element responsive to the Nic gene product (eg, a DNA sequence that binds to a Nic gene product) such as (a) isolated nucleic acids having a sequence according to SEQ ID N °: 1 or a fragment thereof consisting of at least 200 consecutive nucleotides; and (b) a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of (a), wherein the nucleic acid, when coupled to a reporter gene and used to transform N plants tabacum, is capable of promoting the expression of said reporter gene at a level that is at least 1-5 times higher when expressed in Nic + / nic plants^- that when expressed in nic plants^-/ nic^-.

A second aspect of the present invention is a recombinant nucleic acid construct comprising / containing a cis-acting regulatory element such as an element responsive to a Nic gene product as described above, together with the use of said nucleic acid. recombinant for the production of a transgenic plant or host cell as described herein. The construct can be a vector, such as a ballistic nucleic acid transfer particle or an Agrobacterium vector. Plant cells containing such constructs, and preferably multiple copies thereof, are also an aspect of the invention.

A further aspect of the present invention is a process for manufacturing a transgenic tobacco plant having reduced tobacco-specific nitrosamine and / or nicotine content (TSNA). The method comprises introducing an exogenous nucleic acid construct comprising an element responsive to the Nic gene product as described above into said at least one tobacco plant cell to produce at least one transformed tobacco plant cell. The at least one transformed tobacco plant cell contains the exogenous nucleic acid in an amount or number of copies sufficient to reduce the level of nicotine and / or TSNA of a regenerated tobacco plant from this cell or cells compared to the level of nicotine and / or TSNA that would be present in the absence of the exogenous nucleic acid. The method may further include generating a tobacco plant from the cells of the transformed plant, and (optionally) collecting tobacco leaves, stems, or seeds from the tobacco plant. Therefore, tobacco plants, including leaves, stems, and seeds, generated from said process are also aspects of the present invention.

A further aspect of the present invention is a tobacco plant that has reduced levels of nicotine and / or TSNA in its interior, the plant comprising cells that contain an exogenous nucleic acid, exogenous nucleic acid that comprises an element sensitive to the product of the Nic gene. as described above. The exogenous nucleic acid is contained in the cells in a sufficient copy number to reduce the level of nicotine in that tobacco plant compared to the level of nicotine that would be present in that plant in the absence of the exogenous nucleic acid. Again, the leaves, stems, and seeds of said plant are also aspects of the present invention.

Tobacco products are also disclosed but not claimed including, but not limited to, smoking materials (e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, lozenges that are prepared from such tobacco plants. transgenic tobacco, which are also embodiments of the invention. Preferably these tobacco products are made from collected tobacco leaves and stems that have been cut, dried, cured and / or fermented according to conventional techniques in tobacco preparation. However, modified techniques in tobacco curing and processing can also be implemented to further lower TSNA levels. In some embodiments, tobacco that is made substantially without nicotine and / or TSNA is prepared from a variety of Burley tobacco (eg, Burley 21), Oriental tobacco, or hot air cured tobacco. It should be understood, however, that most varieties of tobacco can be manufactured to be free of nicotine and / or TSNA in accordance with the embodiments described herein.

Additional embodiments that are not claimed include tobacco products that have been carefully mixed to achieve the desired levels of nicotine and / or TSNA. For example, tobacco having a reduced level of nicotine and / or TSNA, prepared as described above, can be mixed with conventional tobacco to obtain virtually any amount of nicotine and / or TSNA. In addition, two or more varieties of tobacco having a reduced nicotine and / or TSNA level can be mixed to achieve a desired amount of nicotine and / or TSNA.

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In this way, the differences in variety, aroma, as well as amounts of nicotine and / or TSNA can be adjusted gradually. These mixed tobacco products can be incorporated into tobacco cessation kits and programs designed to reduce or eliminate nicotine dependence and carcinogenic potential. Such kits and programs are also embodiments of the invention.

Also described but not claimed is the reduction of the amount of TSNA and metabolites thereof in humans who smoke, consume or otherwise ingest tobacco. This procedure is practiced by providing a tobacco product having a reduced amount of TSNA, as described above, to said humans, thereby lowering the carcinogenic potential of said product in said humans.

In addition, a process for manufacturing a plant that has an increased or reduced content of a protein of interest in its interior is described but not claimed, in which the protein of interest is regulated by a cis-acting element selected from the group constituted by (i) a cis-acting activator element that binds to an activator compound, an activator compound that increases the expression of said protein of interest in said plant, and (ii), a cis-acting repressor element that binds to a repressor compound, a repressor compound that decreases the expression of said protein of interest in said plant. The method comprises introducing an exogenous nucleic acid construct comprising said cis-acting element into at least one plant cell to produce at least one transformed plant cell, the at least one transformed plant cell containing the exogenous nucleic acid in a number of copies sufficient to increase or reduce the level of said protein of interest in a plant regenerated from said cells compared to the amount of said protein of interest that would be present in the absence of said exogenous nucleic acid.

Also described but not claimed is a plant (and parts thereof) having increased or decreased levels of a protein of interest within it, the plant comprising cells containing an exogenous nucleic acid, exogenous nucleic acid comprising an element of cis-acting selected from the group consisting of (i) a cis-acting activator element that binds to an activator compound, activator compound that increases the expression of said protein of interest in said plant, and (ii), a cis-acting repressor element that binds to a repressor compound, a repressor compound that decreases the expression of said protein of interest in said plant; the cells containing the exogenous nucleic acid in a copy number sufficient to increase or decrease the level of the protein of interest in the plant compared to the amount of the protein of interest that would be present in the absence of the exogenous nucleic acid.

Also described is a general procedure to decrease the expression of a protein of interest in a host cell (prokaryotic or eukaryotic), in which the transcription of the protein of interest is enhanced by a cis-acting activator element that binds to a activator compound, activator compound that increases the expression of the protein of interest in the host cell. The method comprises the steps of (a) providing a decoy recombinant nucleic acid construct comprising the cis-acting activator element; and (b) introducing the decoy construct into the host cell in an amount sufficient to bind the activator compound and reduce expression of the protein of interest.

Also described is a general procedure for increasing the expression of a protein of interest in a host cell, wherein the transcription of the protein of interest is reduced by a cis-acting repressor element that binds to a repressor compound, repressor compound which decreases the expression of said protein of interest in said host cell. The method comprises the steps of (a) providing a decoy recombinant nucleic acid construct comprising said cis-acting activator element; and (b) introducing said decoy construct into the host cell in an amount sufficient to bind to said repressor compound and increase expression of said protein of interest.

The foregoing and other aspects of the present invention are explained in more detail in the figures herein and the specification shown below.

Brief description of the figures

Figure 1 represents the biosynthetic pathway that leads to nicotine biosynthesis. Enzyme activities known to be regulated by Nicl and Nic2 are QPTase (quinolinate phosphoribosyl transferase) and PMTase (rot methyl transferase). QPTase and PMTase are the rate-limiting enzymatic steps in nicotine biosynthesis and therefore nicotine levels are directly proportional to the activities of QPTase and PMTase.

Figure 2 shows a schematic representation of the NtQPTl gene and the NtQPTluidA promoter chimeras. The transcription start site (+1) is indicated and the arrow indicates the transcription of NtQPTl. The ten exons are presented as diagonally hatched bars. The promoter deletion series are also shown as solid bars truncated from the 5 'end of the promoter. The sizes of the promoter fragments fused to the uidA gene, encoding d-glucuronidase (GUS), (ie, Δ2.0, Δ1.4, etc.) in kilo base pairs (kb) are indicated. The chimeric NtQPTl promoter-uidA fusions were cloned into pBI101.

Figure 3 shows jd-glucuronidase (GUS) activity in roots, leaves, and stems of transgenic tobacco plants bearing the CaMV 35S promoter (Ca MV 35S), promoterless GUS (pBI101), and 5 'deletions. nested from the TobRD2 promoter (gene encoding NtQPTl) fused with GUS. The sizes of the fragments of the

ES 2 267 809 T3 promoter fused to the uidA gene (ie Δ2.0, Δ1.4, etc) in kilo base pairs (kb). For each construct at least 20 independent transformants were tested.

Detailed description of preferred embodiments

The term "plants" as used herein refers to vascular plants. Exemplary plants include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa sp.), Alfalfa (Medicago sativa), rice (Oryza sativa), rapeseed (Brassica napus), rye (Secale cereale), sorghum ( Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), Sweet potato (Ipomoea batatus), yucca (Manihot esculenta), coffee (Cofea sp), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus sp), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa sp), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale) macadamia (Macadamia integrifolia), almond (Prunus amygdalus), beetroot (Beta vulgaris ), apple (Malus pumila), blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), vine (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica), plum (Prunus domestica), pear (Pyrus communis), watermelon (Citrullus vulgaris) duckweed ( Lemna), oats, barley, vegetables, decorative plants, conifers, lawns, (for example, for ornamental, recreational or forage purposes). Vegetables include Solanaceae species (for example, tomatoes, Lycopersicon esculentum), lettuce (for example, Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (Apium graveolens), eggplant (Solanum melongena), asparagus ( Asparagus officinalis), okra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima bean (Phaseolus limensis), pea (Lathyrus sp.), Members of the genus Cucurbita such as Hubbard squash (C. hubbard), Butternut squash (C. moschata), zucchini (C. pepo), curve-necked squash (C. crookneck), C. argyrosperma, C. argyrosperma sp sosoria, C. digitata, C. ecuadorensis, C, foetidissima, C. lundelliana, and C. martinetzii, and members of the genus Cucumis such as cucumber (cucumis sativus), cantaloupe (C. cantalupensis), and melon (C. melo). Ornamental plants include azalea (Rhododendron sp.), Hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa sp.), Tulips (Tulipa sp.), Daffodils (Narcissus sp.), Petunia (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and chrysanthemum. Conifers that may be used to practice the present invention include, for example, pine trees such as taeda pine (Pinus taeda) elliotii pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), contorta pine (Pinus contorta), and contorta pine. Monterrey (Pinus radiata); Douglas fir (Pseudotsuga menziesii); Canadian thuja (Tsuga canadensis); white spruce (Picea glauca); redwood (Sequoia sempervirens); authentic fir trees such as Pacific fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as giant thuja (Thuja plicata) and Nootka cypress (Chamaecyparis nootkatensis). Lawns include, but are not limited to, zoysia lawns, bentgrass lawns, fescue lawns, bluegrass lawns, St.Augustinegrass lawns, bermudagrass lawns, buffalograss lawns, ryegrass lawns, and ryegrass lawns. ball of grass. Also included are plants that serve primarily as laboratory models, eg, Arabidopsis. Preferred plants for use in the present processes include (but are not limited to) legumes, solanaceae species (eg, tomatoes), leafy vegetables such as lettuce and cabbage, lawns, and cultivated plants (eg, tobacco, wheat, sorghum. , barley, rye, rice, corn, cotton, cassava, and the like), and laboratory plants (eg, Arabidopsis). Although any plant can be used to carry out the present invention, tobacco plants are particularly preferred.

The plant parts that can be harvested from the plants of the present invention (eg, cut or harvested) include, for example, fruits, flowers, seeds, roots, tubers, leaves, stems, bark, wood, etc. Note that when referring to a particular protein that is increased or decreased in a plant, the amount of that protein can be altered throughout the plant, or only in a particular part.

Taken together, in an illustrative embodiment of the invention, nicotine is produced in tobacco plants by the condensation of nicotinic acid and an N-methylpyrrolinium cation. The biosynthetic pathway that results in nicotine production is illustrated in Figure 1. Two regulatory loci (Nic1 and Nic2) act as co-dominant regulators of nicotine production. Single and double Nic mutant root enzyme analyzes show that the activities of two enzymes, quinolate phosphoribosyl transferase (QPTase) and rot methyl transferase (PMTase), are directly proportional to nicotine biosynthesis levels. A comparison of the enzymatic activity in tobacco tissues (root and callus) with different nicotine synthesis capacities shows that QPTase and PMTase activity are strictly correlated with nicotine content (Wagner and Wagner, Plant 165: 532 (1985)) . Saunders and Bush (Plant Physiol 64: 236 (1979) demonstrated that the level of QPTase in the roots of low nicotine mutants is proportional to the level of nicotine in the leaves.

The present invention is based, in a preferred embodiment, on an isolated nucleic acid (for example, SEQ ID NO: 1 or a fragment thereof consisting of at least 200 consecutive nucleotides) that is or contains at least one regulatory element of cis-acting, which exists upstream of the coding sequences for plant rot quinolate phosphoribosyl transferase (QPTase) and rot methyl transferase (PMTase).

Thus, in some embodiments, the encompassed nucleic acids have a structure that promotes an interaction with one or more transcription factors (eg, Nic1 and Nic2), which are involved in initiation of QPTase and / or PMTase transcription. Accordingly, such nucleic acids are said to be or contain binding sequences to at least one transcription factor (eg, Nic1 and Nic2), which are also referred to as "cis-acting regulatory elements". Introducing multiple copies of these cis-acting regulatory elements

ES 2 267 809 T3 (eg, sequences that interact with Niel and / or Nic2) in a plant cell, the ability of the transcription factor to initiate transcription of the targeted gene (eg, QPTase and / or PMTase genes) it can be reduced or inhibited.

Since QPTase and PMTase activities are strictly correlated with nicotine content, the construction of transgenic tobacco plants in which QPTase or PMTase levels are lowered in plant roots (compared to levels in tobacco plants). wild type), as described above, results in plants having reduced levels of nicotine. Without wishing to be bound by any particular theory, it is contemplated that the creation of tobacco plants, tobacco, and tobacco products that have a reduced amount of nicotine will also have a reduced amount of TSNA. That is, by removing nicotine from tobacco plants, tobacco, and tobacco products, the alkaloid substrate is effectively removed for TSNA formation. The reduction of nicotine in tobacco was found to be directly related to the reduction of TSNA. Unexpectedly, the processes described in this document not only produce tobacco with a reduced addictive potential but, concomitantly, produce tobacco that has a lower carcinogenic potential.

It should be emphasized that the phrase "a reduced amount" is intended to refer to an amount of nicotine and / or TSNA in a transgenic tobacco plant, tobacco, or a tobacco product that is less than that which would be found in a plant. tobacco, tobacco, or tobacco product of the same variety of tobacco processed in the same way, that was not made transgenic for reduced nicotine and / or TSNAs. Therefore, in some contexts, wild-type tobacco of the same variety that has been processed in the same way is used as a control by which to measure whether a reduction in nicotine and / or TSNA has been obtained by the methods of the invention. described in this document.

Wild-type tobacco varies significantly in the amount of TSNA and nicotine depending on the variety and the way it is grown, harvested, and cured. For example, a Burley tobacco leaf has 30,000 parts per million (ppm) of nicotine and 8,000 parts per billion (ppb) of TSNA; a hot air cured Burley leaf has 20,000 ppm of nicotine and 300 ppb of TSNA; and a cured Oriental leaf has 10,000 ppm of nicotine and 100 ppb of TSNA. A tobacco plant or part thereof having a reduced amount of nicotine and / or TSNA, according to the invention, may have non-detectable nicotine and / or TSNA, or may contain some detectable amounts of one or more TSNA and / or or nicotine as long as the amount of nicotine and / or TSNA is less than that found in a control plant of the same variety. That is, a Burley tobacco leaf embodiment of the invention having a reduced amount of nicotine may have between 0 and 30,000 ppm nicotine, and 0 and 8,000 ppb TSNA, desirably between 0 and 20,000 ppm nicotine and 0 and 6,000. ppb TSNA, more desirably between 0 and 10,000 ppm nicotine and 0 and 5,000 ppb TSNA, preferably between 0 and 5,000 ppm nicotine and 0 and 4,000 ppb TSNA, more preferably between 0 and 2,500 ppm of nicotine and 0 and 2,000 ppb of TSNA and even more preferably between 0 and 1,000 ppm of nicotine and 0 and 1,000 ppb of TSNA. Burley leaf embodiments prepared by the procedures described herein may also have between 0 and 1000 ppm of nicotine and 0 and 500 ppb TSNA and some embodiments of Burley leaf prepared by the procedures described herein have an amount practically no detectable nicotine or TSNA.

Similarly, a hot air cured tobacco leaf embodiment of the invention having a reduced amount of nicotine may have between 0 and 20,000 ppm nicotine and 0 and 300 ppb TSNA, desirably between 0 and 15,000 ppm nicotine, and 0 and 250 ppb of TSNA, more desirably between 0 and 10,000 ppm of nicotine and 0 and 200 ppb of TSNA, preferably between 0 and 5,000 ppm of nicotine and 0 and 150 ppb of TSNA, more preferably between 0 and 2,500 ppm of nicotine and 0 and 100 ppb of TSNA and even more preferably between 0 and 1,000 ppm of nicotine and 0 and 50 ppb of TSNA. Embodiments of hot air cured tobacco prepared by the procedures described herein may also have between 0 and 500 ppm nicotine and 0 and 25 ppb TSNA and some embodiments of hot air cured tobacco prepared by the procedures described herein they have a virtually undetectable amount of nicotine or TSNA.

In addition, a cured Oriental tobacco embodiment of the invention having a reduced amount of nicotine may have between 0 and 10,000 ppm nicotine and 0 and 100 ppb TSNA, desirably between 0 and 7,000 ppm nicotine and 0 and 75 ppb TSNa. , more desirably between 0 and 5,000 ppm of nicotine and 0 and 50 ppb of TSNA, preferably between 0 and 3,000 ppm of nicotine and 0 and 25 ppb of TSNa, more preferably between 0 and 1,500 ppm of nicotine and 0 and 10 ppb of TSNA and even more preferably between 0 and 500 ppm of nicotine and no TSNA. Cured Oriental tobacco embodiments prepared by the procedures described herein may also have between 0 and 250 ppm nicotine and no TSNA and some cured Oriental tobacco embodiments prepared by the procedures disclosed herein have a virtually undetectable amount of nicotine or TSNA.

The present invention provides methods and nucleic acid constructs for producing said transgenic plants, as well as said transgenic plants. Such procedures include the development of transgenic cassettes that will reduce (or eliminate) nicotine biosynthesis. Tobacco plants are transformed with an excessive amount of DNA sequences (cis-acting elements) from the promoters of genes encoding, but not limited to, QPTase and PMTase that are regulated in nicotine biosynthesis. These cis-acting elements are preferably integrated into the plant genome to allow them to be passed on to successive generations. Typically, Nicl and Nic2 DNA-binding proteins that interact with these cis-acting DNA sequences are expressed at relatively low levels in the cell, thus excess transgenic cis-acting elements will compete with endogenous elements. associated with the genes that encode, but not limited to,

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QPTase and PMTase per Niel and Nic2 available. Accordingly, these cis-acting DNA sequences (and those of other cis-acting elements) are referred to herein as "decoys" or "molecular decoys." Competition decreases the occupancy of trans-acting DNA-binding proteins on their cis-acting cognate elements, thereby downregulating the synthesis of nicotine biosynthesis enzymes.

The present invention also provides DNA molecules of cis-acting elements of QPTase or PMTase, and vectors comprising those DNA molecules, as well as transgenic plant cells and plants transformed with those DNA molecules and vectors. The transgenic tobacco cells and plants of the present invention are characterized by a lower nicotine content than the control non-transformed tobacco cells and plants.

Tobacco plants with low levels of nicotine production, or basically no nicotine production, are attractive as recipients of transgenes expressing products of commercial value such as pharmaceuticals, cosmetic components, or food additives. Tobacco is attractive as a target plant for a transgene that encodes a desirable product, since tobacco is easily genetically manipulated and produces a lot of biomass per acre (0.4047 hectares); tobacco plants with reduced resources dedicated to nicotine production will therefore have more resources available for the production of transgene products. Procedures for transforming tobacco with transgenes that produce the desired products are known in the art; Any suitable technique can be used with the low nicotine tobacco plants of the present invention.

Tobacco plants of the present invention with reduced QPTase and PMTase expression and reduced nicotine levels will be desirable in the production of tobacco products having reduced nicotine and / or TSNA content. The tobacco plants described in this document are suitable for conventional cultivation and harvesting techniques (for example, cutting the top or not, bagging the flowers or not, growing in a soil rich in compost or without compost) and the leaves and stems collected are suitable for use in any traditional tobacco product including, but not limited to, pipe, cigar and cigarette tobacco, and chewing tobacco in any form including raw tobacco, cut tobacco, or cut tobacco.

It is also contemplated that the low nicotine and / or TSNA tobacco described herein can be processed and mixed with conventional tobacco to create a wide range of tobacco products with varying amounts of nicotine and / or nitrosamines. These mixed tobacco products can be used in tobacco product cessation programs to slowly switch a user from a product high in nicotine and TSNA to a product low in nicotine and TSNA. For example, a smoker may start the program by smoking mixed cigarettes that have 10 mg of nicotine and 1.5 mg of nitrosamine, gradually switch to smoking cigarettes with 7 mg of nicotine and 1 mg of nitrosamine, followed by cigarettes that have 5.0 mg of nicotine and 0.5 mg of nitrosamine, followed by cigarettes that have 2.0 mg of nicotine and 0.25 mg of nitrosamine, followed by cigarettes that have 1.0 mg of nicotine and no TSNA until the consumer chooses to only smoke the cigarettes that have virtually no nicotine and nitrosamines or to quit smoking altogether. Accordingly, the mixed cigarettes described herein provide the basis for an approach to reduce the carcinogenic potential in a human in a stepwise manner.

1. Nucleic acids encoding cis-acting elements such as elements sensitive to the Nic gene product

Elements sensitive to the Nic gene product can be isolated by scanning the promoter region of genes that are transcriptionally activated by the Nic gene product in the same way as described herein, or they can be identified by hybridization with SEQ ID NO. : 1 of this document and subsequently selecting the ability to bind the Nic gene product in the manner described below.

The nucleic acid sequences used to carry out the present invention include SEQ ID NO: 1 or a fragment thereof consisting of at least 200 consecutive nucleotides. This definition is intended to encompass natural allelic variations of the DNA of SEQ ID NO: 1 or such fragments. Therefore, DNA sequences that are at least 90% identical to SEQ ID NO: 1, or their complement, and that, when coupled into a reporter gene, are capable of promoting the expression of said reporter at levels at least 1.5 times higher in Nic + / nic plants^- compared to nic plants^-/ nic^-. Conditions that allow other DNA sequences with sequence similarity to SEQ ID NO: 1 can be routinely determined. For example, hybridization of such sequences can be performed under conditions of reduced stringency or even stringent conditions (for example, conditions represented by a wash stringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1 SDS % at 60 ° C or even 70 ° C to DNA with the sequence given herein as SEQ ID NO: 1 using a standard in situ hybridization assay See J. Sambrook et al., Molecular Cloning, A Laboratory Manual ( 2^to Ed. 1989) (Cold Spring Harbor Laboratory)). In general, such sequences will be at least 65% similar, 75% similar, 80% similar, 85% similar, 90% similar, or even 95% similar or more with the sequence given herein as SEQ ID NO: 1. Sequence similarity determinations are made with the two sequences aligned for maximum match; Gaps in either sequence are allowed to match to maximize matching. Gap lengths of 10 or less are preferred, gap lengths of 5 or less are preferred, gap lengths of 2 or less are even more preferred.

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The DNA sequence of the present invention may essentially consist of the sequence provided herein (SEQ ID NO: 1).

The use of the phrase "substantial sequence similarity" in the present specification and claims means that DNA, RNA or amino acid sequences having slight and inconsequential sequence variations from the exact sequences described and claimed herein , are considered equivalent to the sequences of the present invention. In this regard, "slight and inconsequential sequence variations" means that "similar" sequences (ie, sequences that have substantial sequence similarity to the DNA, RNA, or proteins described and claimed herein) will be functionally equivalent to the sequences described and claimed in the present invention. Functionally equivalent sequences will function in basically the same way to produce essentially the same compositions as the nucleic acid and amino acid compositions described and claimed herein.

The DNA sequences provided herein can be transformed in various host cells, as described below. Various host cells, which have desirable culture and handling properties, are readily available in the art.

The use of the phrase "isolated" or "basically pure" in the present specification and claims as a modifier of DNA, RNA, polypeptides, or proteins means that the so-called DNA, RNA, polypeptides, or proteins have been separated from their cellular environments. in vivo through the efforts of humans. As used herein, a "native DNA sequence" or "natural DNA sequence" means a DNA sequence that can be isolated from non-transgenic cells or tissue. Native DNA sequences are those that have not been artificially altered, such as by site-directed mutagenesis. Once native DNA sequences are identified, DNA molecules having native DNA sequences can be synthesized or chemically produced using recombinant DNA procedures as known in the art. As used herein, a native plant DNA sequence is one that can be isolated from non-transgenic plant cells or tissue. As used herein, a native tobacco DNA sequence is one that can be isolated from non-transgenic tobacco cells or tissue.

2. Nucleic Acid Transfer Vectors and Constructs

The nucleic acid constructs or "cassettes" of the present invention include a cis-acting element such as an element responsive to the Nic gene product as described above, typically as a recombinant construct in a linear or circular nucleic acid serving as a transfer vector for introducing the Nic gene product into plant cells.

The construct or cassette can be provided in a DNA construct that also has at least one replication system. For convenience, it is common to have a functional replication system in Escherichia coli, such as ColE1, pSC101, pACYC184, or the like. In this way, at each stage after each manipulation, the resulting construct can be cloned, sequenced, and the accuracy of the manipulation determined. In addition, or instead of the replication system in E. coli, a wide host range replication system can be employed, such as the replication systems of P-1 incompatibility plasmids, eg, pRK290. In addition to the replication system, frequently at least one marker will be present, which may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be employed for selection in a prokaryotic host, while another marker may be employed for selection in a eukaryotic host, particularly the host plant. The markers can be protection against a biocide, such as antibiotics, toxins, heavy metals, or the like; they can provide complementation, giving prototrophy to an auxotrophic host; or they can provide a visible phenotype through the production of a new compound in the plant.

The nucleic acid constructs of the present invention may include one or more matrix binding regions in position 5 ', 3', or 5 'and 3' to the cis-acting element (s) to enhance stability and / or heritability thereof, as described in US Patent Nos. 5,773,689 to Thompson et al., 5,773,695 to Thompson et al., 6,245,974 to Michalowski et al., 6,239,328 to Thompson et al. ., 6,100,448 to Thompson et al., And 6,037,525 to Thompson et al.

The various fragments comprising the various constructs, cassettes, labels, and the like can be introduced consecutively by restriction enzyme cleavage of an appropriate replication system, and insertion of the particular construct or fragment into the available site. After ligation and cloning the DNA construct can be isolated for further manipulation. All of these techniques are amply exemplified by Sambrook et al., Molecular Cloning, A Laboratory Manual (2^to Ed. 1989) (Cold Spring Harbor Laboratory).

Vectors that can be used to transform plant tissue with nucleic acid constructs of the present invention include ballistic vectors and Agrobacterium vectors, as well as vectors suitable for DNA-mediated transformation. This is described in more detail below.

The nucleic acid construct molecules and vectors used to produce the transformed cells and plants of this invention may further comprise a dominant selectable marker gene. The marked8

ES 2 267 809 Dominant selectable T3 res suitable for tobacco use include, inter alia, antibiotic resistance genes encoding neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), and chloramphenicol acetyltransferase (CAT). Another well known dominant selectable marker suitable for tobacco use is a mutant dihydrofolate reductase gene encoding methotrexate resistant dihydrofolate reductase. DNA vectors containing suitable antibiotic resistance genes, and corresponding antibiotics, are commercially available.

3. Transformation, regeneration and propagation of plants

Transformed cells are selected from the surrounding population of non-transformed cells by placing the mixed population of cells in culture medium containing an appropriate concentration of the antibiotic (or other compound normally toxic to cells) against which the selectable marker gene product Chosen dominant confers resistance. In this way, only the plant cells that have been transformed will survive and multiply.

The methods of making recombinant plants of the present invention, in general, involve first providing a plant cell capable of regeneration (the plant cell typically residing in tissue capable of regeneration). The plant cell is then transformed with a DNA construct comprising a cassette of the present invention (as described herein) and a recombinant plant is regenerated from the transformed plant cell. As explained below, the transformation step is performed by techniques as are known in the art, including but not limited to bombarding the plant cell with microparticles carrying the transcription cassette, infecting the cell with an Agrobacterium tumefaciens containing a Ti plasmid carrying the cassette, or any other suitable technique for the production of a transgenic plant.

Microparticles carrying a DNA construct of the present invention, which microparticle is suitable for ballistic transformation of a plant cell, are also useful for making transformed plants of the present invention. The microparticle is propelled into a plant cell to produce a transformed plant cell, and a plant is regenerated from the transformed plant cell. Any suitable ballistic cell transformation methodology and apparatus can be used to practice the present invention. Exemplary apparatus and methods of the present invention are described in Sandford and Wolf, US Patent No. 4,945,050, and in Christou et al., US Patent No. 5,015,580. When using ballistic transformation procedures, the cassette can be incorporated into a plasmid capable of replicating in or integrating within the cell to be transformed. Examples of suitable microparticles for use in such systems include 1 to 5 micron (um) gold spheres. The DNA construct can be deposited on the microparticle by any suitable technique, such as by precipitation.

Numerous Agrobacterium vector systems useful in carrying out the present invention are known. For example, US Patent No. 4,459,355 describes a process for transforming susceptible plants, including dicots, with an Agrobacterium strain containing the Ti plasmid. Transformation of woody plants with an Agrobacterium vector is described in US Patent No. 4,795,855. In addition, US Patent No. 4,940,838 to Schilperoort et al. describes a binary Agrobacterium vector (i.e., one in which the Agrobacterium contains a plasmid that has the vir region of a Ti plasmid but no T region, and a second plasmid that has a T region but no vir region) useful for performing the present invention.

Since a large number of copies of decoy sequences must typically be present in the genome, copies of the cis-acting element (s) can be inserted in tandem into an Agrobacterium vector, but the preferred method of transforming plants is by bombardment of particles that introduce multiple copies of transgenic DNA into the plant genome. The exact amount of the cis-acting element (whether each is individually present in a vector such as a plasmid, whether multiple copies are counted in a single vector or plasmid, or combinations thereof) that must be inserted into the host cells ( and the progeny or daughter cells thereof) to obtain increased or decreased levels of the protein of interest in the cells and plants of the invention, will depend in part on the particular element, but generally it will be at least 20, 30, or 50 up to about 500, 1,000, or 2,000, or more.

Plant species can be transformed with the DNA construct of the present invention by DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts according to procedures well known in the art. Fusion of tobacco protoplasts with DNA-containing liposomes or by electroporation is known in the art. (Shillito et al., "Direct Gene Transfer to Protoplast of Dicotyledonous and Monocotyledonous Plants by a Number of Methods, Including Electroporation", Methods in Enzymology 153, 313-36 (1987)).

As used herein, "transformation" refers to the introduction of exogenous DNA into cells, to produce transgenic cells stably transformed with the exogenous DNA. By "stably transformed" is meant that the exogenous nucleic acid is passed into the daughter cells or progeny of the initially transformed cells, and is preferably passed to or is inherited by the plants from the progeny of the transformed plants (including plants of the sexually and asexually reproduced progeny).

ES 2 267 809 T3

Transformed cells are induced to regenerate intact plants through the application of cell and tissue culture techniques that are well known in the art. The plant regeneration procedure is chosen to be compatible with the transformation procedure. After regeneration of transgenic plants from transformed cells, the introduced DNA sequence is easily transferred to other plant varieties through conventional plant breeding practices and without undue experimentation.

For example, to analyze the segregation of transgenic DNA, regenerated transformed plants (R₀), assayed for levels of the protein of interest, and self-reproducing to produce Ri plants. A percentage of Ri plants carrying the transgenic DNA are homozygous for the transgenic DNA. To identify R plants₁ homozygous, R plants are grown to full development₁ transgenic and self-reproducing. Plants R₁ homozygous will produce R progeny₂ where each progeny plant carries the transgenic DNA; progeny of R plants₁ heterozygous will secrete 3: 1.

Nicotine serves as a natural pesticide that helps protect tobacco plants from pest damage. It may therefore be desirable to further transform low-nicotine or non-nicotine plants produced by the present methods with a transgene (such as Bacillus thuringiensis) that will confer additional protection against insects.

A preferred plant for use in the present invention is any species of the genus Nicotiana, or tobacco, including N. tabacum, N. rustica, and N. glutinosa. Any strain or variety of tobacco can be used.

Any plant tissue capable of subsequent clonal propagation, either by organogenesis or embryogenesis, can be transformed with a vector of the present invention. The term "organogenesis", as used herein, means a process by which shoots and roots develop sequentially from meristematic centers; the term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a coordinated (not sequential) fashion, either from somatic cells or from gametes. The particular tissue chosen will vary depending on the clonal propagation system available for, and most appropriate for, the particular species being transformed. Exemplary target tissues include leaf discs, pollen, embryos, cotyledons, hypocotyls, callus tissue, existing meristem tissue (eg, apical meristems, axillary buds, and root meristems), and induced meristematic tissue (eg, cotyledon meristem and meristem. hypocotyl).

The plants of the present invention can take various forms. Plants can be chimeras of transformed cells and non-transformed cells; plants can be clonal transformants (eg, all cells transformed to contain the cassette); plants may comprise grafts of transformed and non-transformed tissues (eg, a transformed stem root grafted onto an untransformed stem in citrus species). The transformed plants can be propagated by various means, such as by clonal propagation or classical cultivation techniques. For example, first generation (or T1) transformed plants can be self-reproduced to give homozygous second generation (or T2) transformed plants, and T2 plants further propagated by classical culture techniques. A dominant selectable marker (such as nptII) can be associated with the construct to aid in culture.

In some preferred embodiments of the invention, to help ensure that a sufficient number of decoys or cis-acting elements are inserted into cells and retained for many cell divisions to produce a transgenic plant with altered levels of protein or proteins in its interior, biolistic transformation is used as described above, circular DNA or plasmids are used to carry the cis-acting decoy segments as described above, the circular DNA or plasmids that are used are relatively small (for example, they consist of less than 10,000 or less than 6,000 base pairs ), and a high molar ratio of cis-acting element to selectable marker (eg, 10 to 1) is inserted into host cells.

As used herein, a crop comprises a plurality of plants of the present invention, and of the same genus, planted together in an agricultural field. By "agricultural field" is meant a common plot of land or a greenhouse. Thus, the present invention provides a method of producing a plant culture that have altered levels of a protein of interest, (eg, QPTase and PMTase activity and therefore have reduced nicotine levels), compared to a similar culture. of non-transformed plants of the same species and variety.

Although the invention describes methods for reducing nicotine levels in transgenic tobacco, this method can also be used to copy the phenotype of mutations in trans-acting transcriptional activators and repressors without cloning their respective genomic loci. Promoter regions of a gene can be analyzed, using technology known to those of skill in the art, to define regions of the promoter that respond to transcription factors. Typically, this is done by promoter deletion analysis. Nested promoter deletions are fused to a reporter gene and the expression of the reporter gene is controlled in transgenic organisms. Isolation of transcription factors using current technologies is very difficult; The present invention avoids the need to clone cognate transcription factors for applications where it is desirable to alter any set of genes that are coordinately regulated by one or more transcriptional activators. The other way,

ES 2 267 809 T3 the method could positively regulate the expression of any series of genes that are coordinately regulated by one or more transcriptional repressors.

As indicated above, the present invention could be used to alter gene expression and negatively regulate the expression of a protein of interest that is under the control of a cis-acting activator element in various host cells, including plant cells (particularly of vascular plants such as monocots and dicots), animals (birds, mammals), fungi, or bacteria, both in vivo and in vitro. In bacteria and fungi, multicopy plasmids can be used to increase the molecular decoy copies present in the cell.

The examples that follow are shown to illustrate the present invention, and should not be construed as limiting it.

Example 1

Location of the cis-acting Element in the Promoter of NtQPTl

To characterize the minimum sequence required for the cis-acting element NtQPT1, the promoter region of the NtQPT1 gene was isolated, truncated at the 5 'end, and fused with the gene encoding fl-glucuronidase (GUS) to assess function. as a specific enhancer of nicotine production. The NtQPT1 gene was isolated and sequenced. The onset of transcription was determined by comparing the TobRD2 cDNA sequence with the sequence of the genomic locus. The sequence located 5 'from the transcription start site was defined as the promoter sequence. Using PCR primers and the promoter as a template, truncations were made at the 5 'end of the promoter to determine the minimal cis-acting enhancer sequence (see Figure 2). The truncations were fused to the uidA gene, which encodes GUS. The fusion gene was inserted into a vector and transformed by standard ballistic transformation procedures into Nicotiana tabacum Burley 21.

GUS activity was tested by dividing the plants into roots, stems, and leaves. Each plant tissue, transformed with a different NtQPT1 truncation construct, was ground with a mortar and pestle, proteins were extracted with NaPO buffer.₄pH 7.0, X-Glc (100 µg / ml) was added, and the assay was performed at 37 ° C for 30 min. GUS activity was measured at 595 nm). For each construct at least 20 independent transformants were tested. The mean and standard deviation were determined. GUS activity in truncations was compared to a CaMV 35S-GUS fusion and a promoterless GUS control (pBI101). The maximum GUS activity, which represents the expression of NtQPT1, was obtained when -586 to -2000 bp was fused to the uidA gene (Figure 3). The shorter promoters from -1 to -586 did not maintain high levels of uidA expression. Therefore, the cis-acting element of NtQPT1 is between -586 and -2000 bp 5 'from the transcription start site.

Example 2

Location of the Product Binding Site of the Nic Gene in the NtQPT1 promoter

The NtQPT1 promoter deletion string fused to the uidA reporter gene (encoding GUS) was used to transform homozygous nic N. tabacum plants^-/ nic^-. The transformants R₀ that had transgenic DNA at a single locus were crossed with homozygous nic plants^-/ nic^- or Nic + / Nic + to give homozygous progeny (nic^-/ nic^-) and heterozygous (Nic + / nic^-) that carry the transgene (NtQPT1 -GUS promoter) in the same chromosomal position. GUS activity was quantified in multiple progeny from multiple independent transformants and compared between the Nic + and nic phenotypes.^- (Table 1). Proportions greater than 1.5 were determined to contain the cis-acting elements that responded to activation by the Nic gene product.

ES 2 267 809 T3

TABLE 1

Regulation of GUS Expression Driven by the NtQPT1 Promoter, by Nic Gene Products in Tobacco

<td>Promoter</td><td>Transformants Independent</td><td>Activity of GUS at Nic⁺/ nic '²</td><td>Activity of GUS in nic / nic '²</td><td>Proportion of GUS from Nic / nic</td>

<td> 2,0 (2010)'</td><td> 2</td><td> 111,7 (7)</td><td> 21,2 (5)</td><td> 5,3</td>

<td></td><td></td><td> 46,6 (6)</td><td> 11,9 (8)</td><td> 5,6</td>

<td> 1,3 (1306)¹</td><td> 2</td><td> 92,4 (6) 93,9 (7)</td><td> 16,6 (4) 12,9 (5)</td><td> 5,5 7,3</td>

<td> 1,0 (1042)¹</td><td> 3</td><td> 55,7 (6) 74,1 (6) 78,0 (5)</td><td> 18,3(4) 27,5 (7) 17,2 (7)</td><td> 3,0 2,7 4,5</td>

<td> 734</td><td> 1</td><td> 5,5 (5)</td><td> 3,5 (5)</td><td> 1,5</td>

<td> 586</td><td> 3</td><td> 47,55 (5) 24,3 (3) 29,1 (33)</td><td> 44,9 (5) 16,0 (36) 30,3 (19)</td><td> 1,06 1,5 0,97</td>

<td> 535</td><td> 3</td><td> 71,6 (10) 54,0 (5) 51,9 (5)</td><td> 50,3 (5) 40.7 (3) 67.8 (5)</td><td> 1,4 1,3 0,8</td>

<td>CaMV 35S</td><td> 4</td><td> 32.7 (4) 44.8 (6) 54,8(5) 97 (4)</td><td> 19.6 (4) 47.6 (3) 40.6 (5) 8.6 (3)</td><td> 1,7 0,94 1,3 1.1</td>

<td colspan="5">GUS activity is expressed as pmol MU ^ g protein / min. Έ! actual promoter size (bp) is indicated in parentheses.^¿The number in parentheses indicates the number of plants tested.</td>

These experiments demonstrated that the binding of Nic gene products is between approximately -1000 and -600 or -700 bp from the NtQPT1 promoter as determined by GUS activity in Nic plants.⁺/ nic^- and nic^-/ nic^-.

Example 3

Regulation of NtQPT1 Gene Expression Using Molecular Decoys

The nucleotide sequence between -1000 and -600 or -700 bp of the NtQPT1 promoter is inserted in tandem series into a plant Agrobacterium shuttle vector and subsequently used to transform tobacco by procedures known to one of ordinary skill in the art. Plants stably transformed with said vector are tested for the level of expression of NtQPT1 and to evaluate the content of nicotine and / or TSNA. These experiments will demonstrate that tobacco transformed with molecular decoys that interact with Nic gene products will show a reduced amount of nicotine and / or TSNA. Plants with multiple tandem insertions of the molecular decoy that have reduced NtQPT1 expression and reduced nicotine levels are used for the expression of products of commercial value and the production of tobacco products that have reduced nicotine and / or TSNA content.

ES 2 267 809 T3

Example 4

Regulation of Binding to ASF-l Using a TGACG Molecular Decoy

The TGACG nucleotide sequence is inserted in tandem series into a plant Agrobacterium shuttle vector and used to transform a plant such as pea by methods known to one of ordinary skill in the art. Plants stably transformed with such a vector have reduced binding activity of the trans-acting DNA-binding factor ASF-1 that recognizes the TGACG sequence motif found in plant genes such as histone genes (Mikami and col. (1987) FEBS Lett. 223: 273); genes for enzymes for agropine biosynthesis (Velten et al., EMBO J. 3: 2723-30); the octopine synthase gene (Ellis et al., EMBO J. 6: 3203) and the mannopine synthase gene (DeRita and Gelvin, (1987) Mol. Gen. Genet. 207: 233); as well as the CaMV35S gene, the histone H3 gene and the nopaline synthase gene.

Example 5

Regulation of Spatial and Temporal Expression of Beta-Phaseolin Using Molecular Decoys

The nucleotide sequence corresponding to UAS1 (-295 to -109) of the beta-phaseolin gene is inserted in tandem series into an Agrobacterium plant shuttle vector and used to transform a legume plant by procedures known to a person skilled in the art. The technique. Plants stably transformed with said vector have reduced binding activity of the trans-acting DNA-binding factor PvALF, which recognizes the CATGCAAA and CATGCATG sequences located in UAS1 (Bobb et al. (1997) Nucleic Acids Res 25 ( 3): 641-7). Plants with reduced PvALF binding could have reduced expression of seed-specific betaphaseolin expression mainly in cotyledons and meristematic shoots (Bustos et al. (1991) EMBO J. 10 (6): 1469-1479).

The transformation of tandem series of nucleotide sequences corresponding to the vicilin box (GCCACCTCAA; SEQ ID NO: 2) and site B (CACACGTCAA: SEQ ID NO: 3) of the beta phaseolin gene in a legume plant gives as a result the reduced binding activity of trans-acting DNA binding factors ROM1 and ROM2 leading to premature initiation of beta-phaseolin expression. The ROM1 and ROM2 proteins function as repressors of the expression of beta-phaseolin and the L subunit of phytohaemagglutinin to block the initiation of seed maturation. (US Patent No. 6,160,202 to Bustos; Chern et al. (1996) Plant Cell 8: 305-321; Chern et al. (1996) Plant J. 10: 135-148).

Example 6

Regulation of Plant Gene Expression Using Molecular Decoys

Transformation of tobacco plants with tandem arrays of the root-specific cis-acting element of the tobacco RB7 promoter (US Patent No. 5,459,252 to Conkling et al .; Yamamoto et al. (1991) Plant Cell 12: 3399-3406), which encodes a structural gene, results in reduced binding activity of the trans-acting DNA-binding factor of the cis-acting element RB7.

Similarly, similar tandem arrays of the following cis elements are used to transform plants to reduce the binding activity of the corresponding trans-acting DNA-binding factors: the cis-acting repeat element AATT and its acting factor in corresponding trans PABF (see US Patent Nos. 5,834,236 and 6,191,258); the positive regulatory element poly (dA-dT) and the binding protein and the negative repeat element CCAA and the binding protein (Wang et al. (1992) Mol. Cell Biol. 12: 3399-3406); the tobacco phytochrome AI promoter root tip regulatory element (Adam et al. (1995) Plant Mol. Biol. 29: 983993); the anaerobic sensitive element of the maize glyceraldehyde-3-phosphate dehydrogenase 4 gene (Geffers et al. (2000) Plant Mol. Bio. 43: 11-21); and the seed-specific regulatory region of an Arabidopsis oleosin gene (see US Patent No. 5,792,922).

Example 7

Tobacco Having Reduced Nicotine and / or TSNA Levels Generated Using Molecular Lures

Multiple copies of an approximately 300 or 400 nucleotide long fragment of the NtQPTl promoter (for example, including the nucleotide sequence between -1000 and -600 or -700 bp of the NtQPTl promoter, such as SEQ ID N °: 1) to microparticles (eg, by precipitation) that are suitable for ballistic transformation of a plant cell (eg, 1 to 5 pm gold spheres). The microparticles are propelled into tobacco plant cells (eg Burley 21 LA) to produce transformed plant cells, and the plants regenerate from the transformed plant cells. Burley 21 LA is a variety of Burley 21 with considerably reduced nicotine levels compared to Burley 21 (i.e. Burley 21 LA has 8% of the nicotine levels of Burley 21, see Legg et al., Can J Genet Cytol , 13: 287-91 (1971); Legg et al., JHered, 60: 213-217 (1969)).

ES 2 267 809 T3

Any suitable ballistic cell transformation methodology and apparatus can be used. Examples of suitable apparatus and procedures are described in Sanford and Wolf, US Patent No. 4,945,050, and in Christou et al., US Patent No. 5,015,580.

Optionally, the transformed nucleic acid can include a gene encoding a selectable marker (eg, a marker that allows positive or negative selection of transformants) or the molecular decoys can be co-transferred with a selectable marker gene. In this way, positive transformants can be easily identified.

The transformed cells, tissues, and seedlings are grown in Murashige-Skoog (MS) medium (with or without the selection compound, eg, antibiotic, depending on whether a selectable marker was used). One hundred Burley 21 LA (To) independent transformants are allowed to self-reproduce. The progeny of self-reproducing plants (Ti) germinate. The nicotine levels of the Ti progeny are qualitatively measured using a microassay technique. About ~ 200 mg of fresh tobacco leaves are collected and minced in 1 ml of extraction solution (Extraction solution: 1 ml of Acetic Acid in 100 ml of H₂OR). The homogenate is centrifuged for 5 min at 14,000 xg and the supernatant is removed to a clean tube, to which the following reagents are added: 100 µl of NH₄OAC (5 g / 100 ml H₂O + 50 pl de Brij 35); 500 μl of Cyanogen Bromide (Sigma C-6388, 0.5 g / 100 ml of H₂O + 50 pl de Brij 35); 400 μl of Aniline (0.3 ml of Aniline buffered in 100 ml of NH₄OAC + 50 pl de Brij 35). Prepare and dilute a 10 mg / ml nicotine standard stock solution in extraction solution to create a standard series for calibration. The absorbance at 460 nm is read and the nicotine content of the test samples is determined using the standard calibration curve.

To progeny T₁ that has less than 10% of the Burley 21 LA parent's nicotine levels is allowed to self-reproduce to produce the T progeny₂. The progeny T is then identified₂ homozygous. Nicotine levels in homozygous and heterozygous T2 progeny are also qualitatively determined using the microassay. Leaf samples from homozygous T2 progeny can also be submitted to the Southern Research and Testing Laboratory in Wilson, NC for quantitative analysis of nicotine levels using Gas Chromatography / Flame Ionization Detection (GC / FID). Progeny T₂ homozygous will have nicotine levels that are considerably reduced compared to unprocessed tobacco (eg ~ 70 ppm). Since the nicotine levels in these plants are considerably reduced, the levels of TSNA in these plants are concomitantly reduced.

These experiments will demonstrate that tobacco transformed with molecular decoys that interact with Nic gene products will show a reduced amount of nicotine and / or TSNA. Plants with multiple tandem insertions of the molecular decoy that have reduced NtQPT1 expression and reduced nicotine levels are used for the expression of products of commercial value and the production of tobacco products that have reduced nicotine and / or TSNA content.

Example 8

Low Nicotine and TSNA Blended Tobacco

The following example describes various ways to create tobacco products that have specific amounts of nicotine and / or TSNA through mixing. Some blending approaches start with tobacco made from varieties that have extremely low amounts of nicotine and / or TSNA. Mixing tobacco made from a low-nicotine / TSNA variety (for example, undetectable levels of nicotine and / or TSNA) with a conventional tobacco (for example, Burley, which has 30,000 parts per million (ppm) of nicotine and 8,000 parts per billion (ppb) of TSNA; Hot Air Cure, which has 20,000 ppm of nicotine and 300 ppb of TSNA; and Oriental, which has 10,000 ppm nicotine and 100 ppb TSNA), tobacco products can be made that have virtually any desired amount of nicotine and / or TSNA. Tobacco products containing varying amounts of nicotine and / or TSNA can be incorporated into tobacco cessation programs and kits to help tobacco users reduce or eliminate their dependence on nicotine and reduce carcinogenic potential.

For example, a stage 1 tobacco product can be comprised of about 25% low nicotine / TSNA tobacco and 75% conventional tobacco; a stage 2 tobacco product can be comprised of about 50% low nicotine / TSNA tobacco and 50% conventional tobacco; a stage 3 tobacco product can be comprised of about 75% low nicotine / TSNA tobacco and 25% conventional tobacco; A stage 4 tobacco product can be comprised of approximately 100% low nicotine / TSNA tobacco and 0% conventional tobacco. A tobacco use cessation kit may comprise a quantity of tobacco product from each of the aforementioned blends to satisfy a consumer during a single month program. That is, if the consumer is a daily pack smoker, for example, a single month kit could provide 7 packs of each stage, a total of 28 cigarette packs. Each tobacco use cessation kit could include a set of instructions that specifically guide the consumer through the procedure step by step. Of course, tobacco products that have specific amounts of nicotine and / or TSNA could be provided in suitably sized amounts (cigar boxes, cigarette packs, snuff cans, and gum bags or rolls) so that consumers could select the amount of nicotine and / or TSNA they want individually. There are many ways to obtain various low-nicotine / low-nicotine tobacco blends.

ES 2 267 809 T3 in TSNA using the teachings described herein and the following is only intended to guide one of ordinary skill in the art to a possible approach.

To obtain a stage 1 tobacco product, which is a low nicotine / 25% TSNA mixture, tobacco prepared from a tobacco of approximately 0 ppm nicotine / TSNA can be mixed with conventional Burley, Hot Air Curing, or Oriental in a ratio of 25% / 75% respectively to obtain a Burley tobacco product that has 22,500 ppm of nicotine and 6,000 ppb of TSNA, a Hot Air Cured product that has 15,000 ppm of nicotine and 225 ppb of TSNA, and an Oriental product that has 7,500 ppm of nicotine and 75 ppb of TSNA. Similarly, to obtain a stage 2 product, which is a low nicotine / 50% TSNA blend, tobacco prepared from approximately 0 ppm nicotine / TSNA tobacco can be blended with Burley, Hot Air Curing, or Oriental conventional in a 50% / 50% ratio respectively to obtain a Burley tobacco product that has 15,000 ppm of nicotine and 4,000 ppb of TSNA, a hot air-cured product that has 10,000 ppm of nicotine and 150 ppb of TSNA, and an Oriental product that has 5,000 ppm of nicotine and 50 ppb of TSNA. In addition, a stage 3 product, which is a low nicotine / 75% TSNA / 25% blend, can be prepared by blending tobacco prepared from approximately 0 ppm nicotine / TSNA tobacco with Burley, Hot Air Cure, or Oriental conventional in a 75% / 25% ratio respectively to obtain a Burley tobacco product that has 7,500 ppm of nicotine and 2,000 ppb of TSNA, a hot air-cured product that has 5,000 ppm of nicotine and 75 ppb of TSNA, and an Oriental product that has 2,500 ppm of nicotine and 25 ppb of TSNA.

It should be understood that tobacco products are often a mixture of many different types of tobacco, which were grown in many different parts of the world under various growing conditions. As a result, the amount of nicotine and TSNA will differ from culture to culture. However, using conventional techniques, an average amount of nicotine and TSNA per culture used can easily be determined to create a desired mixture. By adjusting the amount of each type of tobacco that makes up the blend, one skilled in the art can balance the amount of nicotine and / or TSNA with other considerations such as appearance, aroma, and shot. In this way, various types of tobacco products can be created that have a variable level of nicotine and / or nitrosamine, as well as appearance, aroma and strength.