Polynucleotides and polypeptides involved in plant fiber development and methods of using same

ES 2 635 546 T3

DESCRIPTION

Polynucleotides and polypeptides involved in the development of plant fiber and methods of using them

Field and background of the invention

The present invention relates to methods of use of polynucleotides and polypeptides involved in the development of plant fiber, as characterized in the appended claims.

Cotton and cotton by-products provide raw materials that are used to produce a wealth of consumer products in addition to textiles including cotton, food, livestock feed, fertilizers, and paper. The production, marketing, consumption and trade of cotton-based products generates in excess of $ 100 billion annually in the US alone, making cotton the number one value-added crop.

The use of cotton as a fiber by humans is estimated to date back 7000 years in Central America and 5000 years in India. Even with the growth of synthetic fibers in the last 50 years, cotton is still responsible for approximately 50% of the world's textile fiber [Agrow Reports, World Seed Market DS208, October 2000].

Although 90% of the value of cotton as a crop resides in the fiber (yarn), the yield and quality of the fiber has declined, especially in the last decade [Meredith (2000), Proc. World Cotton Research Conference II, Athens, Greece pgs. 97-101]. This decline has been attributed to general erosion in the genetic diversity of cotton varieties, and an increased vulnerability of the crop to environmental conditions [Bowman et al., Crop Sci. 36: 577-581 (1996); Meredith, supra].

There are many varieties of cotton plant, from which cotton fibers with a range of characteristics can be obtained and used for various applications. Cotton fibers can be characterized according to a variety of properties, some of which are considered highly desirable within the textile industry for the production of increasingly high-quality products and the optimal use of modern spinning technologies. Commercially desirable properties include length, length uniformity, fineness, maturity ratio, decreased fuzz fiber production, micronaire, beam strength, and individual fiber strength. Much effort has been put into improving the characteristics of cotton fibers by focusing primarily on fiber length and fiber fineness. In particular, there is a great demand for cotton fibers of specific lengths.

Methods for improving the characteristics or performance of cotton fibers can be classified into the following three categories:

1. Improvement of the variety by crossing

This method has been widely used until now. At present, almost all cultivated varieties of cotton plant are bred by this method. However, the improvement of cotton lint yield using traditional crossing is relatively slow and inefficient and the degree of variability that can be achieved is limited.

two. Treatment with plant hormones.

Plant hormones such as auxin, gibberellin, cytokinin, and ethylene have been widely used in field crops and horticultural products. The influence of plant hormones, particularly gibberellin, auxin and brassinolide, on the characteristics of cotton plant fibers [p. ex. US Patent No. 5880110 produces cotton fibers with improved fiber characteristics by treatment with brassinosteroids]. However, no measurable effects have been documented, making practical use of these hormones on a large scale highly unlikely.

3. Variety improved by genetic engineering.

The wide acceptance of genetically modified cotton in major producing countries and the fact that it is a non-food crop makes it an attractive candidate for genetic engineering to improve fiber quality and / or yield.

In recent years, remarkable advances have been made in plant genetic engineering as a result of several successful cases of breeding a variety of commercially important crop plants (eg, cotton, soybean, corn, rapeseed, tomato) have been reported. For example, methods of improving insect resistance by introducing a gene encoding BT toxin (ie, the insecticidal toxin protein produced by Bacillus thuringiensis) into a cotton plant have been developed and put into practical use. Furthermore, cotton plants with improved resistance to herbicide (Glyphosphate) have been genetically engineered by introducing a gene encoding 5-enol-pyruvyl-shichemical acid 3-phosphate synthetase.

ES 2 635 546 T3

The availability and success of plant genetic engineering combined with the fact that cotton is an excellent candidate for genetic manipulation through recombinant techniques have led researchers to postulate that if a gene associated with a property can be identified improved cotton fiber, can be up-regulated using recombinant techniques thus improving the characteristics or performance of cotton fibers.

Conversely, if a gene associated with a decrease in a cotton fiber property can be identified, it could be down-regulated using gene silencing methods. For this purpose, the mechanisms of fiber elongation and formation at the genetic level must be clarified and genes closely associated with these mechanisms must be identified.

A cotton fiber is made up of a single cell that has differentiated from an epidermal cell of the seed coat, developing through four stages, i.e. initiation, elongation, secondary cell wall thickening, and stages of maturation. More specifically, elongation of a cotton fiber begins in the epidermal cell of the ovule immediately after flowering, after which the cotton fiber rapidly elongates for approximately 21 days. The elongation of the fiber is then terminated, and a secondary cell wall is formed and grows through maturation to become a mature cotton fiber.

Several candidate genes have been isolated that are associated with the elongation and formation of cotton fibers. For example, five cotton plant genes that are specifically expressed in the elongation stage of cotton fiber have been identified by the differential screening method and the differential visualization method, [US Patent No. 5,880,100 and applications US Patent Serial Nos. 08 / 580,545, 08 / 867,484 and 09 / 262,653].

WO0245485 describes methods and means to modulate fiber quality in fiber-producing plants, such as cotton, by modulating the activity and / or expression of sucrose synthase (a sugar important for cell wall synthesis) in said plants.

US Patent No. 6,472,588 and WO0117333 provide methods for increasing the quality of cotton fiber produced from a cotton plant by transformation with a DNA encoding sucrose phosphate synthase. Fiber qualities include strength, length, fiber maturity ratio, immature fiber content, fiber uniformity, and micronaire.

WO9508914 describes a fiber-producing plant that comprises a heterologous genetic construct in its genome. The genetic construct comprises a fiber-specific promoter and a coding sequence that encodes a plant peroxidase, such as a cotton peroxidase.

WO9626639 provides methods in which an ovarian-specific promoter sequence is used to express hormones that modify plant growth in cotton egg tissue. The methods allow modification of the characteristics of the boll assembly in cotton plants and provide a mechanism to alter the quality characteristics of the fiber such as dimension and strength of the fiber.

US Patent No. 5,981,834, US Patent No. 5,597,718, US Patent No. 5,620,882, US Patent No. 5,521,708, and US Patent No. 5,495,070 describe all a method of engineering a fiber-producing plant and the identification of cDNA clones useful for identifying fiber genes in cotton. The cDNA clones are useful in the development of corresponding genomic clones of fiber-producing plants to carry out genetic engineering of cotton and other plants using these genes. The coding sequences of these isolated genes are used in sense or anti-sense orientation to alter the fiber characteristics of transgenic fiber-producing plants.

US patent applications 2002049999 and US 2003074697 describe both cotton plants of the genus Gossypium with improved cotton fiber characteristics. The cotton plant has an expression cassette containing a gene encoding an enzyme selected from the group consisting of endoxyloglucan transferase, catalase, and peroxidase so that the gene is expressed in cotton fiber cells to enhance the characteristics of the cotton fiber. cotton.

WO 01/40250 provides methods for improving cotton fiber quality by modulating transcription factor gene expression.

WO 96/40924 provides novel DNA constructs that can be used as molecular probes or alternatively inserted into a host plant to provide for transcriptional modification of a DNA sequence of interest during various stages of cotton fiber development. The DNA constructs comprise a gene-associated cotton fiber transcriptional initiation regulatory region, which is expressed in cotton fiber. A new cotton having a cotton fiber having a natural color is also provided. Color is achieved by the introduction and expression in the cotton fiber cell of a pigment gene construct.

ES 2 635 546 T3

EP0834566 provides a gene that controls the fiber formation mechanism in the cotton plant and which can be used for industrially useful breeding.

However, alongside Sacrosa Sintase, there is no evidence to date that the expression of any particular gene plays an essential role in cotton fiber formation or improved fiber characteristics.

Thus, there remains a need to identify other genes associated with cotton plant fiber characteristics and a more exhaustive search for quality-related genes is required.

But reducing the present invention to practice, the present inventors devised and employed a new computational approach that uses comparative genomics to identify genes that play a critical role in fiber development. As has been demonstrated in this specification, the expression of said genes correlates with fiber length and their overexpression is sufficient to modify tomato seed hair, a recent model for cotton fibers. These results suggest that polynucleotides can be used to generate transgenic cotton plants that are characterized by fibers of desired length.

Compendium of the invention

According to one aspect of the present invention there is provided a method for improving the fiber elongation of a fiber-producing plant, which method comprises expressing in the fiber-producing plant an exogenous polynucleotide comprising a nucleic acid sequence having at least 95% of sequence identity with the complete polynucleotide set forth in SEQ ID NO: 19, thereby improving the fiber elongation of the fiber-producing plant. According to another aspect of the present invention there is provided a method for improving the fiber elongation of a fiber-producing plant, which method comprises expressing in the fiber-producing plant an exogenous polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 121, thereby improving the elongation of the fiber of the fiber-producing plant.

According to additional features in preferred embodiments, said exogenous polynucleotide is ligated into a nucleic acid construct comprising a promoter capable of regulating the expression of said polynucleotide in a plant cell, wherein said promoter is heterologous to said plant cell.

According to still additional features in the described preferred embodiments said exogenous polynucleotide is set forth in SEQ ID NO: 19.

According to still additional features in the described preferred embodiments said promoter is set forth in SEQ ID NO: 74, 75, 85 or 91 or a functional equivalent thereof.

According to still additional features in the described preferred embodiments said promoter is a constitutive promoter.

According to still additional features in the described preferred embodiments said promoter is an inducible promoter.

According to still further features in the described preferred embodiments said promoter is a developmental stage specific promoter or a tissue specific promoter.

According to still additional features in the described preferred embodiments the plant is a monocotyledonous plant.

According to still additional features in the described preferred embodiments the plant is a dicotyledonous plant.

The present invention successfully solves the deficiencies of the currently known configurations by providing genes involved in cotton fiber development and methods for using them.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which said invention belongs.

In case of conflict, it will send the patent application, including the definitions.

Brief description of the drawings

In the drawings:

FIG. 1 is an illustration depicting the bioinformatics methodology of the present invention carried out to identify genes that can be used to improve cotton fiber yield and quality.

FIGs. 2a-d are bar graphs showing the expression patterns of fiber-specific genes (CT_11 Figure 2b), genes associated with elongation (CT_1, Figure 2c) and genes associated with initiation

ES 2 635 546 T3 (CT_22, Figure 2d).

FIG. 3 is a graph representing the expression of CT_76 in cotton plant varieties (G. hirsutum var Tamcot, Coker and Acala, and G. barbadense var Prima S5), as determined by RT-PCR.

FIG. 4 is a schematic illustration of the binary plasmid pPi.

FIGs. 5a-l are photographs of wild and transgenic arabidopsis plants that overexpress genes of the present invention. Figure 5a shows a two-week rosette of wt plants; Figure 5b shows a two-week rosette from arabidopsis plants over-expressing CT11; Figure 5c shows two week old roots of CT11; Figure 5d shows three week old wild arabidopsis; Figure 5e shows three week CT_20; Figure 5f shows three week old CT-22; Figure 5g shows 30-day rosettes of wt and CT_9; Figure 5h shows the 30-day influorescence of wt and CT_9; Figure 5i shows two week old roots of CT9; Figure 5j shows 30-day rosettes of wt and CT_40; Figure 5k shows a 5 week old rosette from wt and overexpressing CT81 plants; Figure 5l shows a leaf of wt arabidopsis plants that overexpress CT81.

FIGs. 6a-e are photographs representing wild and transgenic tomato plants that overexpress CT_20. Figure 6a shows a leaf of a wild plant; Figure 6b shows a leaf from a CT_20 transgenic tomato; Figure 6c shows seed hairs of tomato plants WT and overexpressing CT_20; Figure 6d shows a section of a WT tomato seed; Figure 6e shows a section of a tomato seed that over-expresses CT_20; Figure 6f seed hairs from WT and CT_82.

FIGs. 7a-b are photographs representing transgenic tomato plants that overexpress GUS under the expression of the CT_2 promoter. Figure 7a is a transgenic tomato fruit cross section, which overexpresses GUS under the CT2 promoter in the mature green stage (5x magnification). Figure 7b similar to Figure 7a shows 25x magnification;

FIGs. 8a-b are photographs representing various increases of tomato fruits or wild and transgenic tomato seeds. Figure 8a is a single wild tomato seed covered with 10x magnification seed hairs; Figure 8b shows tomato seed that over-expresses expansin under 35S (10x magnification).

Detailed description

The present invention is about a method for improving fiber elongation comprising expressed polypeptides and polynucleotides that encode the same ones that are involved in plant fiber development, as characterized in the appended claims.

The principles and operation of the present invention may be better understood with reference to the accompanying drawings and descriptions.

Cotton and cotton by-products provide the raw materials used to produce a large number of consumer products; In addition to textiles, cotton is used to produce food products, livestock feed, fertilizers, and paper. The production, marketing, consumption and trade of cotton-based products generate in excess of $ 100 billion a year in the US alone, making cotton the number one value-added crop.

During the past decade, cotton lint production has drastically declined, prompting growers and researchers to seek other approaches, which can be used to improve lint quality and yield.

Increasing fiber quality and / or yield under various environmental conditions will increase the profitability of cotton crop production and provide a new spectrum of material properties for exploitation by processing industries.

But by reducing the present invention to practice, the present inventors have configured a new computing approach that uses comparative genomics to identify genes that play a role in fiber development. Genes identified using this approach can be used successfully to generate transgenic plants that are characterized by fibers of desired properties.

Thus, a method of identifying genes that are involved in the development of cotton fiber is described herein.

As used herein the term "cotton" refers to a wild type (eg hybrid) cultivar or a transgenic cotton plant (Gossypium).

As used herein the phrase "fiber development" refers to the development of cottonseed hair.

As used herein the term "development" when used in the context of cotton fibers is

ES 2 635 546 T3 refers to the initiation of the fiber and / or its elongation, as well as to the thickening and maturation of the secondary cell wall of the fiber.

The method is carried out by:

(a) providing expressed nucleic acid sequences derived from cotton fibers;

(b) providing expressed nucleic acid sequences derived from an ovule tissue (i.e., a tissue developed from an ovary of a seed plant. Examples include, but are not limited to, carpels, seed coat, embryo, endosperm);

(c) assembling the expressed nucleic acid sequences of (a) and (b) to generate clusters; and (d) identifying clusters of such clusters comprising expressed nucleic acid sequences from (a) and (b), thereby identifying genes that are involved in cotton fiber development.

Expressed nucleic acid sequences used as a potential source to identify genes involved in cotton fiber development in accordance with this aspect of the present invention are preferably libraries of

Expressed messenger RNA [ie, expressed sequence tags (ESTs), cDNA clones, contigs, premRNAs, etc.] obtained from tissue or cell line preparations that may include genomic or cDNA sequence.

Expressed nucleic acid sequences can be retrieved from publicly available pre-existing databases (see Example 1 in the Examples section below) or from private databases.

Alternatively, the expressed nucleic acid sequences used can be generated from sequence libraries (eg, cDNA libraries, EST libraries, mRNA libraries, and others).

CDNA libraries are suitable sources of expressed sequence information.

In such a case generating a sequence database is typically accomplished by tissue or cell sample preparation, RNA isolation, cDNA library construction, and sequencing.

It will be appreciated that such cDNA libraries can be constructed from RNA isolated from whole plant, specific tissues, or cell populations.

Once the expressed sequence data from both cotton fibers and ovule tissue is obtained, the sequences can be grouped to form contigs. See Example 1 in the Examples section that follows.

Such contigs are then assembled to identify homologous sequences (from cotton fibers and ovule tissue) present in the same cluster, such contigs are considered to be involved in cotton fiber development.

A number of commonly used computer software fragment reading assemblers capable of forming clusters of expressed sequences are commercially available. These packages include, but are not limited to, The TIGR Assembler [Sutton G. et al. (1995) Genome Science and Technology 1: 9-19], GAP [Bonfield JK. et al. (1995) Nucleic Acids Res. 23: 4992-4999], CAP2 [Huang X. et al. (1996) Genomics 33: 21-31], The Genome Construction Manager [Lawrence CB. Et al. (1994) Genomics 23: 192-201], Bio Image Sequence Assembly Manager, SeqMan [Swindell SR. and Plasterer JN. (1997) Methods Mol. Biol. 70: 75-89], LEADS and GenCarta (Compugen Ltd. Israel).

Once the genes that are involved in the development of cotton fiber are identified, their expression pattern can be analyzed as described in Example 2 of the Examples section that follows, to thereby identify genes that are differentially expressed. in cotton fiber (ie, specific expression) or during cotton fiber development (ie change in expression during cotton fiber development).

Methods for identifying differentially expressed genes are well known in the art.

Using the above methodology, the present inventors were able to identify genes that are involved in the development of cotton fiber.

As illustrated in the Examples section that follows, the genes identified using the teachings of the present invention can be classified into 6 functional categories according to their sequence homology to known proteins and enzymes (Table 3, below). Two genes were classified into a category of cellular fate compromise: homologous to the MYB transcription factor and GL3 that are known to be involved in the development of trichome in arabidopsis. The expression patterns of both genes and the phenotype of the CT20 transgene both in

ES 2 635 546 T3 arabidopsis and tomato T1 plants support their involvement mainly in the initiation phase. The other two genes (Table 3, above) are transcription factors of the MYB and MADS BOX families. Many studies have demonstrated the function of these two families of transcription factors as homeotic genes with key roles in different developmental processes, among them is the morphogenesis of trichomes and fiber (Suo. J. et. Al. 2003, Ferrario S et. . al. 2004). Their role in the early stages of fiber development is also supported by their RNA expression patterns, which are induced before and during the day of anthesis. One gene belongs to the starch and sucrose metabolism pathways. Recent work shows that another gene (SUS), which belongs to this pathway, is a limiting factor in both fiber initiation and development. Another gene (Table 3, below) is classified as lipid transport whose RNA expression is highly induced during the early stage of fiber elongation conforms to the fact that lipids are key components in fiber formation. Several genes (Table 3, below) were classified as either genes involved in desiccation, response to abscisic acid-stimulated salinity, and genes involved in electron transfer. Of these, 3 genes were selected by RNA expression pattern that were induced in the elongation stage.

In view of the above and in conjunction with the experimental results that correlate gene expression with fiber length, it is suggested that the genes of the present disclosure can be used to generate fiber-producing plants of commercially desired fiber quality.

Therefore, polynucleotides identified using the above-described methodology and their encoded polypeptides as well as functional equivalents of the polypeptides identified herein (i.e., polypeptides that are capable of regulating cotton fiber growth) are also described herein. , as can be determined based on the assays described in the Examples section below) and their coding sequences. Such functional equivalents can be at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least

<td>approximately</td><td> 83%,</td><td>to the</td><td>less</td><td>approximately</td><td> 84%,</td><td>to the</td><td>less</td><td>approximately</td><td> 85%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 86%,</td><td>to the</td><td>less</td><td>approximately</td><td> 87%,</td><td>to the</td><td>less</td><td>approximately</td><td> 88%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 89%,</td><td>to the</td><td>less</td><td>approximately</td><td> 90%,</td><td>to the</td><td>less</td><td>approximately</td><td> 91%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 92%,</td><td>to the</td><td>less</td><td>approximately</td><td> 93%,</td><td>to the</td><td>less</td><td>approximately</td><td> 94%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 95%,</td><td>to the</td><td>less</td><td>approximately</td><td> 75%,</td><td>to the</td><td>less</td><td>approximately</td><td> 75%,</td><td>to the</td><td>less</td>

about 75%, at least about 75%, said 100% homologous to SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 , 121, 122, 123, 124, 125, 126, 95 or 96.

Polynucleotides encoding functional equivalents can be at least about 70%, at least

<td>approximately</td><td> 75%,</td><td>to the</td><td>less</td><td>approximately</td><td> 80%,</td><td>to the</td><td>less</td><td>approximately</td><td> 81%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 82%,</td><td>to the</td><td>less</td><td>approximately</td><td> 83%,</td><td>to the</td><td>less</td><td>approximately</td><td> 84%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 85%,</td><td>to the</td><td>less</td><td>approximately</td><td> 86%,</td><td>to the</td><td>less</td><td>approximately</td><td> 87%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 88%,</td><td>to the</td><td>less</td><td>approximately</td><td> 89%,</td><td>to the</td><td>less</td><td>approximately</td><td> 90%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 91%,</td><td>to the</td><td>less</td><td>approximately</td><td> 92%,</td><td>to the</td><td>less</td><td>approximately</td><td> 93%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 94%,</td><td>to the</td><td>less</td><td>approximately</td><td> 95%,</td><td>to the</td><td>less</td><td>approximately</td><td> 75%,</td><td>to the</td><td>less</td>

about 75%, at least about 75%, at least about 75%, said to be 100% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 27.

Homology (eg, percent homology) can be determined using any homology comparison software, including, for example, the BlastP software from the National Center for Biotechnology Information (NCBI) as using the default parameters.

Identity (eg, percent homology) can be determined using any homology comparison software, including, for example, the BlastN software from the National Center for Biotechnology Information (NCBI) as using the default parameters.

As used herein the phrase "an isolated polynucleotide" refers to single or double stranded nucleic acid sequences that are isolated or provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a sequence genomic polynucleotide and / or composite polynucleotide sequences (eg, a combination of the foregoing).

As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from the reverse transcription of a messenger RNA using a reverse transcriptase or any other RNA-dependent DNA polymerase. Such a sequence can be further amplified in vivo or in vitro using a DNA-dependent DNA polymerase.

As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and therefore represents a contiguous portion of a chromosome.

As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exon sequences required to encode the polypeptide, as well as some intronic sequences interposed between them. Intronic sequences can be from any source, including other genes,

ES 2,635,546 T3 and will typically include conserved splice signal sequences. Such intron sequences may further include cis-acting expression regulatory elements.

According to a preferred aspect of the present description, the nucleic acid sequence is as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14 , 15, 16, 17, 19, 21, 22, 23, 24, 25 or 26.

According to another preferred aspect of the present description, the sequence of the isolated polynucleotide is as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25 or 27.

According to yet another preferred aspect of the present disclosure, the polypeptide is as set forth in SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 95 or 96.

According to yet another preferred aspect of the present disclosure, the amino acid sequence is as set forth in SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 95 or 96.

Isolated polynucleotides can also be scored using a hybridization assay by incubating the isolated polynucleotides described above in the presence of oligonucleotide probe or primer under moderate to stringent hybridization conditions.

Moderate to stringent hybridization conditions are characterized by a hybridization solution as containing 10% dextran sulfate, 1M NaCl, 1% SDS, and labeled probe. ³²P to 5x10⁶ cpm, at 65 ° C, with a final wash solution of 0.2x SSC and 0.1% SDS and final wash at 65 ° C and yet moderate hybridization is carried out using a hybridization solution containing dextran sulfate at 10%, 1M NaCl, 1% SDS, and labeled probe ³²P to 5x10⁶ cpm, at 65 ° C, with a final wash solution of 1x SSC and 0.1% SDS and final wash at 50 ° C.

Therefore, the present disclosure includes nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable to them, sequences homologous to them, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, whether of origin natural or man-induced, either randomly or in a targeted way.

Since the polynucleotide sequences of the present disclosure encode previously unidentified peptides, the present disclosure also includes novel polypeptides or portions thereof, which are encoded by the isolated polynucleotides and the respective nucleic acid fragments thereof described above herein. memory.

Therefore, the present disclosure also includes polypeptides encoded by the polynucleotide sequences of the present disclosure. The amino acid sequences of these new polypeptides are set forth in SEQ ID NO: 26, 106, 107, 109, 110, 112, 114, 115, 118, 119, 122, 123, 124, 126, 95 or 96.

The present description also includes homologues of these polypeptides, such homologues can be at least

<td>approximately</td><td> 70%,</td><td>to the</td><td>less</td><td>approximately</td><td> 75%,</td><td>to the</td><td>less</td><td>approximately</td><td> 80%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 81%,</td><td>to the</td><td>less</td><td>approximately</td><td> 82%,</td><td>to the</td><td>less</td><td>approximately</td><td> 83%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 84%,</td><td>to the</td><td>less</td><td>approximately</td><td> 85%,</td><td>to the</td><td>less</td><td>approximately</td><td> 86%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 87%,</td><td>to the</td><td>less</td><td>approximately</td><td> 88%,</td><td>to the</td><td>less</td><td>approximately</td><td> 89%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 90%,</td><td>to the</td><td>less</td><td>approximately</td><td> 91%,</td><td>to the</td><td>less</td><td>approximately</td><td> 92%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 93%,</td><td>to the</td><td>less</td><td>approximately</td><td> 93%,</td><td>to the</td><td>less</td><td>approximately</td><td> 94%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 95%,</td><td>to the</td><td>less</td><td>approximately</td><td> 96%,</td><td>to the</td><td>less</td><td>approximately</td><td> 97%,</td><td>to the</td><td>less</td>

about 98%, at least about 99%, or rather 100% homologous to SEQ ID NO: 26, 106, 107, 109, 110, 112, 114, 115, 118, 119, 122, 123, 124, 126, 95 or 96.

The present disclosure also includes fragments of the polypeptides described above and polypeptides that have mutations, such as deletions, insertions, or substitutions of one or more amino acids, either naturally occurring or man-induced, either randomly or in a targeted fashion.

The ability of the polynucleotides of the present disclosure and their products to regulate cotton fiber development can be determined directly or at least one structural parameter of a cotton fiber such as fiber length or fiber fineness, or speed of fiber growth (described later in this specification). However, cotton fiber development can also be determined indirectly such as by plant model systems for cotton fiber development. For example, it is well established that trichome cells and root hairs share common characteristics with cotton fiber cells, and as such can be used as a model for cotton fiber development [Reviewed in Wagner GJ et al. (2004)], as demonstrated in details in Example 12 of the Examples section that follows. By analyzing the expression profiles, the present inventors were able to determine the involvement of the

ES 2 635 546 T3 biomolecular sequences (ie, polynucleotides and polypeptides) of the present disclosure in fiber initiation and / or elongation. These results were further supported by establishing a correlation between gene expression and fiber length (see Example 7).

These results suggest that the biomolecular sequences of the present disclosure (eg, polynucleotides, polypeptides, promoters, oligonucleotides, antibodies, also referred to herein as agents) can be used to improve fiber quality and / or performance of a fiber-producing plant.

Therefore, according to one aspect of the present invention there is provided a method for improving the fiber quality and / or yield of a fiber producing plant, as characterized in the appended claims.

The method of this aspect of the present invention is carried out by regulating a level of expression or activity of at least one polynucleotide or polypeptide of the present description (described above) in the fiber-producing plant as characterized in the appended claims, thus improving the quality and / or performance of the fiber producing plant.

As used herein the phrase "fiber producing plant" refers to plants that share the common characteristic of having an elongated shape and abundant cellulose in thick cell walls, typically referred to as secondary walls. Such walls may or may not be lignified, and the proptoplast of such cells may or may not be viable at maturity. Such fibers have many industrial uses, for example, in wood and manufactured wood products, paper, textiles, packaging and packaging material, cordage, brushes and brooms, filling and filling, sealing, reinforcing other materials, and manufacturing of derivatives. of cellulose.

According to a preferred aspect of the present description, the fiber-producing plant is cotton.

The term "fiber" is that it typically includes thick-walled conductive cells such as vessels and tracheids to fibrillar aggregates of many individual fiber cells. Therefore, the term "fiber" refers to (a) thick-walled conductive and non-conductive cells of the xylem; (b) fibers of extraxillary origin, including those from phloem, bark, ground tissue, and epidermis; and (c) fibers from stems, leaves, roots, seeds, and flowers or inflorescences (such as those of Sorghum vulgare used in the manufacture of brushes and brooms).

Examples of fiber-producing plants include, but are not limited to, agricultural crops such as cotton, silk cotton tree (Kapok, Ceiba petandra), desert willow, creosote bush, winter blubber, balsa tree, kenaf, roselle , jute, abaca sisal, flax, corn, sugar cane, hemp, ramie, kapok, coconut fiber, bamboo, Spanish moss and Agave spp. (eg sisal).

As used herein the phrase "fiber quality" refers to at least one fiber parameter that is agriculturally desired, or required in the fiber industry (described later herein). Examples of such parameters include, but are not limited to, fiber length, fiber strength, fiber suitability, fiber weight per unit length, maturity ratio, and uniformity (further described hereinafter).

Cotton fiber (yarn) quality is typically measured according to fiber length, strength, and fineness. Therefore, the strand quality is considered higher when the fiber is longer, stronger and finer.

As used herein the phrase "fiber yield" refers to the quantity and quantity of fibers produced from the fiber producing plant.

As used herein the term "improvement" refers to at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, change in fiber quality / yield compared to the native plant (ie, not modified with the biomolecular sequences of the present disclosure).

As used herein the term "regulation" refers to up regulating, down regulating or a combination thereof. For example, when an increase in fiber number is desired, the present invention can be accomplished by upregulating at least one polynucleotide of the present disclosure, which is involved in fiber initiation (eg, SEQ ID NOs: 4, 10, 9, 12, 16 and 25). Alternatively, when short fibers are desired as for example in corn, then the present invention is effected by down-regulating at least one polynucleotide of the present disclosure that is involved in fiber elongation (eg, SEQ ID Nos. : 1, 2, 3, 5, 6, 7, 17, 18, 19, 20, 21, 22, 23, 24 and 27). Alternatively, the present invention can be effected by upregulating the expression of at least one polynucleotide (as one involved in fiber elongation) and down-regulating at least one polynucleotide (as one involved in fiber initiation) of the polynucleotides of the present description. In this way it is feasible to obtain a fiber producing plant with improved fiber yield of each short length.

Upregulating an expression level of at least one of the polynucleotides of the present disclosure can be effected at the genomic level (eg, activation of transcription by means of promoters, enhancers, and others.

ES 2 635 546 T3 regulatory elements), at the transcriptional level, or at the protein level.

The following is a non-exhaustive list of agents capable of up-regulating the level of expression and / or activity of the biomolecular sequences (ie, nucleic acid or protein sequences) of the present disclosure.

An agent capable of upregulating the expression of a polynucleotide of interest can be an exogenous polynucleotide sequence designed and constructed to express at least a functional portion thereof (e.g., fiber quality / yield improvement, increased biomass, etc.). Accordingly, the exogenous polynucleotide sequence can be a DNA or RNA sequence encoding a polypeptide molecule, capable of improving fiber yield or quantity. Alternatively, the exogenous polynucleotide can be a cis-acting regulatory region (eg, SEQ ID NO: 74, 75, 85, 88, or 91) that can be introduced into the plant to increase the expression of any polynucleotide that it is involved in fiber development (eg, sucrose phosphate synthase, as described in US Pat. No. 6,472,588).

To express exogenous polynucleotides in plant cells, a polynucleotide sequence is preferably ligated into a nucleic acid construct suitable for expression in plant cell. Such a nucleic acid construct includes a regulatory region that acts in cis as a promoter sequence to direct transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner. The promoter can be homologous or heterologous to the transformed plant / cell.

Preferred promoter sequences that can be used in accordance with this aspect of the present invention are endothelial cell promoters.

For example, promoter sequences from each of the polynucleotide sequences described herein may preferably be used in the nucleic acid constructs described herein.

According to a preferred embodiment of this aspect of the present invention the promoter is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least

<td>approximately</td><td> 87%,</td><td>to the</td><td>less</td><td>approximately</td><td> 88%,</td><td>to the</td><td>less</td><td>approximately</td><td> 89%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 90%,</td><td>to the</td><td>less</td><td>approximately</td><td> 91%,</td><td>to the</td><td>less</td><td>approximately</td><td> 92%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 93%,</td><td>to the</td><td>less</td><td>approximately</td><td> 94%,</td><td>to the</td><td>less</td><td>approximately</td><td> 95%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 96%,</td><td>to the</td><td>less</td><td>approximately</td><td> 97%,</td><td>to the</td><td>less</td><td>approximately</td><td> 98%,</td><td>to the</td><td>less</td>

approximately 99%, or 100% identical to SEQ ID NO. 85 or 91, which is capable of regulating the expression of at least one polynucleotide sequence operably linked thereto in an ovule endothelial cell (ie, capable of exerting a regulatory effect on the coding sequence linked thereto).

As clearly illustrated in the Examples section that follows, such promoter sequences are capable of regulating the expression of a coding nucleic acid sequence (eg, GUS) operably linked thereto.

Other examples of cotton fiber augmentation promoters include the cotton fiber E6 expressed genes (John et al., Plant Mol. Biol., 30: 297-306 (1996) and John et al., Proc. Natl. Acad Sci., 93: 12768-12773 (1996) e), H6 (John et al., Plant Physiol., 108: 669-676, (1995)), FbL2A (Rinehart et al., Plant Physiol., 112: 13311341 (1996) and John et al., Proc. Natl. Acad. Sci. USA, 93: 12768-12773 (1996)), rac (Delmer et al., Mol. Gen. Genet., 248: 43-51 (1995 )); CelA (Pear et al., Proc. Natl. Acad. Sci. USA, 93: 12637-12642 (1996)); CAP (Kawai et al., Plant Cell Physiol. 39: 1380-1383 (1998)); ACP (Song et al., Biochim. Biophys. Acta 1351: 305-312 (1997); and LTP (Ma et al., Biochim. Biophys. Acta 1344: 111-114 (1997)). Other specific promoters of cotton fiber in US Pat. No. 5,495,070.

Other promoters that can be used in accordance with this aspect of the present invention are those that ensure expression only in specific organs, such as the leaf, root, tuber, seed, stem, flower, or specific cell types such as parenchyma, epidermal cells. , trichome or vascular.

Preferred promoters for enhancing expression in trichome cells are described in WO 2004/111183, to Evogene Ltd.

Preferred promoters that enhance expression in vascular tissue include the CAD 2 promoter (Samaj et al., Planta, 204: 437-443 (1998)), the Pt4C11 promoter (Hu et al., Proc. Natl. Acad. Sci. USA, 95: 5407-5412 (1998)), the C4H promoter (Meyer et al., Proc. Natl. Acad. Sci. USA, 95: 6619-6623 (1998)), the PtX3H6 and PtX14A9 promoters (Loopstra et al. ., Plant Mol. Biol., 27: 277-291 (1995)), the RolC promoter (Graham, Plant Mol. Biol., 33: 729-735 (1997)), the Hvhsp17 promoter (Raho et al., J . Expt. Bot., 47: 1587-1594 (1996)), and the COMT promoter (Capellades et al., Plant Mol. Biol., 31: 307-322 (1996)).

Preferred promoters that enhance expression in stem tissue include marrow promoters (Datta, Theor. Appl. Genet., 97: 20-30 (1998) and Ohta et al., Mol. Gen. Genet., 225: 369) -378 (1991)), and the anionic peroxidase promoter (Klotz et al., Plant Mol. Biol., 36: 509-520 (1998)). Preferred promoters that enhance expression in phloem, cortex, and cork, but not in xylem or pith, include the Psam-1 promoter.

ES 2 635 546 T3 (Mijnsbrugge et al., Plant and Cell Physiol., 37: 1108-1115 (1996)).

Preferred promoters that enhance expression in seeds include the phas promoter (Geest et al., Plant Mol. Biol. 32: 579-588 (1996)); the GluB-1 promoter (Takaiwa et al., Plant Mol. Biol. 30: 1207-1221 (1996)); the gamma-zein promoter (Torrent et al., Plant Mol. Biol. 34: 139-149 (1997)), and the oleosin promoter (Sarmiento et al., The Plant Journal 11: 783-796 (1997)).

Other promoter sequences that mediate constitutive, inducible, tissue specific or developmental stage specific expression are described in WO 2004/081173 to Evogene Ltd.

Truncated or synthetic promoters can be used including specific nucleotide regions that confer improved expression in tissue, as exemplified by the identification of regulatory elements within larger promoters that confer increased expression in the xylem (Seguin et al., Plant Mol. Biol., 35: 281-291 (1997); Torres-Schumann et al., The Plant Journal, 9: 283-296 (1996); and Leyva et al., The Plant Cell, 4: 263271 (1992) ).

The nucleic acid construct can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome. Preferably, the nucleic acid construct of the present invention is a plasmid vector, more preferably a binary vector.

The phrase "binary vector" refers to an expression vector that carries a modified T region of a Ti plasmid, capable of multiplying in both E. Coli and Agrobacterium cells, and that normally comprises the marker gene (s). (s) for plant transformation between the two host regions. A suitable binary vector for the present invention includes pBI2113, pBI121, pGA482, pGAH, pBIG, pBI101 (Clonetech), pPI (see Example 5 of the Examples section that follows) or modifications thereof.

The nucleic acid construct can be used to transform a host cell (eg, bacterium, plant) or plant.

As used herein, the terms "transgenic" or "transformed" are used interchangeably in reference to a cell or plant into which cloned genetic material has been transferred.

In stable transformation, the nucleic acid molecule integrates within the plant genome, and as such represents a stable and inherited trait. In transient transformation, the nucleic acid molecule is expressed by the transformed cell but does not integrate within the genome, and as such represents a transient characteristic.

There are several methods of introducing foreign genes into monocotyledonous and dicotyledonous plants (Potrykus, I. (1991). Annu Rev Plant Physiol Plant Mol Biol 42, 205-225; Shimamoto, K. et al. (1989). Fertile plants of transgenic rice regenerated from transformed protoplasts. Nature (1989) 338, 274-276).

The main methods of stable integration of exogenous DNA into plant genomic DNA include two main approaches:

(i) Agobacterium-mediated gene transfer. See: Klee, HJ et al. (1987). Annu Rev Plant Physiol 38, 467486; Klee, HJ and Rogers, SG (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pg. 2-25, J. Schell and LK Vasil, eds., Academic Publishers, San Diego, Cal .; and Gatenby, AA (1989). Regulation and Expression of Plant Genes in Microorganisms, pg. 93-112, Plant Biotechnology, S. Kung and CJ Arntzen, eds., Butterworth Publishers, Boston, Mass.

(ii) Direct DNA uptake. See, p. eg: Paszkowski, J. et al. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pg. 52-68, J. Schell and LK Vasil, eds., Academic Publishers, San Diego, Cal .; and Toriyama, K. et al. (1988). Bio / Technol 6, 1072-1074 (methods of direct DNA uptake within protoplasts). See also: Zhang et al. (1988). Plant Cell Rep 7, 379-384; and Fromm, ME et al. (1986). Stable transformation of corn after gene transfer by electroporation. Nature 319, 791-793 (Plant cell electrical shock induced DNA uptake). See also: Klein et al. (1988). Bio / Technol 6, 559-563; McCabe, DE et al. (1988). Stable transformation of soybean (Glycine max) by particle acceleration. Bio / Technology 6, 923-926; and Sandford, JC (1990). Biolistic plant transformation. Physiol Plant 79, 206-209 (Injection of DNA into plant cells or tissues by particle bombardment). See also:

Neuhaus, JM et al. (1987). Theor Appl Genet 75, 30-36; and Neuhaus, JM and Spangenberg, GC (1990). Physiol

Plant 79, 213-217 (use of micropipette systems). See Pat. US No. 5,464,765 (transformation into whiskers of glass fibers or silicon carbide of cell cultures, embryos or callus tissue). See also: DeWet, JMJ et al. (1985). "Exogenous gene transfer in corn (Zea mays) using pollen treated with DNA",

Experimental Manipulation of Ovule Tissue, GP Chapman et al., Eds., Longman, New York-London, pg. 197-209; and Otha, Y. (1986). High Yield Genetic Transformation of Corn by a Mix of Pollen and Exogenous DNA.

Proc Natl Acad Sci USA 83, 715-719 (direct incubation of DNA with germinating pollen).

The Agrobacterium-mediated system includes the use of plasmid vectors containing DNA segments

ES 2 635 546 T3 defined that are integrated into plant genomic DNA. Plant tissue inoculation methods vary depending on the plant species and the Agrobacterium delivery system. A widely used approach is the leaf-disc procedure, which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation (Horsch, RB et al. (1988). "Disc sheet transformation." Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). A complementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially useful in creating transgenic dicot plants.

There are several methods of direct transfer of DNA into plant cells. In electroporation, proptoplasts are briefly exposed to a strong magnetic field, opening mini-pores to allow DNA to enter. In microinjection, DNA is mechanically injected directly into cells using micropipettes. In microparticle bombardment, DNA is adsorbed onto microprojectiles such as magnesium sulfate crystals or tungsten particles, and microprojectiles are physically accelerated within plant cells or tissues.

After stable transformation, propagation of the plant occurs. The most common method of propagation is by seed. However, the disadvantage of regeneration by seed propagation is the lack of uniformity in cultivation due to heterozygosity, since the seeds are produced by plants according to the genetic variances governed by Mendel's rules. In other words, each seed is genetically different and each will grow with its own specific characteristics. Therefore, it is preferred that the regeneration is effected in such a way that the regenerated plant has identical traits and characteristics as the parent transgenic plant. The preferred method of regenerating a transformed plant is by micropropagation, which provides rapid, consistent reproduction of the transformed plants.

Micropropagation is a second generation plant culture process from a single tissue sample excised from a selected parent plant or cultivar. This process allows the mass reproduction of plants that have the preferred tissue and that express a fusion protein. The newly generated plants are genetically identical to, and have all the characteristics of the original plant. Micropropagation enables the mass production of quality plant material in a short period of time and offers rapid multiplication of selected cultivars while preserving the characteristics of the original transgenic or transformed transformed plant. Advantages of this method of plant cloning include the speed of plant multiplication and the quality and uniformity of the plants produced.

Micropropagation is a multi-stage process that requires alteration of culture medium or growth conditions between stages. The micropropagation process involves four basic stages: stage one, initial tissue culture; stage two, multiplication of tissue culture; stage three, differentiation and formation of the plant; and stage four, greenhouse cultivation and consolidation. During stage one, the tissue culture is established and certified to be free of contaminants. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the newly grown tissue samples are divided and grown into individual seedlings. In stage four, the transformed seedlings are transferred to a greenhouse for consolidation where the plants' tolerance to light is gradually increased so that they can continue to grow in the natural environment.

Although stable transformation is currently preferred, transient transformation of, for example, leaf cells, meristematic cells, or the whole plant is also envisioned.

Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.

Viruses that have been shown to be useful for transforming host plants include cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV), and baculovirus (BV). Transformation of plants using plant viruses is described in, for example: US Pat. US No. 4,855,237 (Jewish Golden Mosaic Virus, BGMV); EPA

67,553 (TMV); Japanese Published Application No. 63-14693 (TMV); EPA 194,809 (BV); EPA 278,667 (BV); and Gluzman, Y. et al. (1988). Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pg. 172-189. The use of pseudovirus particles in the expression of foreign DNA in many hosts, including plants, is described in WO 87/06261.

The construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences in plants is demonstrated in the above references as well as in: Dawson, WO et al. (1989). A hybrid tobacco mosaic virus expresses and loses an added gene. Virology 172, 285-292; French, R. et al. (1986) Science 231, 1294-1297; and Takamatsu, N. et al. (1990). Production of enkephalin in tobacco protoplasts using a tobacco mosaic virus RNA vector. FEBS Lett 269, 73-76.

If the transforming virus is a DNA virus, one of skill in the art can make suitable modifications to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid to facilitate construction of the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then

ES 2 635 546 T3 replicates by the bacteria. Transcription and translation of the DNA will produce the coat protein, which will encapsulate the viral DNA. If the virus is an RNA virus, normally the virus is cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all the plant genetic constructs. The RNA virus is then transcribed from the viral sequence of the plasmid, followed by translation of the viral genes to produce the envelope proteins that encapsulate the viral RNA.

The construction of plant RNA viruses for introduction and expression in plants of non-viral exogenous nucleic acid sequences, such as those included in the construct of the present disclosure, is also demonstrated in the above references as well as in US Pat. US No. 5,316,931.

In one aspect of the disclosure, a plant viral nucleic acid is provided for insertion, comprising a deletion of the coding sequence for the native coat protein of the viral nucleic acid, a coding sequence for the non-native plant viral coat protein (foreign ), and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, and capable of expression in the host plant, packaging of the recombinant plant viral nucleic acid, and which ensures a systemic infection of the host by the recombinant plant viral nucleic acid. Alternatively, the native coat protein coding sequence can be made non-transcribable by inserting the non-native nucleic acid sequence into it, such that a non-native protein is produced. The recombinant plant viral nucleic acid construct may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the host plant and incapable of recombination with each other and with native subgenomic promoters. In addition, the recombinant plant viral nucleic acid construct may contain one or more cis-acting regulatory elements, such as enhancers, that bind to a trans-acting regulator that regulates transcription of a downstream coding sequence. the same. Non-native nucleic acid sequences can be inserted adjacent to the native plant viral subgenomic promoter or non-native plant viral subgenomic promoters if more than one nucelic acid sequence is included. Non-native nucleic acid sequences are transcribed or expressed in the host plant under the control of the subgenomic promoter (s) to produce the desired products.

In a second aspect, a recombinant plant viral nucleic acid construct is provided as in the first aspect except that the coding sequence for the native coat protein is located adjacent to one of the subgenomic promoters of the non-native coat protein instead. of to the non-native coat protein coding sequence.

In a third aspect, a recombinant plant viral nucleic acid construct is provided comprising a native coat protein gene located adjacent to its subgenomic promoter and to one or more non-native subgenomic promoters inserted within the viral nucleic acid construct. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a host plant and are incapable of recombination with each other and with native subgenomic promoters. Non-native nucleic acid sequences can be inserted adjacent to non-native subgenomic plant viral promoters such that such sequences are transcribed or expressed in the host plant under the control of the subgenomic promoters to produce the desired product.

In a fourth aspect, a recombinant plant viral nucleic acid construct is provided as in the third aspect except that the sequence encoding the native coat protein is replaced by a non-native coat protein coding sequence.

Viral vectors are encapsulated by expressed coat proteins encoded by recombinant plant viral nucleic acid constructs as described hereinbefore, to produce a recombinant plant virus. The recombinant plant viral nucleic acid construct or the recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral nucleic acid construct is capable of replicating in a host, spreading systemically within the host, and transcribing or expressing one or more foreign genes (isolated nucleic acid) in the host to produce the desired protein.

In addition to the above, the nucleic acid molecule can also be introduced into the chloroplast genome thereby allowing expression in the chloroplast.

A technique is known for introducing exogenous nucleic acid sequences into the chloroplast genome. This technique involves the following procedures. First, the plant cells are chemically treated in order to reduce the number of chloroplasts per cell to about one. The exogenous nucleic acid is then introduced into the cells preferably through particle bombardment, with the aim of introducing at least one exogenous nucleic acid molecule into the chloroplasts. Exogenous nucleic acid is selected by one of skill in the art to be capable of integration into the chloroplast genome through homologous recombination, which is readily accomplished by enzymes inherent to the chloroplast. To this end, the nucleic acid comprises, in addition to the gene of interest, at least one nucleic acid sequence derived from the chloroplast genome. Furthermore, the exogenous nucleic acid comprises a selectable marker, which by sequential selection procedures serves to enable a craftsman to verify that all or substantially all copies

ES 2 635 546 T3 of the chloroplast genome that follow such selection include exogenous nucleic acid. More details regarding this technique are found in US Pat. US Nos. 4,945,050 and 5,693,507, which are incorporated herein by reference. In this way, a polypeptide can be produced by the chloroplast protein expression system and integrated into the inner membrane of the chloroplast.

Downregulation of a gene of interest can be accomplished at the genomic and / or transcriptional level using a variety of molecules that interfere with transcription and / or translation (eg, antisense, siRNA), or at the level of protein using e.g. eg, antibodies, immunization techniques, and the like.

For example, an agent capable of downregulating an activity of a polypeptide of interest is an antibody or an antibody fragment capable of specifically binding a polypeptide of the present invention. Preferably, the antibody specifically binds to at least one epitope of the polypeptide of interest. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.

Downregulation at the RNA level can be effected by RNA-based silencing strategies that are effective in plants. See for example, Kusaba (2004) RNA interference in crop plants. Curr. Opin. Biotechnol. 15 (2): 139-43; Matzke (2001) RNA based silencing strategies in plants. Curr. Opin. Genet. 11: 221-7.

For example, an agent capable of downregulating a polynucleotide of interest is a small interfering RNA molecule (siRNA) in the RNA interference process (RNAi).

DsRNAs can be delivered into plants in a number of ways (reviewed in Waterhouse P, Helliwell C. 2003. Exploring plant genomes by RNA-induced gene silencing. Nature Genet 4: 29-38): microprojectile bombardment with vectors expressing dsRNA or intron-containing hairpin RNA (ihpRNA); infiltration of plant tissue with an Agrobacterium strain bearing a T-DNA that expresses an ihpRNA transgene; virus-induced gene silencing (VIGS); wherein the target sequence is integrated into viral sequences that are used to infect the plant, or are expressed from transgenes introduced into Agrobacterium, and by stable transformation with ihpRNA-expressing transgenes. The various iRNA techniques each have advantages and disadvantages with respect to how persistent its effect is and the range of plants to which it can be applied, e.g. ex. Bombardment can be applied to any plant, but it only produces transitory effects. Alternatively, transformation with ihpRNA-expressing transgenes provides stable and heritable gene silencing, but requires efficient plant transformation techniques. IhpRNA transgenes have been shown to be highly efficient for a wide range of target genes in various plant species (reviewed in Waterhouse P, Helliwell C. 2003. Exploring plant genomes by RNA-induced gene silencing. Nature Genet 4: 29-38; Wesley S, Helliwell C, Smith N, et al. 2001. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27: 581-590), indicating that the RNAi mechanism is probably conserved in all plant species. This is supported by a recent report of RNAi in the non-vascular moss Physcomitrella patens (Bezanilla M, Pan A, Quatrano R. 2003. RNA interference in the moss Physcomitrella patens. Plant Physiol 133: 470-474).

Antisense genetic constructs for fiber-specific promoters (eg, for SEQ ID NO: 85, 91) can be used to inhibit or decrease the expression of one or more fiber genes in fiber cells. The use of antisense constructs is described in US Pat. US No. 5,495,070 and in Smith, et al. Nature 334: 724-726, 1988; Bird, et al. Bio / Technology 9: 635-639, 1991; Van der Krol, et al. Gene 72: 45-50, 1988.

It will be appreciated that the generation of the fiber-producing plant of desired characteristics can be accomplished by crossing each of the above genetically modified plants with the wild-type, hybrid, or transgenic plants, using methods that are well known in the art.

Once the transgenic plants are generated, the fibers are harvested (eg by mechanical harvesting and / or hand plucking) and fiber quality and yield are determined.

Cotton fiber grading methods are described below.

Fiber Length- Instruments such as a fibrograph and HVI (High Volume Instrumentation) systems are used to measure fiber length. The LVH instruments calculate length in terms of "mean" and "upper half" (UHM) length. The mean is the average length of all fibers while UHM is the average length of the longest half of the fiber distribution.

Fiber Strength- As mentioned, fiber strength is generally defined as the force required to break a bundle of fibers or a single fiber. In the HVI test the breaking force is converted to “grams of force per unit text”. This is the force required to break a fiber bundle that is one tex unit in size. In the HVI test the resistance is given in grams per tex units (grams / tex). Fibers can be classified as low strength (eg. g., 19-22 grs / tex), medium resistance (eg, 23-25 ​​grs / tex), high resistance (eg, 26-28 grs / tex), and very high resistance (eg. eg, 29-36 grs / tex).

Micronaire- The micronaire reading of a fiber is obtained from an air flow porosity test. The test was carried out as follows. A weighed sample of cotton is compressed to a given volume and passed

ES 2 635 546 T3 controlled air flow through the sample. The resistance to air flow is read as mlchronous units. The mlcronal readings reflect a combination of maturity and finesse. Since the diameter of fibers within a variety of cotton is quite consistent, the mlcronal index will most likely indicate variation in maturity rather than variations in fineness. A mlcronal reading of 2.6-2.9 is low while 3.0-3.4 is below average, 3.5-4.9 is normal, and 5.0 and above are high. For most textile applications a level of 3.5-4.9 is used. Anything higher than this is normally not desirable. It will be appreciated however, that different applications require different fiber properties. Therefore, it is understood that a fiber property that is disadvantageous in one application could be advantageous in another.

As illustrated in the Examples section, which follows, bomolecular sequences are capable of increasing number and length of trichoma / leaf hair, as well as seed hair. As such bomolecular sequences of the present invention can be used to generate transgenic plants with increased number / length of trichomes that better adapt to herbivores, guide the pathway of pollinators, or affect photosynthesis, leaf temperature, or water loss at high temperatures. through increased light reflection. Furthermore such transgenic plants can be used for the compartmentalized production of recombinant proteins and chemicals in trichomes, as described in detail in WO 2004/111183 to Evogene Ltd.

Interestingly and unexpectedly, the present Inventors found that the polynucleotide sequences of the present disclosure are capable of increasing a plant mass. It will be appreciated that the ability of the polypeptides of the present disclosure to increase plant yield / mass / weight is inherent in their ability to promote increase in plant cell size or volume (as described herein).

Therefore, also described herein is a method of increasing a mass / vlgor / yield of a plant (coniferous, moss, algae, monocotlledon or dicotyledonous plants, as well as other plants listed at www.natlonmaster.com/encvclopedla/ Plantae). This is done by regulating the expression and / or activity of at least one of the polynucleotides of the present disclosure, as described above.

As used herein the phrase "plant biomass" refers to the amount or amount of tissue produced from the plant in one growing season, which could also determine or affect plant yield or yield per area. cultivation.

As used herein the phrase "plant vigor" refers to the amount or amount of tissue produced from the plant at any given time. Therefore increasing vigor could determine or affect plant performance or performance by growing time or growing area.

As used herein the phrase "plant yield" refers to the amount or amount of tissue produced and harvested as the plant product produced. Therefore increasing the yield could affect the economic benefit that one can obtain from the plant in a certain growth area and / or growth time.

Therefore, the present disclosure is of high agricultural value in promoting the yield of commercially desired crops (eg, vegetative organ mass such as poplar wood, or reproductive organ such as number of seeds or seed mass).

As used herein the term "about" refers to ± 10%.

Examples

Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Blology" Volumes l-lll Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Blology", John Willey and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Gulde to Molecular Cloning", John Willey &amp; Sons, New York (1988); Watson et al., "Recomblnant DNA", Sclentiflc American Books, New York; Blrren et al. (eds) "Genome Analyzes: A Laboratory Manual Serles", Vols. 1-4, Coid Sprng Harbor Laboratory Press, New York (1998); methodologies as set forth in US Pat. from USA Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Blology: A Laboratory Handbook", Volumes l-lll Cellls, JE, ed. (1994); "Current Protocols in Immunology" Volumes l-lll Coligan JE, ed. (1994); Stltes et al. (eds), "Basic and Clinical Immunology" (8^to Edition), Appleton &amp; Lange, Norwalk, CT (1994); Mlshell and Shllgl (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); Available immunoassays are widely described in the patent and scientific literature, see, for example, US Pat. from USA Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Ollgonucleotlde Synthesls" Galt, MJ, ed. (1984); "Nuclelc Acld Hybrldlzatlon" Hames, BD, and Hlgglns SJ, eds. (1985); "Transcrlptlon and Translatlon" Hames, BD, and Hlgglns SJ, Eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immoblized Cells and Enzymes" IRL Press, (1986); "A Practical Gulde to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Gulde To Methods and Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategles for Protein Purification and Characteristics-A Laboratory Course Manual" CSHL Press (1996). Other general references are provided throughout this document. The procedures in it

ES 2 635 546 T3 are believed to be well known in the art and are provided for the convenience of the reader.

Example 1

In siiico identification of cotton genes involved in fiber formation

Experimental procedures

Comparison between species of expressed sequences.- Two main tools were used during the data extraction stage. A large number of gene profiles were collected from an ORACLE database that houses Compugen's GeneCarta platform (Compugen Ltd. Israel). These data were loaded into Microsoft Excel spreadsheets for further manual refinement. Using these data, a genomic comparison across species was carried out, with the aim of defining organs of other plant species for which publicly available EST libraries can be used as models and as new sources of information to define new genes with paper. key in fiber formation (Figure 1). This comaparaclon analysis used mainly the cotton, arabdopsl, and tomato databases.

Grouping and grouping between species of EST sequences.- The cotton genomic database included less than 50,000 ESTs (Genebank publication # 135) originating mainly from two species Gossypium arboreum (-35,000 ESTs) and Gossypium hirsutum L. (- 9,000 ESTs, Table 1, below). These ESTs are grouped and assembled using the LEADS ™ software platform (Compugen Ltd, Israel) in two alternative approaches.

In the first approach, the ESTs of two species were grouped and assembled together (thus mimicking the evolutionary process since G. arboreum is an ancestor of G. hirsutum). This process revealed 6,478 clusters including 3,243 new clusters (no mRNA in the public database) that were defined as high-quality clusters (Table 1, below).

In the second approach, ESTs of each species were grouped and assembled separately. The comparison between the two approaches showed that using the first approach adds information to the cotton clusters without significant bias in the analysis. The tomato database contains 126,156 ESTs that originate from approximately 30 well-defined libraries that through the clustering and assembly process revealed 14,034 clusters of which a large group of 12,787 clusters were high-quality clusters (Table 1). The arabldopsls genomic database includes 99,417 ESTs (ftp://ftp.ncbl.nlh.gov/aenbank/). 8,573 full-length cDNA (Rlkken and genbank mRNAs ftp://ftp.ncbl.nlh.gov/aenbamk/) and the entire DNA sequence. Using LEADS software, 23,148 clusters and 6,777 slngletons were revealed (unique ESTs that do not have another EST clustered within it), all of which were supported by sequence ESTs, against the public consortium (TAIR, www.arabldopsls.org/).

EST libraries from other plants and organs that share biological processes similar to those of cotton fiber were searched. Such ESTs are expected to serve as models and as new sources of information for the identification of genes that are involved in fiber development. To this end, a list of known genes suspected of being involved in fiber development was generated. These genes originate from arabldopsls and are shown in several studies to have a key role in trichome formation (i.e. GL2, CPC, bHLH, TTG1, GL1, reviewed in Larkln JC et al. 2003, Schellmann S. et al. 2002). Comprehensive comparative genomic analysis revealed that tomato genes, with high homology to cotton fiber genes and to trichome genes of Arabdopsls, have significant EST content in any of the developmental and trichome leaf libraries of the flower. A further analysis compared the genomic database of these three species - cotton, Arabdopsls and tomato (focusing on the tomato libraries mentioned above) as key parameters in the present database search (Figure 1).

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Table 1

Cotton, Tomato and Arabidopsis genomic databases

<td>Species</td><td>EST Bibl Description</td><td>EST count</td><td>mRNA</td><td>After LEADS (groupings)</td>

<td>G. arboreum</td><td>6DPA fiber</td><td> 37.276</td><td> 12</td><td rowspan="3">16,294 clusters in mixed production *</td>

<td>G. hirsutum</td><td>Fiber 7-10 DPA</td><td> 7.944</td><td> 236</td>

<td>G. hirsutum</td><td>Flower ovule 1 DPA</td><td> 1.272</td><td> 870</td>

<td>L. esculentum</td><td>All libraries</td><td> 115.859</td><td> 7</td><td rowspan="3">25,678 clusters in mixed production</td>

<td>L. hirsutum</td><td>Libraries Trichome</td><td> 2.409</td><td> 7</td>

<td>L. pennellii</td><td>Libraries Trichome</td><td> 2.723</td><td> 24.450</td>

<td>A. thaliana</td><td>All libraries</td><td> 160.698</td><td>mRNA</td><td>25,678 clusters</td>

* groupings derived from different species, cotton G. arboreum and G. hirsutum, tomato L. esculentum, L. hirsutum and L. pennellii

In silico identification of cotton genes with a role in fiber development.- To find if tomato genomic data can be used as a relevant source of genomic data to study cotton fiber development, a genomic comparison was made. extensive to identify both tomato and cotton genes that have high homology to key genes that determine trichome development in arabidopsis (eg, GL2, CPC, bHLH, TTG1, GL1).

Homologous genes were identified in cotton and tomato. Since almost all cotton ESTs are produced from cotton fibers, in-silico prediction of the expression profile of these genes was impossible. However, many tissue sources used for the production of the tomato EST database allowed the identification of tissues in which trichome-specific genes were expressed.

In tomato it was revealed that both trichome and ovule ESTs are enriched in clusters representing trichome-specific genes. Interestingly, cotton fibers were found to be produced from cells lining the ovum. As tomato seeds are covered with downy tissue, similar to cotton seeds, it was postulated that these hairs are linked in terms of development to the formation of trichome and cotton fiber.

~ 1,100 clusters were found in tomato that include at least one EST from trichome libraries. Among them approximately 1,000 sequences included sequences that also originate from libraries of the tomato flower (in which ovule tissue is present). Comparison of this group of genes with the cotton data revealed ~ 2,300 cotton genes with high homology to tomato trichome genes. Database extraction using these two gene clusters along with other bioinformatics information [homology across species, Gene Ontology (GO)] revealed 80 cotton clusters predicted to play a key role in fiber formation. Those genes were selected based on the following criteria:

Cotton clusters with at least 2 ESTs;

Homology with tomato clustering with e-score greater than 1e-5;

Tomato cluster homology with at least one EST originating from trichome libraries or one EST originating from ovule-containing tissues;

The following criteria were considered advantageous although not necessary:

Large number of ESTs in a cluster;

Transcription factor / signal transduction proteins;

Annotation of genes related to cell expansion, turgor pressure, cell wall synthesis.

ES 2 635 546 T3

The new genes were further analyzed along with the cotton control genes known to be involved in fiber formation by their RNA expression profile in cotton plants.

Example 2

Analysis of mRNA expression of genes identified according to the teachings of the present invention

To study the RNA expression profile of candidate genes identified as described in Example 1 above, reverse transcription followed by real-time PCR (RT-qPCR) was performed.

Experimental procedures

Quantitative analysis by Real Time PCR (qRT PCR) .- To verify the levels of specific expression and associated with the characteristic, a Reverse Transcription was carried out followed by quantitative PCR (in Real Time) (RTqPCR). Total RNA was extracted at different stages of fiber development (from before day to post-before day 20). To study expression specificity, RNA was collected from other cotton plant tissues and analyzed for control expression (ie, young leaves, young stems, mature stems, young roots, sepals, petals, and stamen). For this purpose, RNA was extracted from cotton tissue using the Hot Borate Extraction protocol according to www.eeob.lastate.edu/facultv/WendelJ/ultramlcrorna.html. Reverse transcription was performed using 1.5 pg of total RNA, using 300 U of Super Script II Reverse Transcrlptase enzyme (Invitrogen), 225 ng of random deoxynucleotide hexamers (Invitrogen), mixture of dNTPs (Takara, Japan) 500 μΜ , 0.2 volumes of 5x RT buffer (Invitrogen), 0.01 M DTT, 60 U RNAsin (Promega), double distilled water treated DEPC was added to 37.5 µl. RT reactions were Incubated for 50 mln at 42 ° C, followed by 70 ° C for 15 mln. The cDNA was diluted 1:20 in Tris EDTA, pH = 8. 5 ml of diluted cDNA was used for qRT-PCR.

Quantitative RT-PCR was performed on the cDNA (5 µl), using SYBR GREEN PCR 1x standard mix (Applied Blosystems), 0.3 µΜ Forward and Inverse primers of each. The ABI7000real-tlme PCR machine was used with the following conditions 50 ° C for 2 mln, 95 ° C for 10 mln, 40 times of 95 ° C for 15 sec and 1 mln at 60 ° C, followed by 95 ° C for 15 sec, 60 ° C for 60 sec, and 70 times of 60 ° C for 10 sec + 0.5 ° C Increment in each cycle. For each gene, a standard curve was prepared from a group of RTs from all samples, in 5 dilutions (dilutions - 1:60, 1: 200, 1: 600, 1: 2000. 1: 10000). The standard curve plot [ct (threshold cycle) vs. log (concentration)] must have R&gt; = 0.98 with an efficiency in the Range of 100% +/- 5%. Expression levels (Qty) measured in qPCR were calculated using the efficiency (E) of the amplification reaction and the corresponding CT (the cycle at which the samples cross the threshold) Qty = EC.T .. The dissociation curves obtained were examined for the absence of additional PCR products or unwanted primer dimers. The reactions were repeated at least twice. The method is based on the fact that the efficiencies of the reactions of GOl (gene of interest) and housekeeping genes are similar.

To normalize the level of expression between the different tissues, specific primers were designed to specifically hybridize with the following constitutive genes: Actin (GenBank Accession No. D88414 SEQ ID NO: 28, forward and reverse primers are set out in SEQ ID NO: 68 and 69, respectively), GAPDH (GenBank Accession No. COTCWPPR, partial sequence, SEQ ID NO: 29, forward and reverse primers are set forth in SEQ ID NO: 97 and 98, respectively), and RPL19 (GenBank Accession No. ΑΙ729179, SEQ ID NO: 30, forward and reverse primers are set forth in SEQ ID NO: 99 and 100, respectively).

Using this methodology it was possible to identify genes that exhibited high expression during fiber elongation, as well as genes that exhibited unique cotton fiber specificity. Genes that showed high expression during anthesis that decreased during fiber elongation were considered good candidates to be involved in fiber differentiation and initiation. The quantification methodology described above did not remarkably provide absolute expression levels, but did provide good parameters for evaluating relative gene expression throughout fiber development despite the fact that differences as high as more than 1000-fold were detected in the maximum expression levels achieved by different genes (Table 2, below).

Results

88 cotton genes were evaluated by expression profile in different cotton fabrics (Gossypium hirsutum, var Acala). Based on the results of gene expression, 23 genes were predicted to improve fiber quality and yield. The expression profiles of all candidate genes are presented in Table 2.

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Table 2

<td>stems youths</td><td> 0,202</td><td> 0,025</td><td> 0,063</td><td>εοο'ο</td><td> 0,044</td><td> 0,049</td><td> 0,012</td><td>OOO'O</td><td> ¿90‘0</td><td> 0,762</td><td>ooo'o</td><td> 0,007</td><td> 0,004</td><td> 0,047</td><td> 1,294</td><td> 0,480</td><td> 0,464</td><td> 2,759</td><td>OOO'O</td><td>εοο'ο</td><td> 1,089</td>

<td>estate youths</td><td> 0,002</td><td> 0,068</td><td> 0,037</td><td>εοο'ο</td><td> 0,037</td><td>OOO'O</td><td>OOO'O</td><td>OOO'O</td><td> 0,069</td><td> 1,308</td><td></td><td> 0,001</td><td> 0,001</td><td> 0,005</td><td> 1,177</td><td> 0,004</td><td>εοο'ο</td><td> 0,872</td><td>OOO'O</td><td> 0,001</td><td> 0,004</td>

<td>sheets youths</td><td> 0,347</td><td> 0,021</td><td> 0,142</td><td> 0,001</td><td> 0,148</td><td> 0,055</td><td>00 or or or'</td><td>OOO'O</td><td> 0,037</td><td> 0,463</td><td></td><td> 0,001</td><td> 0,005</td><td> 0,060</td><td> 1,904</td><td> 1,301</td><td>οεο'ο</td><td> 8,534</td><td></td><td> 0,023</td><td> 6,614</td>

<td>stamen</td><td> 0,277</td><td> 0,01</td><td> 0,020</td><td> 0,044</td><td> 0,026</td><td> 0,001</td><td> 0,004</td><td> 0,068</td><td> 0,572</td><td> 0,521</td><td> 0,001</td><td> 0,001</td><td> 0,005</td><td> 0,007</td><td> 1,207</td><td> 6,599</td><td> 0,136</td><td> 28,659</td><td>OOO'O</td><td></td><td> 0,021</td>

<td>sepals</td><td> 0,336</td><td>00 or or or'</td><td> 0,086</td><td>OOO'O</td><td> 0,085</td><td> 0,007</td><td> 0,032</td><td>OOO'O</td><td> 0,076</td><td> 0,408</td><td></td><td> 0,005</td><td> 0,022</td><td> 0,007</td><td> 0,671</td><td> 1,268</td><td> 0,015</td><td> 6,317</td><td>ooo'o</td><td> 0,009</td><td> 0,913</td>

<td>petals</td><td> 9,368</td><td> 0,001</td><td> 0,038</td><td> 0,004</td><td> 0,037</td><td> 0,125</td><td> 0,019</td><td>OOO'O</td><td> 0,459</td><td> 0,168</td><td> 0,006</td><td>00 or or or'</td><td> 0,002</td><td> 0,011</td><td> 9,976</td><td> 1,258</td><td> 0,203</td><td> 83,72</td><td>ooo'o</td><td> 0,020</td><td> 1,165</td>

<td>stems mature</td><td> 0,029</td><td>ooo'o</td><td> 0,032</td><td> 0,001</td><td> 0,028</td><td> 0,001</td><td>OOO'O</td><td>OOO'O</td><td> 0,051</td><td> 0,636</td><td></td><td> 0,007</td><td>OOO'O</td><td> 0,002</td><td> 0,492</td><td> 1,708</td><td> 0,002</td><td> 3,644</td><td>ooo'o</td><td> 0,026</td><td> 26,444</td>

<td>sheets mature</td><td> 0,53</td><td> 0,014</td><td> 0,109</td><td> 0,001</td><td> 0,113</td><td> 0,066</td><td> 0,012</td><td>OOO'O</td><td> 0,051</td><td> 0,541</td><td></td><td></td><td> 0,007</td><td> 0,031</td><td> 1,065</td><td> 0,627</td><td> 0,017</td><td> 4,473</td><td>ooo'o</td><td> 0,016</td><td> 9,477</td>

<td>9-11 dpa</td><td> 00</td><td> 0,267</td><td> 0,092</td><td> 0,774</td><td> 0,110</td><td> 0,263</td><td> 2,095</td><td> ¿60‘0</td><td>εεο'ο</td><td> 1,589</td><td> 0,017</td><td>εοο'ο</td><td> 0,017</td><td> 0,332</td><td> 4,796</td><td> 608‘0</td><td> 6,983</td><td> 20,295</td><td> 0,036</td><td> 0,123</td><td> 1,153</td>

<td>6-8 dpa</td><td> 1,347</td><td> 0,238</td><td> 0,116</td><td> 0,757</td><td> 0,104</td><td> 0,228</td><td> 1,103</td><td> 0,163</td><td> 0,032</td><td> 1,017</td><td> 0,039</td><td> 0,005</td><td> 0,016</td><td> 0,315</td><td> 7,361</td><td> 1,139</td><td> 4,272</td><td></td><td> 0,015</td><td> 0,166</td><td> 0,515</td>

<td>4-5 dpa</td><td> 0,976</td><td> 0,183</td><td> 0,084</td><td> 999‘0</td><td> 0,095</td><td> 0,219</td><td> 1,265</td><td> 0,131</td><td> 0,042</td><td> 1,268</td><td> 0,028</td><td> 0,005</td><td> 0,013</td><td> 0,283</td><td> 5,313</td><td>S89'0</td><td> 4,301</td><td> 8,935</td><td> 0,020</td><td> 0,161</td><td> 0,354</td>

<td>2-3 dpa</td><td> 2,295</td><td> 0,060</td><td> 0,057</td><td> 0,622</td><td> 0,066</td><td> 0,112</td><td> 086‘0</td><td> 0,142</td><td> 0,045</td><td> 1,693</td><td> 0,017</td><td>00 or or or'</td><td> 0,012</td><td> 0,161</td><td> 2,152</td><td> 0,338</td><td> 3,135</td><td> 3,812</td><td> 0,007</td><td> 0,057</td><td> 0,210</td>

<td>18-20 dpa</td><td> 2,477</td><td> 0,819</td><td> 0,819</td><td> 0,419</td><td> 0,916</td><td> 0,197</td><td> 096‘0</td><td> 0,121</td><td> 0,016</td><td> 0,749</td><td> 0,018</td><td> 0,010</td><td> 0,024</td><td>or'</td><td> 10,709</td><td> 2,670</td><td> 5,859</td><td> 20,171</td><td>OR co or or'</td><td> 1,434</td><td> 14,028</td>

<td>15-17 dpa</td><td> 2,138</td><td> 0,735</td><td> 0,632</td><td> 0,561</td><td> 0,732</td><td> 0,297</td><td> 0,715</td><td> 0,163</td><td> 0,013</td><td> 0,838</td><td> 0,001</td><td> 0,009</td><td> 0,023</td><td> 0,131</td><td> 7,782</td><td> 2,079</td><td> 4,398</td><td> 15,856</td><td> 0,039</td><td> 0,892</td><td>OR co co • sf</td>

<td>12-14 dpa</td><td> 2,034</td><td> 0,870</td><td> 0,511</td><td> 0,389</td><td> 0,580</td><td> 0,362</td><td> 1,166</td><td> 0,132</td><td> 0,021</td><td> 0,870</td><td> 0,017</td><td> 0,009</td><td> 0,014</td><td> 0,156</td><td> 8,460</td><td> 1,736</td><td> 3,474</td><td> 16,012</td><td> 0,041</td><td> 0,555</td><td> 3,455</td>

<td>0-1 dpa</td><td> 0,049</td><td> 0,040</td><td> 0,070</td><td> 0,719</td><td> 0,075</td><td> 0,055</td><td> 086‘0</td><td> 0,163</td><td> 0,035</td><td> 1,631</td><td> 0,001</td><td> 0,009</td><td> 0,016</td><td> 0,114</td><td> 2,247</td><td> 0,403</td><td> 2,555</td><td> 0,282</td><td> 0,002</td><td> 0,011</td><td> 0,196</td>

<td>-DPA *</td><td> 0,053*</td><td> 0,025</td><td> 0,082</td><td> 1,313</td><td> 0,093</td><td> 0,074</td><td> 0,276</td><td> 0,148</td><td> 0,074</td><td> 2,989</td><td> 0,022</td><td> 0,010</td><td> 0,016</td><td> 0,056</td><td> 1,406</td><td> 0,095</td><td> 2,971</td><td> 1,727</td><td>OOO'O</td><td> 0,005</td><td> 0,161</td>

<td>Gen ID / SEQ ID NO.</td><td>CT1 / 1</td><td>CT2 / 2</td><td>CT3 / 3</td><td>CT4 / 4</td><td>S / 9 ± O</td><td>CO 1- OR</td><td>CT9 / 7</td><td>00 1- OR</td><td>CT20 / 9</td><td>OR CJ CN 1— or</td><td>CT26 / 11</td><td>CT27 / 12</td><td>CO δ Ν ' 1- OR</td><td>CT49 / 17</td><td>CT70 / 18</td><td>CT71 / 19</td><td>CT74 / 20</td><td>CT75 / 21</td><td>CT76 / 22</td><td>CO CN Γ-1— OR</td><td>CT81 / 24</td>

ES 2 635 546 T3

CT82 / 25 0.024 0.022 0.005 0.004 0.006 0.018 0.016 0.014 0.011 0.053 0.034 0.017 0.045 0.036 0.004 - 0.000

CT84 / 27 0.007 0.005 0.136 0.167 0.371 0.004 0.014 0.027 0.031 0.036 0.346 0.034 0.196 0.101 0.061 0.071 0.035

CT88 / 13 0.002 0.371 0.841 2.978 3.045 4.947 14.725 17.514 28.290 0.001 0.034 0.005 0.000 0.005 0.004 0.007 ω

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ES 2 635 546 T3

Two main criteria were used to select cotton genes as candidates that may be involved in fiber development based on their RNA profile. Genes showing a high degree of fiber expression specificity and genes showing expression levels that change concomitantly with fiber development (Table 3, below).

Twenty-three genes meet these selection criteria:

CT-1 (SEQ ID NOs. 1 and 106), CT_2 (SEQ ID NOs. 2 and 107), CT_3 (SEQ ID NOs. 3 and 108), CT_4 (SEQ ID NOs. 4 and 109) CT_6 (SEQ ID NOs. 5 and 110), CT_7 (SEQ ID NOs. 6 and 111), CT_9 (SEQ ID NOs. 7 and 112), CT_11 (SEQ ID NOs. 8 and 113), CT_20 (SEQ ID NOs. 9 and 114), CT_22 (10 and 115), CT_26 (SEQ ID NOs. 11 and 116), CT_27 (SEQ ID NOs. 12 and 117), CT_40 (SEQ ID NOs. 16 and 118), CT_49 (SEQ ID NOs. 17 and 119) , CT_70 (SEQ ID NOs. 18 and 120), CT_71 (SEQ ID NOs. 19 and 121), CT_74 (SEQ ID NOs. 20 and 122), CT_75 (SEQ ID NOs. twenty-one and 123), CT_76 (SEQ ID NOs. 22 and 124), CT_77 (SEQ ID NOs. 23 and 125), CT_81 (SEQ ID NOs. 24 and 126), CT_82 (SEQ ID NOs. 25 and 95), CT_84 ( SEQ ID NOs. 27 and 96) and CT_88 (SEQ ID NOs. 13 and 26).

CT-4, 22, 20, 27, 40, 82 (SEQ ID Nos: 4, 10, 9, 12, 16 and 25, respectively) were chosen mainly as candidate genes that may have a role in fiber initiation ( Table 3) while CT 27 (SEQ ID NO: 12), which is a homologous gene of GL3, was also used as a control (CT 22, SEQ ID NO: 10 is shown in Figure 2d).

CT-1, 2, 3, 6, 7, 9, 49, 70, 71, 74, 75, 76, 77, 81, 84 (SEQ ID Nos. 1, 2, 3, 5, 6, 7 , 17, 18, 19, 20, 21, 22, 23, 24 and 27, respectively, see Figures 2a, c) would be involved in fiber elongation and quality (strength and fineness) according to their expression patterns (Table 3, CT is shown in Figure 2C).

CT11, 40, 74 and CT 26 (SEQ ID NOs. 8, 16, 20 and 11, respectively, see Figures 2a, b) which are homologous to

Arabidopsis glabrousl (GeneBank Accession No. AbO06078) are fiber-specific genes that showed uniform and fiber-specific expression during all stages of fiber development (Table 3, CT 11 is shown as an example in Figure 2B) . The expression profiles of all the genes chosen are shown in Table 2, above.

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Table 3

<td>Biological Process</td><td>Carbohydrate metabolism</td><td>A stranger</td><td>TRNA processing</td><td>A stranger</td><td>A stranger</td><td>A stranger</td><td>Proteolysis and peptidolysis</td><td>Water deprivation</td><td>Desiccation</td><td></td><td>Amino acid phosphorylation of protein</td><td></td><td>wall organization and biogenesis mobile</td><td>amino acid family biosynthesis aromatic</td><td>electron transport</td><td>regulation of transcription</td><td>a stranger</td>

<td>Fiber Specific</td><td>w</td><td></td><td>w</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>w</td><td>w</td><td></td><td></td><td>w</td><td></td><td></td>

<td>Stable and Specific Fiber Expression</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Quality &amp; Fiber Elongation</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td></td><td></td><td></td>

<td>Initiation</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &gt;</td><td> &gt;</td><td> &gt;</td>

<td>Gene annotation</td><td>Sucrose-6-phosphate hydrolase sour</td><td>Supposed acyltransferase</td><td>Hypothetical protein</td><td>Hypothetical protein</td><td>Lipase / hydrolase-like protein with GDSL motif</td><td>Supposed mitochondrial protein</td><td>Aspartyl protease</td><td>Cysteine ​​protease</td><td>Dehydration response protein</td><td>Putative lipid transfer protein</td><td>Putative receptor kinase</td><td>Hypothetical protein</td><td>APETAL2-like protein</td><td>Hypothetical protein</td><td>or IT Q_ OR AND or or or or or Q. (or or I heard</td><td>MYB-related homologous protein</td><td>Hypothetical protein</td>

<td>CT #</td><td>CT_2</td><td>L ± 0</td><td> 6 ±0</td><td>CT_49</td><td>CT_1</td><td>CT_3</td><td> 9 ±0</td><td>CT_70</td><td>CT_71</td><td>CT_75</td><td>CT_76</td><td>LL ± 0</td><td>CT_81</td><td>CT_84</td><td>CT_4</td><td>CT_20</td><td>CT_22</td>

ES 2 635 546 T3

<td>regulation of transcription</td><td>regulation of transcription</td><td>regulation of transcription</td><td>cell fate compromise</td><td>lipid transport</td><td>organization and biogenesis of the cell wall</td>

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

<td></td><td></td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt;</td>

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

<td> &gt;</td><td> &gt;</td><td></td><td></td><td></td><td></td>

<td>Factor-type protein bHLH transcription</td><td>MADS pritein type box</td><td>MADS box transcription factor agamous type</td><td>MYB-related homologous protein</td><td>Lipid Transfer Precursor Protein 3 (LTP 3)</td><td>Type transposon protein EN / SPM</td>

<td>CT_27</td><td>CT_82</td><td>CT_11</td><td>CT_26</td><td>CT_40</td><td>CT_74</td>

ES 2 635 546 T3

The selected genes were over-expressed in transgenic arabidopsis and tomato, using the constitutive 35S CaMV promoter (SEQ ID NO. 31). Transgenic plants were further evaluated for epidermal modifications, trichome density and length, and seed hair yield (as further described herein below).

Example 3

Gene expression analysis using publicly available mlcrochlps

Additional information on the expression of the selected genes (Example 2, above) was collected by statistical analysis of data from arabidopsis mlcrochlps. Essentially, the best homologues of the new candidate genes in arabidopsis were compared with a set of 77 experimental mlcrochlps from different Arabidopsis tissues (AtGenExpress databases, Principal Investigator for AFGN: Prof. Dr. Lutz Nover, Botanlsches Instituí, Molekulare Zellblologie, FB Biologle und Informatlk der JW Goethe Unlversltat Frankfurt; Blozentrum N200 3OG, Marle-Curle-Strasse 9, 60439 Frankfurt am Main, www.arabldopsls.ora / lnfo / expresslon / ATGenExpress.ispj.

Polynucleotide sequences that were highly expressed in elongated cells or inflorescence merls were selected for further analysis.

Table 4 below lists the tissues that exhibit the highest levels of expression.

Table 4

<td></td><td>Fabrics with high expression</td><td>&lt;change in times / specificity</td><td>Fiber related</td>

<td>CT_1</td><td>Seed, siliques</td><td> 10-20</td><td>Elongated cells</td>

<td>CT_11</td><td>carpels, flower, seed, siliques</td><td>Tissue specific</td><td>Flower specific</td>

<td>CT_2</td><td>root, seedling and sepals</td><td>Tissue specific</td><td>Elongated cells</td>

<td>CT_22</td><td>carpels, flower, Inflorescence, bud</td><td> 4-10</td><td>Inflorescence</td>

<td>CT_4</td><td>Petals, stamen</td><td> &gt;10</td><td>Elongated cells</td>

<td>CT49</td><td>siliques</td><td> &gt;2</td><td>Elongated cells</td>

<td>CT_7</td><td>carpels, flower, Inflorescence, petals, bud, siliques</td><td> 10-30</td><td>Inflorescence</td>

<td>CT_70</td><td>flower, root, stamen</td><td>Near tissue specific</td><td></td>

<td>CT_76</td><td>carpels, flower, Inflorescence, bud, siliques</td><td> &gt;2</td><td>Elongated Cells &amp; Inflorescence</td>

<td>CT_77</td><td>seeds, pollen, stamen, petals, sepals, siliques</td><td> 10-50</td><td>Elongated cells</td>

<td>CT_82</td><td>Inflorescence, shoot stem</td><td> 3-6</td><td>Inflorescence</td>

<td>CT_88</td><td>petals, stamen</td><td></td><td>Elongated cells</td>

Example 4

Establish a correlation between candidate gene expression and fiber length

In order to define the correlations between the RNA expression levels of the selected genes and the fiber length, fibers from 4 different cotton lines were analyzed. These fibers were selected which showed very good fiber quality and a high lint index (PIma types, from other species of cotton, this is G. barbadense) and different quality levels and lint indices from various lines of G. hirsutum: good quality

ES 2 635 546 T3 and high lint index (Acala type), medium lint index (Coker type) and poor quality and low lint index (Tamcot type).

Experimental procedures

RNA extraction.- Samples of fiber development stages, representing different fiber characteristics, were taken at 5, 10, 15 and 20 DPA and the RNA was extracted as described in Example 2.

Fiber evaluation.- The fiber length of the previous lines was measured using the fibrograph. The fibrograph system was used to calculate the length in terms of "Top Half Half" length (UHM). The Upper Half Mean (UHM) is the mean length of the longest half of the fiber distribution. The fibrograph measures length in span lengths at a given percentage point (www.cotton¡nc.com / Class¡f¡cat¡onofCotton /? Pq = 4 # Lenqth.)

Results

Four different lines of cotton were grown in Rehovot, Israel during the summer of 2004, and their fiber lengths were measured. The UHM values ​​of the fibers are summarized in Table 5, below:

Table 5

<td></td><td>Length (UHM)</td>

<td>Premium S5</td><td>1.40 ± 0 a</td>

<td>Acala</td><td>1.23 ± 0.01 b</td>

<td>Coker 310</td><td>1.18 ± 0.01 c</td>

<td>Tamcot</td><td>1.15 ± 0.02 c</td>

Five genes were tested for the correlation between gene expression and fiber length (presented for CT_76 in Figure 3). The results are summarized in Table 6 below:

Table 6

<td colspan="2" rowspan="3"></td><td colspan="7">Tissue Sampling Day (DPA)</td>

<td> 0</td><td colspan="2"> 5</td><td colspan="2"> 10</td><td colspan="2"> 15</td>

<td>Relative amounts of mRNA</td><td>Relative amounts of mRNA</td><td>Relative expression in relation to T0</td><td>Relative amounts of mRNA</td><td>Relative expression in relation to T0</td><td>Relative amounts of mRNA</td><td>Relative expression in relation to T0</td>

<td rowspan="3">CT_1</td><td>Tamcot</td><td> 0,75</td><td> 2,99</td><td> 4,0</td><td> 4,71</td><td></td><td></td><td></td>

<td>Coker 310</td><td> 0,51</td><td> 4,80</td><td> 9,3</td><td> 7,56</td><td></td><td></td><td></td>

<td>Acala</td><td> 0,64</td><td> 5,08</td><td> 7,9</td><td> 8,01</td><td></td><td></td><td></td>

<td rowspan="4">CT 2</td><td>Tamcot</td><td> 0,03</td><td> 0,19</td><td> 7,6</td><td> 8,17</td><td></td><td></td><td></td>

<td>Coker 310</td><td> 0,03</td><td> 0,35</td><td> 11,4</td><td> 15,04</td><td></td><td></td><td></td>

<td>Acala</td><td> 0,02</td><td> 0,36</td><td> 17,7</td><td> 15,28</td><td></td><td></td><td></td>

<td>Pima S5</td><td> 0,02</td><td> 0,41</td><td> 23,6</td><td> 17,58</td><td></td><td></td><td></td>

<td rowspan="4">CT 40</td><td>Tamcot</td><td> 0,28</td><td></td><td></td><td></td><td></td><td> 0,47</td><td> 1,67</td>

<td>Coker 310</td><td> 0,37</td><td></td><td></td><td></td><td></td><td> 0,46</td><td> 1,24</td>

<td>Acala</td><td> 0,30</td><td></td><td></td><td></td><td></td><td> 0,67</td><td> 2,25</td>

<td>Pima S5</td><td> 0,37</td><td></td><td></td><td></td><td></td><td> 1,03</td><td> 2,75</td>

ES 2 635 546 T3

<td rowspan="4">CT 76</td><td>Tamcot</td><td> 0,01</td><td> 0,03</td><td> 5,4</td><td> 0,01</td><td> 2,3</td><td> 0,00</td><td> 0,10</td>

<td>Coker 310</td><td> 0,01</td><td> 0,08</td><td> 8,9</td><td> 0,04</td><td> 5,1</td><td> 0,00</td><td> 0,10</td>

<td>Acala</td><td> 0,01</td><td> 0,12</td><td> 16,6</td><td> 0,06</td><td> 9,1</td><td> 0,00</td><td> 0,12</td>

<td>PIma S5</td><td> 0,01</td><td> 0,13</td><td> 122,4</td><td> 0,18</td><td> 177,9</td><td> 0,12</td><td> 99,51</td>

<td rowspan="3">CT_81</td><td>Tamcot</td><td> 0,50</td><td> 1,33</td><td> 2,68</td><td> 5,03</td><td> 10,15</td><td> 1,11</td><td> 2,24</td>

<td>Coker 310</td><td> 0,31</td><td> 2,64</td><td> 8,65</td><td> 4,51</td><td> 14,76</td><td> 0,84</td><td> 2,75</td>

<td>Acala</td><td> 0,49</td><td> 4,38</td><td> 8,98</td><td> 6,36</td><td> 13,05</td><td> 3,65</td><td> 7,49</td>

Reverse transcription followed by quantitative PCR using real time PCR was carried out in tissues of 0, 5, 10 and 15 DPA of cotton plants (G. hirsutum var Tamcot, Coker and Acala, and G. barbadense var PIma S5) . Relative amounts of mRNA and relative TO-related expression of each gene in all tissues examined are presented.

Example 5

Cloning of the selected genes in a binary vector under constitutive regulation and recombinant expression thereof

ORF Analysis.- The gene sequences of the present invention were analyzed by ORFs using Gene Runner software version 3.05 (Hastlng Software, Inc: www.generunner.com/). The ORFs of each gene were compared with the Genbank database, using Blast (www.ncbl.nlm.nlh.gov/BLAST/). The position of the ATG Initiation codon was determined, comparing with ORFs of higher homology. All sequences described herein were shown to have a full length ORF listed and Include the ATG Initiation codon listed.

Cloning into the expression vector pPL- To clone genes of the present invention, total RNAs were extracted from the various stages of fiber-producing cell development using Borate RNA Extraction

String of cotton fabric according to www.eeob.lastate.edu/facultv/WendelJ/rnaextractlon.html. Complement DNA (cDNA) molecules were produced from mRNA using the Reverse transcription (RT) enzyme M-MuLV (Roche) and the Τ-ιβΝΝ DNA primer, following the protocol provided by the manufacturer. The cDNA amplification of 19 genes was carried out, of the previous sequences, that is, the CT clones numbers 1,2, 3, 6, 7, 9, 11, 20, 22, 27, 40, 71, 74, 75, 76 , 81, 82, 84 and 88, by PCR using the PFU correcting DNA polymerase enzyme (Promega www.promega.com/pnotes/68/7381 07/7381 07.html) following the protocol provided by the manufacturer. Primers for each gene were designed to span the entire ORF. Additional endonuclease restriction sites were added to the 5 'end of each primer to facilitate further cloning of the CTs into the binary vector (pPI). Table 7 below lists the primers used for the cloning of each of the genes:

ES 2 635 546 T3 _Ω (O ω

oo

c 'O (Λ &lt;D ω

σ or

&lt;υ c

&lt;υ n

υ nj ΐΛ υ

¢ 13 υ

iB you u

fix you rj 'you cú

Table 7

<td colspan="2">or restriction &lt;ba</td><td rowspan="2">nj ε</td><td rowspan="2">Jl &lt;Ü X</td><td rowspan="2">1—1 Í0 ε tea</td><td rowspan="2">1—1 íC £ Cu</td><td rowspan="2">H &lt;Ü 1</td><td rowspan="2"> 1—1 &lt;0</td><td rowspan="2">ll i ω</td><td rowspan="2">1—1 V3</td><td rowspan="2">1—1 ε «</td><td rowspan="2">l · - » Π5 ε</td><td rowspan="2">b tt ε w</td><td rowspan="2">1—1 tC .Ώ X</td><td rowspan="2">M¹ &lt;0 ε</td><td rowspan="2">M Ü1</td><td rowspan="2">M tea</td><td rowspan="2">H V)</td><td rowspan="2">Fl (5 ε v&gt;</td><td rowspan="2">M ro ε w</td>

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

<td></td><td></td><td></td><td></td><td>Γ-</td><td></td><td> (—1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>re</td><td></td><td>EC</td><td></td><td>b</td><td></td><td></td><td></td><td></td><td></td><td></td><td>EU</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>re</td><td></td><td>X</td><td></td><td>X</td><td>re</td><td>i ± e</td><td></td><td></td><td></td><td></td><td>Ul</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>X</td><td></td><td>OR</td><td>on</td><td>CJ</td><td></td><td>'r</td><td></td><td>0Ί</td><td>Lil</td><td>EC</td><td>X</td><td>p-</td><td></td><td></td><td>re</td><td>m</td><td>r-.</td>

<td></td><td></td><td>CJ</td><td></td><td>b</td><td>re</td><td>CJ</td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>EU</td><td>t&gt;</td><td>Lfl</td><td>EC</td><td></td><td>IT</td><td>IT</td><td>IT</td>

<td></td><td></td><td>&lt;j</td><td></td><td>rt</td><td>X</td><td>H</td><td> 13</td><td>OR</td><td>r *</td><td>X</td><td>or</td><td>X</td><td> ( &gt;</td><td>X</td><td>a</td><td></td><td>X</td><td>X</td><td>X</td>

<td></td><td></td><td>c</td><td></td><td>OR</td><td>OR</td><td>rt</td><td>t3</td><td>c</td><td>&lt;ξρ</td><td>L?</td><td>Ε — l</td><td>Rh</td><td>OR</td><td>L</td><td>X</td><td>IT</td><td>OR</td><td>C</td><td>or</td>

<td></td><td></td><td>b</td><td></td><td>d</td><td> &lt;</td><td>υ</td><td>AND-·</td><td>Y</td><td>X</td><td>L</td><td>rt</td><td>b</td><td>rt</td><td>OR</td><td>OR</td><td>X</td><td>rt</td><td>rt</td><td>(D</td>

<td></td><td></td><td></td><td></td><td>b</td><td>or</td><td>or</td><td>OR</td><td>rt</td><td>t-&gt;</td><td>rt</td><td>CJ</td><td>H</td><td>IT</td><td>b</td><td>LJ</td><td>tD</td><td>(D</td><td>b</td><td>rt</td>

<td></td><td></td><td>rt</td><td>ιΛ</td><td> &lt;5</td><td>rt</td><td>t- «</td><td>rt</td><td>b</td><td>t_&gt;</td><td>F-</td><td>CD</td><td>t)</td><td>LT</td><td>to</td><td>or</td><td>rt</td><td>id</td><td>&lt;D</td><td>f 1</td>

<td></td><td></td><td>or</td><td>re</td><td> &lt;</td><td>rt</td><td>b</td><td>Y</td><td>or</td><td>Ei</td><td>H</td><td>CD</td><td>OR</td><td>or</td><td> &lt;0</td><td rowspan="2">Cl</td><td>rt</td><td>TD</td><td rowspan="2">OR rt</td><td>b</td>

<td></td><td></td><td>LL »</td><td>X</td><td>rt</td><td>rt</td><td>OR</td><td>rt</td><td>you</td><td>rt</td><td>He</td><td>Hey</td><td>rt</td><td>rt</td><td>rt</td><td>í 1</td><td>Id</td><td>b</td>

<td>or</td><td></td><td>OR</td><td>OR</td><td>or</td><td>rt</td><td>rt</td><td>L&gt;</td><td>OR</td><td>or</td><td>rt!</td><td>gave</td><td rowspan="2">3 b</td><td>b</td><td>b</td><td>rf</td><td>b</td><td>b</td><td>rt</td><td>t&gt;</td>

<td>H</td><td></td><td></td><td> £</td><td>b b</td><td>or c)</td><td>Ei OR</td><td>OR rt</td><td>OR H</td><td>H Y</td><td>L&gt;</td><td>or rt</td><td>b CJ</td><td>b r</td><td> 5</td><td>OR b</td><td>s</td><td>b rt</td><td>OR b</td>

<td>Q</td><td></td><td>IL?</td><td>rt</td><td>H</td><td>L'J</td><td>&lt;J</td><td>t6</td><td> &lt;</td><td>rt</td><td> &lt;3</td><td>Γ3</td><td>OR</td><td>rt</td><td>fD</td><td>ΓΪ</td><td>b</td><td>b</td><td>CD</td><td>t * l</td>

<td></td><td></td><td>b</td><td>or</td><td>OR</td><td>or</td><td>Y</td><td>rt</td><td></td><td>H</td><td>rt</td><td>AND-</td><td>d</td><td>b</td><td></td><td>F)</td><td>í</td><td>rt</td><td>Cl</td><td>b</td>

<td>OR</td><td></td><td>rt</td><td>Li</td><td>rt</td><td>fc- »</td><td>rt</td><td>rt</td><td>rt</td><td>H</td><td>or</td><td></td><td>t</td><td>HEY</td><td>b</td><td>lt</td><td rowspan="2"> 3</td><td>CU</td><td>CD</td><td>Fi</td>

<td>LU</td><td></td><td>rt</td><td>rt</td><td>CJ</td><td>rt</td><td>b</td><td>rt</td><td>OR</td><td>OR</td><td></td><td>H</td><td>TO</td><td>b</td><td>b</td><td>rt</td><td>or</td><td>LD</td><td>L5</td>

<td rowspan="2">W</td><td></td><td>rt</td><td>rt</td><td>rt</td><td>or</td><td>Ly</td><td>or</td><td>or</td><td>H</td><td></td><td>b</td><td>L&gt;</td><td>b</td><td>OR</td><td>C&gt;</td><td>b</td><td>rt</td><td>b</td><td>Cl</td>

<td></td><td>t_5</td><td>or</td><td>b</td><td>H</td><td> &lt;</td><td></td><td>H</td><td>rt</td><td>CJ</td><td>F</td><td rowspan="2">OR rt</td><td>H</td><td>b</td><td>or</td><td>b</td><td>rt</td><td>rt</td><td>b</td>

<td></td><td></td><td>b</td><td>d</td><td>b</td><td>b</td><td>or</td><td>or</td><td>OR</td><td>or</td><td>Cl</td><td>&lt;j</td><td>rt</td><td>LD</td><td>F</td><td>tD</td><td>b</td><td>P?</td><td>&lt;D</td>

<td>OR (fl</td><td></td><td>rt</td><td>b</td><td>n;</td><td>rt</td><td>rt</td><td>rt</td><td>br</td><td>or</td><td>Cl</td><td>rt</td><td> 3</td><td>OR</td><td>LD</td><td>b</td><td>b</td><td>b</td><td>t)</td><td>í I</td>

<td></td><td></td><td></td><td>OR</td><td>or</td><td>H</td><td>Li</td><td>or</td><td>H</td><td>H</td><td>(Ί</td><td>ΓΪ</td><td>or</td><td>LU</td><td>¿R</td><td></td><td>b</td><td>f I</td><td>CJ</td><td>b</td>

<td>ω</td><td></td><td>OR</td><td>Y</td><td>b</td><td>rt</td><td>d</td><td>rt</td><td>H</td><td>OR</td><td>CJ</td><td>t3</td><td>b</td><td>rt</td><td>rf</td><td>rf</td><td>b</td><td>TD</td><td>rt</td><td>rt</td>

<td> &gt;</td><td></td><td>or</td><td>rt</td><td>OR</td><td>(j</td><td>H</td><td>rt</td><td>OR</td><td>rt</td><td>AND-·</td><td>l 1</td><td>rt</td><td>t&gt;</td><td>rt</td><td></td><td>CJ</td><td>rt</td><td>b</td><td>b</td>

<td></td><td></td><td>b</td><td>CJ</td><td>b</td><td>b</td><td>rt</td><td>or</td><td>H</td><td>or</td><td>OR</td><td>AND-"</td><td>V</td><td>Cl</td><td>or</td><td>or</td><td>or</td><td>OR</td><td>or</td><td>or</td>

<td>i—</td><td></td><td>L&gt;</td><td>b</td><td>or</td><td>or</td><td>b</td><td>Fh</td><td>Q</td><td>H</td><td>H</td><td>rt</td><td>b</td><td>b</td><td>b</td><td>b</td><td>b</td><td>b</td><td>b</td><td>b</td>

<td>or</td><td></td><td>LD</td><td> %</td><td></td><td>d</td><td>rt</td><td>OR</td><td>OR</td><td>or</td><td>OR</td><td>H</td><td>or</td><td>cl</td><td>t&gt;</td><td>rt</td><td>c?</td><td>CI</td><td>c?</td><td>r&gt;</td>

<td>CD</td><td></td><td>rt</td><td>fi</td><td> £</td><td>rt</td><td>or</td><td>and?</td><td>rt</td><td>or</td><td></td><td>rt</td><td>(D</td><td>CD</td><td>TD</td><td>b</td><td>CD</td><td>tea</td><td>tD</td><td rowspan="2">OR rt</td>

<td>_Q</td><td></td><td>U&gt;</td><td></td><td></td><td></td><td>d</td><td>rt</td><td>L?</td><td>rt</td><td>rt</td><td>OR</td><td></td><td>rt</td><td>rt</td><td>rt</td><td>rt</td><td>rt</td><td>rt</td>

<td>ω</td><td></td><td>or</td><td>rt</td><td>H</td><td>or</td><td>b</td><td>Cl</td><td>H</td><td>or</td><td>Í9</td><td>LD</td><td>to</td><td>rD</td><td>LD</td><td rowspan="2"> §</td><td rowspan="2">or rt</td><td>or</td><td>d</td><td>or</td>

<td rowspan="2">OR</td><td></td><td>or</td><td>d</td><td>OR</td><td>b</td><td>b</td><td>rt</td><td>rt</td><td>He</td><td>t_&gt;</td><td>HEY</td><td> &lt;_)</td><td>b</td><td>OR</td><td>í »</td><td>L&gt;</td><td>b</td>

<td></td><td>LD</td><td>b</td><td>d</td><td>d;</td><td>b</td><td></td><td></td><td>HEY</td><td>L</td><td>b</td><td>b</td><td>OR</td><td>rt</td><td>rt</td><td>rt</td><td>u&gt;</td><td>d!</td><td>OR</td>

<td></td><td></td><td>Cl</td><td></td><td></td><td></td><td></td><td>C4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>tea</td><td></td><td></td><td> 00</td><td></td><td>Tf</td><td></td><td></td><td></td><td>or</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>x</td><td>re</td><td>IT</td><td>EC</td><td>or</td><td>X</td><td></td><td></td><td>CO</td><td>iO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>tD</td><td>X</td><td>&lt;e</td><td>X</td><td>'tt</td><td>or</td><td></td><td></td><td> «*</td><td>X</td><td>CN</td><td></td><td></td><td> 00</td><td>OR</td><td></td><td></td><td></td>

<td></td><td></td><td>tD</td><td>or</td><td>X</td><td>Γ&gt;</td><td>X.</td><td>UJ</td><td>X</td><td></td><td>X</td><td>OR</td><td>EU</td><td></td><td>"or</td><td>ιΛ</td><td>IT</td><td></td><td>Ό</td><td>tO</td>

<td></td><td></td><td>rt</td><td>ci</td><td> &lt;3</td><td>d</td><td>OR</td><td>H</td><td> (3</td><td>'Ώ »</td><td> ¿ &gt;</td><td>OR</td><td>X</td><td></td><td>iO</td><td>X</td><td>X</td><td>ex</td><td>X</td><td>IT</td>

<td></td><td></td><td>rt</td><td>or</td><td>rt</td><td>b</td><td>and-</td><td>í&gt;</td><td>t?</td><td>X</td><td>or</td><td>b</td><td>tea</td><td></td><td>X</td><td>CD</td><td>or</td><td>IT</td><td>ω</td><td>X</td>

<td></td><td></td><td>Y</td><td>b</td><td>or</td><td>d</td><td> &lt;5</td><td> (3</td><td>H</td><td>or</td><td></td><td>OR</td><td>Cl</td><td> 4*</td><td>OR</td><td>LD</td><td>c_&gt;</td><td>X</td><td>b</td><td>CJ</td>

<td></td><td></td><td>rt</td><td>b</td><td>b</td><td>b</td><td>i 1</td><td>rt</td><td>rt</td><td>H</td><td>OR</td><td>LD</td><td>or</td><td>EU</td><td> 0</td><td>LD</td><td></td><td>tD</td><td>b</td><td>CJ</td>

<td></td><td></td><td>you</td><td> 3</td><td>b</td><td>OR</td><td>tea</td><td>or</td><td> 3</td><td>H</td><td>LD</td><td>H</td><td>b</td><td>X</td><td>tD</td><td>tD</td><td></td><td>or</td><td></td><td>b</td>

<td></td><td></td><td>or</td><td>rt</td><td>d!</td><td>H</td><td>e&gt;</td><td></td><td>or</td><td>Ei</td><td>rt</td><td>b</td><td>b</td><td>LD</td><td>rt</td><td></td><td>hey</td><td>rt</td><td>rf</td><td>r \</td>

<td></td><td></td><td>rt</td><td>or</td><td>or</td><td>b</td><td>rt</td><td>H</td><td>rt</td><td>OR</td><td>ID</td><td>OR</td><td>OR</td><td>rt</td><td>or</td><td>rt</td><td>you¹</td><td>b</td><td>rf</td><td>b</td>

<td>or</td><td></td><td>b</td><td>b</td><td>what</td><td>CJ</td><td>Cl</td><td>you</td><td>OR</td><td>OR</td><td>tD</td><td>Ei</td><td></td><td>rt</td><td>rt</td><td>rf</td><td rowspan="2">OR</td><td>rt</td><td>rf</td><td>fD</td>

<td></td><td></td><td>and</td><td>V</td><td>rt</td><td>d</td><td>d</td><td>rt</td><td></td><td>TV</td><td>fl</td><td>H</td><td>H</td><td>tD</td><td>or</td><td>rf</td><td>v</td><td>or</td><td>OR</td>

<td></td><td></td><td>b</td><td>LD</td><td>ld</td><td>LD</td><td>tea</td><td>TO</td><td>rt</td><td>H</td><td>H</td><td>rt</td><td>t ►</td><td>b</td><td>LD</td><td>L5</td><td>Do you¹</td><td>LD</td><td>Φ</td><td rowspan="2">í?</td>

<td>Q</td><td></td><td>b</td><td>rt</td><td>b</td><td>b</td><td>b</td><td>Ό</td><td>rt</td><td>k £</td><td>rt</td><td>rt</td><td>LD</td><td>rt</td><td>b</td><td>b</td><td>írf</td><td></td><td>or</td>

<td> —</td><td></td><td>rt</td><td rowspan="2">rt or</td><td>or</td><td>fC</td><td>rt</td><td> £-</td><td>(J</td><td>OR</td><td>OR</td><td>b</td><td>LD</td><td> 3</td><td>rt</td><td>rt</td><td> 3</td><td> 3</td><td>rt</td><td>rf</td>

<td>σ</td><td></td><td>b</td><td>rt</td><td>or</td><td>(X</td><td></td><td>H</td><td>L3</td><td>rt</td><td>OR</td><td>H</td><td>rt</td><td>LÍ</td><td>rn</td><td>b</td><td></td><td>d</td><td></td>

<td>LU</td><td></td><td>b</td><td>rt</td><td>rt</td><td>b</td><td>rt</td><td>rt</td><td>rt</td><td>H</td><td> (7</td><td>b</td><td>rt</td><td>b</td><td>rt</td><td>b</td><td>b</td><td>cT</td><td>rt</td><td>rt</td>

<td>or)</td><td></td><td>or</td><td>or</td><td>d</td><td>d</td><td>or</td><td>b</td><td></td><td></td><td>rt</td><td>rt</td><td>OR</td><td>c</td><td>CD</td><td>rt</td><td rowspan="2"> §</td><td>rf</td><td>CD</td><td>CD</td>

<td></td><td></td><td>Cf</td><td>rt</td><td>ci</td><td>He</td><td>t * j</td><td>HEY</td><td>H</td><td>rt</td><td>LD</td><td>LD</td><td></td><td>b</td><td>rt</td><td>LD</td><td></td><td>b</td><td>b</td>

<td></td><td></td><td>b</td><td>b</td><td>b</td><td>H</td><td>rt</td><td>rt</td><td> [»&gt;</td><td>Η</td><td>rt</td><td>b</td><td>rt</td><td>b</td><td>LJ</td><td></td><td>rt</td><td>b</td><td>^c?</td><td></td>

<td></td><td></td><td>rt</td><td>(I</td><td>*OR</td><td>OR</td><td>rt</td><td>&lt;j</td><td></td><td>CJ</td><td>b</td><td>Ci</td><td> 3</td><td>tD</td><td>rt</td><td>rt</td><td>r&gt;</td><td>tD</td><td></td><td>OR</td>

<td>OR</td><td></td><td>or</td><td>rt</td><td>LÜ</td><td>fcH</td><td>you</td><td>tj</td><td>rt</td><td>OR</td><td>rt</td><td>b</td><td>or</td><td>b</td><td>b</td><td></td><td>IM</td><td>b</td><td>b</td><td>or</td>

<td></td><td></td><td>OR</td><td>rt</td><td>b</td><td>and-</td><td>OR</td><td>rt</td><td>t&gt;</td><td>ΐ&gt;</td><td>t'j</td><td>rt</td><td>rt</td><td>í i</td><td>rt</td><td>LD</td><td>or</td><td>or</td><td>rt</td><td>H</td>

<td>-Q</td><td></td><td>b</td><td>d</td><td>Cl</td><td> (5</td><td></td><td>P</td><td>OR</td><td>Hey</td><td>rt</td><td>f&gt;</td><td>b</td><td>t 1</td><td>or</td><td>rt</td><td></td><td>rt</td><td> 3</td><td>b</td>

<td></td><td></td><td>rt</td><td>rt</td><td>LD</td><td>tn</td><td></td><td></td><td>OR</td><td>or</td><td>ID</td><td>L'J</td><td>LD</td><td>b</td><td>or</td><td>LD</td><td>fb</td><td>(D</td><td>OR</td><td>OR</td>

<td>OR</td><td></td><td>ld</td><td>d</td><td>or</td><td>tr</td><td>or</td><td>W</td><td> 0</td><td>c</td><td>tD</td><td>CD</td><td>LD</td><td>OR</td><td>tD</td><td>tea</td><td>(X</td><td>TD</td><td>Id</td><td>Id</td>

<td>OR</td><td></td><td>1</td><td>rt</td><td>or</td><td>ci</td><td>CJ</td><td> &lt;¿&gt;</td><td>or</td><td>LD</td><td>t?</td><td>or</td><td> &lt;7</td><td>rt</td><td>tD</td><td>LD</td><td>Φ</td><td>or</td><td>L5</td><td>LD</td>

<td> (0</td><td></td><td>t5</td><td>b</td><td>or</td><td>υ</td><td>or</td><td>OR</td><td> (1</td><td>i J</td><td> 11</td><td>(í</td><td>OR</td><td>Q</td><td>Cl</td><td>OR</td><td>c</td><td>c?</td><td>OR</td><td>OR</td>

<td>_Ω</td><td></td><td>or</td><td>or</td><td>Cl</td><td>or</td><td>rt</td><td>c</td><td>or</td><td>CJ</td><td>CJ</td><td>Y</td><td>or</td><td>b</td><td>you</td><td>c?</td><td>or</td><td>c?</td><td>(J</td><td>OR</td>

<td rowspan="2">OR</td><td></td><td>Cl</td><td>b</td><td>or</td><td>b</td><td>or</td><td>Y</td><td>or</td><td>or</td><td>tj</td><td>or</td><td>or</td><td>LD</td><td>you</td><td>t 1</td><td rowspan="2">Cl rt</td><td>YOU</td><td>il</td><td>í 1</td>

<td></td><td>or</td><td> 5</td><td>rt</td><td>HEY</td><td rowspan="2">or</td><td>rt</td><td>or</td><td>or</td><td>LD</td><td>or</td><td>b</td><td>rt</td><td>rt</td><td>OR</td><td>rt</td><td>b</td><td>b</td>

<td></td><td></td><td>rt</td><td>rt</td><td>ci</td><td>b</td><td>He</td><td>rt</td><td>or</td><td>d.</td><td>b</td><td>b</td><td>b</td><td>b</td><td>rt</td><td>rt</td><td>LD</td><td>rt</td><td>rt</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>«-H</td><td>Q</td><td>N</td><td>(x</td><td>or</td><td> 1</td><td>-'T</td><td>YOU</td><td>LP</td><td></td><td>IN</td><td> 4*</td>

<td>ω</td><td></td><td>i — 1</td><td>OJ</td><td>EC</td><td>IT</td><td></td><td></td><td> 1-4</td><td>(N</td><td>N</td><td></td><td>'T</td><td></td><td>r-</td><td>Γ *</td><td>Γ *</td><td> &lt;□</td><td>CD</td><td>CD</td>

<td colspan="2">or 1—</td><td>b</td><td>He</td><td>E-I</td><td>He</td><td>b</td><td>b</td><td>AND-</td><td>AND-¹</td><td>b *</td><td>b ^</td><td>b¹</td><td>b '</td><td>b¹</td><td>b¹</td><td>b</td><td>b</td><td>b</td><td>1 b</td>

<td colspan="2">HO</td><td>OR</td><td>or</td><td>OR</td><td>OR</td><td>or</td><td>or</td><td>or</td><td>Cl</td><td>OR</td><td>or</td><td>OR</td><td>OR</td><td>OR</td><td>ci</td><td>or</td><td>υ</td><td>or</td><td>OR</td>

ES 2 635 546 T3

The resulting blunt PCR products were purified using the PCR Purification Kit (Qiagen, Germany), digested with the appropriate restriction endonucleases (Roche) and cloned into the binary vector pPI (Figure 4), while replacing the existing GUS marker gene. pPI is a modified version of pBI101.3 (Clontech, Accession No. U12640). pPI was constructed by inserting a synthetic poly (A) signal sequence, which came from the plasmid vector pGL3 Basic (Promega, Acc No. U47295, where the synthetic poly (A) signal sequence is located between base pairs 4658-4811 ), within the HindIII restriction site of pBI101.3 (while rebuilding the HindIII site, downstream of the poly (A) insert), to avoid the possibility of upstream Nos promoter revision effect. To replace the GUS gene with each of the CT genes in the binary vector pPI, pPI was digested with the appropriate restriction enzymes [the 5 'restriction enzyme is either SmaI or XbaI and the 3' restriction enzyme is either SacI or EcoRV (Roche- using the protocol provided by the manufacturer)]. The open binary vector was purified using the PCR Purification Kit (Qiagen, Germany). 5-75 ng of PCR product from each of the CT genes and 100 ng of the open pPI plasmid vector were ligated into 10 gl of ligation reaction volume using the enzyme T4 DNA ligase (Roche), following the protocol provided by the maker. The ligation products were introduced into E. coli cells.

Recombinant expression in bacteria.- 60 gl of competent cells of E. coli, strain DH5-a (approximately 10⁹ cells / ml), using 1 gl of electroporation ligation reaction mix, using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and the EC-2 electroporation program (Biorad). E. coli cells were grown in 0.8 ml of LB liquid medium at 37 ° C for 1 h and 0.2 ml of cell suspension were plated on LB-agar plates supplemented with the antibiotic kanamycin at 50 mg / l (Sigma ). The plates were then incubated at 37 ° C for 16 h. Bacterial colonies were grown and expression was confirmed by PCR amplification using primers that were designed to span the inserted sequence in the binary vector. The primers used for DNA amplification of the inserts in the binary vector pPI were:

5'-GGTGGCTCCTACAAATGCCATC-3 '(direct, SEQ ID NO. 70) and 5'-AAGTTGGGTAACGCCAGGGT-3' (reverse, SEQ ID NO. 71).

The PCR products were separated on 1.5% agarose gels and the sizes of the products were estimated by comparing with a DNA ladder (MBI Fermentas). The PCR products with the predicted sizes were sequenced using the same primers previously used for PCR amplification (See Table 7, above).

Additional primers, which were designed based on the sequence of each insert gene, were used to complete the sequencing of the full-length ORF insert.

Sequencing of the inserted sequence was performed to verify that the clones were introduced in the correct orientation, and to eliminate the possibility of sequence errors being included during PCR amplification. DNA sequences were determined using the ABI 377 sequencer (Amersham Biosciences Inc.).

The constitutive Cauliflower Virus Mossaic 35S promoter was cloned into each of the 19 pPI binary constructs harboring the CT genes.

The Cauliflower Mosaic Virus 35S promoter sequence, originating from vector pBI121 (Clontech, Accession No. AF485783), was cloned by digesting vector pBI121 with restriction endonucleases HindIII and BamHI (Roche) and ligated into the constructs binary, digested with the same enzymes (SEQ ID NO. 31).

Example 6

Transformation in Agrobacterium of binary plasmids that harbor the genes of interest and expression in Arabidopsis and tomato plants.

Each of the nineteen binary constructs, comprising the 35S promoter upstream of each of the CTs genes, were transformed into Arabidopsis or tomato plants through Agrobacterium tumefaciens transformation.

60 gl of Agrobacterium tumefaciens GV301 or LB4404 competent cells were transformed (approximately 10⁹ cells / ml) with 20 ng of binary plasmid via electroporation, using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program (Biorad).

Agrobacterium cells were grown in 0.8 ml of LB liquid medium at 28 ° C for 3 h and 0.2 ml of the cell suspension were plated on LB-agar plates supplemented with the antibiotics gentamicin at 50 mg / l (for strains of Agrobacterium GV301) or streptomycin at 300 mg / l (for Agrobacterium strain LB4404) and kanamycin at 50 mg / l (Sigma). The plates were then incubated at 28 ° C for 48 h. Agrobacterium colonies were grown and PCR amplification was performed in Agrobacterium cells, using primers that were designed to span the inserted sequence in the binary vector.

The primers used for PCR amplification were: 5'-GGTGGCTCCTACAAATGCCATC-3 '(direct, SEQ ID NO. 70) and 5'-AAGTTGGGTAACGCCAGGGT-3' (reverse, SEQ ID NO. 71).

ES 2 635 546 T3

The PCR products were separated on 1.5% agarose gels and the sizes of the products were determined by comparing with a DNA ladder (MBI Fermentas). The PCR products with the predicted sizes were sequenced using the primers that were used for PCR amplification. Sequencing of the inserted sequence was performed to verify that the correct clones were introduced into the Agrobacterium cells.

DNA sequencing was performed using the ABI 377 sequencer (Amersham Biosciences Inc.).

Plant transformation and cultivation:

Transformation of Arabidopsis thaliana plants with presumed cotton genes.- Arabidopsis thaliana Columbia plants (T0 plants) were transformed using the Floral Dip procedure described by Clough and Bent and by Desfeux et al., With minimal modifications. Briefly, T0 Plants were sown in 250 ml pots filled with the wet peat based growth mix. The pots were covered with aluminum foil and a plastic dome, kept at 4 ° C for 3-4 days, then uncovered and incubated in a growth chamber at 18-24 ° C under light / dark cycles of 8/16 h. The T0 plants were ready for transformation six days before anthesis. Single Agrobacterium colonies bearing the binary constructs were grown in LB medium supplemented with kanamycin (50 mg / l) and gentamicin (50 mg / l). The cultures were incubated at 28 ° C for 48 h under vigorous shaking and then centrifuged at 4,000 rpm for 5 min. The precipitates comprising Agrobacterium cells were resuspended in transformation medium containing Murashig-Skoog (Duchefa) of medium strength (2.15 g / l); benzylamino purine (sigma) 0.044 µΜ; 112 µ-g / l of Gambourg B5 vitamins (Sigma); 5% sucrose; and 0.2 ml / l of Silwet L-77 (OSI Specialists, CT) in double distilled water, at pH 5.7. Transformation of T0 plants was effected by inverting each plant in an Agrobacterium suspension, such that the above-ground plant tissue was submerged for 3-5 sec. Each T0 inoculated plant was immediately placed in a plastic tray, then covered with a clear plastic dome to maintain humidity and kept in the dark at room temperature for 18 h, to facilitate infection and transformation. The transformed (ie, transgenic) plants were then discovered and transferred to a greenhouse for recovery and maturation.

Transgenic T0 plants were grown in the greenhouse for 3-5 weeks until the siliques were brown and dry. The seeds were collected from the plants and kept at room temperature until sowing. To generate T1 transgenic plants harboring the genes, seeds collected from T0 transgenic plants were surface sterilized by immersing them in 70% ethanol for 1 minute, followed by immersion in 5% sodium hypochlorite and 0.05% newt for 5 minutes. . The surface sterilized seeds were thoroughly washed in sterile distilled water then placed in culture plates containing Murashig-Skoog (Duchefa) of medium strength; 2% sucrose; 0.8% plant agar; 50 mM kanamycin; and cerbenicillin (Duchefa) 200 mM. The culture plates were incubated at 4 ° C for 48 h, then transferred to a growth room at 25 ° C for an additional week of incubation. Vital Arabidopsis T1 plants were transferred to fresh culture plates for another week of incubation. After incubation, the T1 plants were removed from the culture plates and planted in growth mix contained in 250 ml pots. The transgenic plants were grown in a greenhouse to maturity.

Transformation of Micro-Tom tomato plants with presumed cotton genes.- The transformation and cultivation of tomato (Lycopersicon esculentum, var MicroTom) of transgenic plants was carried out according to Curtis et al. 1995, and Meissner et al. 2000.

Example 7

Transformed Arabidopsis Plant Growth and Phenotype Characterizations

Arabidopsis T1 plants were grown as described above and the phenotypes were characterized.

PCR analysis of transgenic plants.- T2 Arabidopsis seeds were sown directly in growth mix contained in 250 ml pots. Transgenic plants positive for kanamycin resistance on two week old leaves were monitored by PCR. The primers used for kanamycin PCR amplification were: 5'-CTATTCGGCTATGACTGGGC-3 '(forward, SEQ ID NO. 72) and 5'ATGTCCTGATAGCGGGTCCGC-3' (reverse, SEQ ID NO. 73).

Root operation.- In order to visualize the root operation, T2 seeds were surface sterilized by immersing them in 70% methanol for 1 minute, followed by immersion in 5% sodium hypochlorite and 0.05% triton during 5 minutes. The surface sterilized seeds were thoroughly washed in sterile distilled water and then placed in culture plates containing Murashig-Skoog (Duchefa) of medium strength; 2% sucrose; 0.8% plant agar; 50 mM kanamycin; and cerbenicillin (Duchefa) 200 mM. The culture plates were incubated at 4 ° C for 48 h then transferred to a growth room at 25 ° C until they reached the correct size for phenotypic characterization.

ES 2 635 546 T3 ω

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<td>T2 phenotype</td><td>Curved and narrow leaves, with long petioles, longer and denser roots (Figures 5a-c)</td><td>The rosette leaves and inflorescences are longer and larger compared to the control. The roots are longer and denser. The phenotype resembles the phenotype of Arabidopsis plants that overexpress expansin as characterized by Hyung-Taeg Cho and Daniel J. Cosgrove in PNAS or August 15, 2000. (Figures 5g-i)</td><td>Irritating and hairy small leaves (Figures 5d and e)</td><td>Longer and curved leaves (Figure 5j)</td><td>Narrower leaves, with long petioles (Figures 5d and f)</td><td>The rosette leaves are almost double than those of the type savage (Figures 5k and l)</td><td>Narrow leaves, with long petioles (same as CT_22, not shown)</td>

<td>No. of floors independent</td><td>IT</td><td>IT</td><td> -</td><td>IT</td><td></td><td> -</td><td>CO</td>

<td>Generation of T</td><td>CM</td><td>CM</td><td> -</td><td>CM</td><td></td><td> -</td><td> -</td>

<td>Assumed gene function</td><td>MADS box transcription factor agamous type</td><td>Hypothetical protein</td><td>MYB-related protein</td><td>Lipid transfer protein 3</td><td>Hypothetical protein</td><td>APETAL2-type protein</td><td>Hydrolase-type protein</td>

<td>CT</td><td>CT_11</td><td> 6 10</td><td>CT_20</td><td>CT_40</td><td>CT_22</td><td>CT_81</td><td>CT_1</td>

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Example 8

MicroTom Transformed Plant Growth and Phenotype Characterizations Experimental Procedures

Transgenic tomato plants.- Plants were transformed as described in Example 6, above. 5 After transformation, MicroTom T1 tomato plants were grown in mixture contained in pots of

1000 ml.

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Results

Table 9 - Analyzing Micro-Tom T1 and T2 tomato plants and seeds that take care of the putative cotton genes

<td>T2 phenotype</td><td>Small, wrinkled leaves, the trichomes on the leaves are longer and denser (Figures 6a-b)</td><td>Large inflorescence</td><td></td><td>Normal plants</td>

<td>Seed hair length T1 (wt 0.3mm)</td><td>0.366 ± 0.006 mm (Figures 6c-e)</td><td>0.347 ± 0.019 mm</td><td> 0,343±0,019</td><td>0.423 ± 0.013 mm (Figure 5f)</td>

<td>No. of floors independent</td><td>OR</td><td>CM</td><td> -</td><td>CO</td>

<td>Generation of T</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>Assumed gene function</td><td>Homologous protein related to MY B</td><td>Putative lipid transfer protein</td><td>Aspartyl protease</td><td>MADS Protein Type Box</td>

<td>CT</td><td>or CM 1- OR</td><td>CT75</td><td> 9 10</td><td>CT_82</td>

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Discussion (Examples 1-8)

In siiico identification of genes involved in cotton fiber development. Little is known about the genetic control of cotton fiber initiation and elongation. Since both cotton fiber and Arabidopsis trichomes develop from single epidermal cells it is assumed that they share similar genetic regulation (reviewed in Wagner GJ et al. 2004). In Arabidopsis, a large number of studies have revealed extensive information on the genetic mechanisms that regulate the initiation and lengthening of the trichome. Several studies demonstrated the similarities between trichome and fiber by showing that cotton fiber-specific promoters in arabidopsis and tobacco plants confer trichome-specific expression (Kim and Triplett, 2001; Hsu et al. 1999; Liu et. Al. 2000 , Wang et al. 2004). Most of the research studying fiber development uses the arabidopsis trichome as a model system to identify cotton genes on a small scale (Kim and Triplett, 2001; Wang et al. 2004).

In this study the present inventors have used EST libraries of tomato flower and trichome as model systems to study cotton fiber development. Profile analysis of the EST libraries of tomato homologous clusters with known arabidopsis trichome genes showed that the trichome and tomato flower EST libraries contributed significantly to this set of clusters.

This result was confirmed while profiling the EST libraries of the new cotton clusters that were selected for their pattern of RNA expression as cotton fiber genes. 9 and 10 clusters contained ESTs that came from flower and trichome libraries, respectively. In addition, the group of tomato trichome clusters (trichome ESTs / total ESTs&gt; 0.1) comprises a large part of the tomato genes that present a high degree of homology with cotton (-50%) despite the fact that their percentage in the total population is only -5%. This may indicate that both organs share common developmental processes. Although there is a large group of studies on the genetic control of the development of tomato fruit and trichome, there are no publications that use these organs as a source of genomic data to study the development of cotton fiber. All 23 cotton genes were compared with unique EST data produced separately from the developing embryo and seed suspensors of the Scarlet Runner bean (www.mcdb.ucla.edu/Research/Goldberg/ests/intro-index.htmi . All the sequences, except one, share high homologies with sequences from the suspensor, which is a maternal tissue. This result supports the in siiico results and identifies the role of these cotton clusters in fiber development, which also originated from maternal cells.

Identify cotton genes with a role in fiber development through RNA expression profile analysis.- The differentiation / initiation phase is represented by gene expression in or before anthesis. The elongation phase mainly in hirsutum cultivars is represented by a very rapid growth during 5 to 20 DPA. One pattern is represented by genes such as CT 1, 2, 3 expressed at their highest levels, slightly before and during the fiber expansion peak period at approximately 20 DPA. Another pattern of gene expression is shown by CT40, 11 or 70 having the same level of expression throughout the fiber development. Similarly, known genes encoding actin, endoxyglucan transferase, or Suc synthase also show invariant RNA levels throughout fiber development (Shimizu et al., 1997).

Since initiation occurs mainly before anthesis up to 1 DPA, it suggests that genes with a peak of expression during this time may play a role in fiber initiation. CT 4, 20, 22 and 11 have expression patterns that indicate their involvement in this stage.

A limitation of the current cotton EST database is the absence of ESTs that were extracted from the flower in the initiation stage (there is a library that was extracted from the ovary 1DPA but of poor quality) most of the ESTs were taken only later, between 6 and 20 DPA. This EST composition may explain why most of the chosen genes have an expression pattern that indicates their association with the elongation stage.

Role of selected genes in fiber development, possible mechanisms.- The 23 fiber-associated clusters can be classified into 6 functional categories according to their sequence homologies with known proteins and enzymes (Table 3, above). The classification was made according to the GO consortium (www.qeneontoloqy.orq / L) The largest group comprises unique sequences without homology to any known protein. The rest of the groupings were classified according to categories known to be associated with fiber development. Two genes (Table 3, above) were classified into a category of compromise of cell fate: a new gene that belongs to the MYB transcription factor and a cotton gene homologous to GL3 that are known to be involved in the development of trichome in arabidopsis. The expression pattern of both genes and the phenotype of the CT20 transgene both in T1 arabidopsis plants and in tomato support their involvement mainly in the initiation phase.

Cumulative tests link cotton MYB genes with fiber development (Suo. J. et. Al. 2003, Cerdoni. ML et. Al. 2003, Loguerico LL et al 1999). Overexpression of a number of genes that function in the same pathway related to the initiation phase can further induce initiation. Kirik et al. (2004) showed that over33

ES 2 635 546 T3 expressing two or three genes of the Initiation phase increase the number of trichomes and root hairs. Genes that relate to the Initiation phase can be used for fiber initiation uniformity in cottonseed, isolating more of the epidermal seed cells into fibers. Overexpression of these genes can be used in vegetative meristems such as stems and leaves as protection against insects (as shown in rapeseed, www.westerngrains.com/news/nr 050413.html) and ab¡ót¡ stresses. cos. However, there is no substantial evidence to prove the direct involvement of any MYB gene in fiber development.

Two other genes (Table 3, above) are transcription factors of the MYB and MADS BOX families. Many studies have demonstrated the role of these two families of transcription factors as homeotic genes with key roles in different developmental processes, among them are trichome and fiber morphogenesis (Suo. J. et. Al. 2003, Ferrado. S . et. al. 2004). Its role in the early stages of fiber development is also supported by its RNA expression pattern, which is induced before and during the day of anthesis. One gene (CT 2, Table 3, above) was classified into the starch and sucrose metabolism pathways. Recent work shows that another gene (SUS), which belongs to this pathway, is a limiting factor in both fiber initiation and development. CT_40, 75 were classified as transporting lipids whose RNA expression is highly induced during the early stage of fiber elongation linked to the fact that lipids are key components in fiber formation. Several genes (Table 3, above, CT_4, 70, 71) were classified either as genes involved in desiccation, abscisic acid-stimulated salinity response, or as genes involved in electron transfer. Of these, 3 genes (CT 7, 9 and 49) were selected per RNA expression pattern to be induced in the elongation stage. Several studies consider changes in proton and potassium pump mechanisms as a key factor in the rapid growth rate of fiber (Smart LB et. Al. 1998). Combine the overexpression of several genes that are related to fiber elongation as genes related to starch and sucrose metabolism that will increase cell wall formation, with lipid transport genes or genes related to desiccation that may influence cell pressure could result in longer fibers than over-expressing a single gene.

Example 9

Cloning and Analysis of Promoter Sequences Upstream of the Genes of the Present Invention

Differential gene expression in fiber fabrics versus other fabrics in cotton is the result of complicated gene regulation. The genomic regions upstream of the 23 selected genes were predicted to possess promoter activities that direct quantitatively and qualitatively unique gene expression in fiber cells. Precise gene expression, targeting fiber cells, is crucial for the development of cotton plants with improved fiber yield, without adversely affecting other plant tissues.

Experimental procedures

Promoter Sequence Cloning.- The upstream genomic sequence of CT2 and CT6 were cloned from cotton genomic DNA (Gossypium hirsutum L. var Acala), as follows. Total genomic DNA was extracted from tissues from the leaves of the 4-week-old planter of cultivated cotton plants (Gossypium hirsutum L. var Acala), using the DNA Extraction Kit (Dneasy plant minkit, Qiagen , Germany). Reverse PCR (IPCR), DNA digestion, auto-ligation, and PCR reaction on genomic DNA were performed, following the common protocol (www.pmci.unimelb.edu.au/core fac¡l¡t¡es / manual / mb390.aspí with the following modifications To avoid errors in the IPCR, the genomic sequence of the 5 'sequence of a relevant cDNA (ie, including introns) was first identified to produce Genomic Island (Gl). The desired region of genomic DNA was amplified by PCR using direct oligonucleotide primers designed based on the sequence of the cDNA cluster (for CT_2 and CT_6, the Gl sequences are, respectively, as stated in SEQ ID NOs. 74 and 75 for CT_2 and CT_6 The primers are as set forth in SEQ ID NOs. 14-15 (CT_2) and 101-102 CT_6). The PCR reaction was performed in a DNA thermal cycler, using common PCR protocols. For example:

92 ° C / 3 min -&gt; 31 x [94 ° C / 30 sec -&gt; 56 ° C / 30 sec -&gt; 72 ° C / 3 min] -&gt; 72 ° C / 10 min).

The PCR products were purified using the PCR purification kit (Qiagen) and the sequencing of the PCR products was performed, using the ABI 377 sequencer (Amersham Biosciences Inc.).

In some cases, a different technique [UP-PCR (Domínguez and López-Larrea 1994)] was used when IPCR results in poor amplification. The UP-PCR technique was used in order to amplify an unknown upstream region of known cluster sequences. Typically, the procedure involves four oligonucleotide primers: two sequence-specific primers (SPs, external and internal) (listed below), both with the same orientation of the 3 'end towards the unknown, but desired, 5' region of the gene, and two universal walking primers (WP28 5'-TTTTTTTTTTGTTTGTTGTGGGGGTGT (SEQ ID NO. 76 and sWP 5-TTTTTGTTTGTTGTGGG, SEQ ID NO. 77). Reactions were carried out using the following reaction mixtures: Sample Mix (SM) - Cotton species genomic DNA (30-40 ng), WP28 primers (20 pmol), and double distilled water were added to volume final 10 μΙ. Polymerase mix (PM) - dNTPs (Roche, Switzerland, 10 nmol each), Expand Long Template Enzyme mix (Roche, Switzerland, 1 U), 10x buffer supplemented with the enzyme and double distilled water were added to a volume 8 μΙ final.

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The SMs were placed in a thermal cycler (Biometra, USA), where they were subjected to a 1 minute amplification program at 90 ° C, kept (pause) at 80 ° C until PM was added, 30 sec at 15 ° C , 10 minutes at 25 ° C, 3 minutes at 68 ° C, they were kept at 90 ° C until external SP (2 µl of concentration 10 µΜ) was added. The process was followed by external PCR reaction for 30 seconds at 92 ° C, 10 seconds at 94 ° C, 30 seconds at 65.5 ° C, 3 minutes at 68 ° C, for 30 cycles followed by a final extension of 10 minutes. at 68 ° C.

The 5,000-25,000-fold diluted external PCR product was used as a template, and PCR amplification was performed using specific internal primers sWP and SP (30 pmol each), 1 U of Ex Taq (Takara), in a 50 µl reaction volume. The internal PCR reaction was subjected to an amplification program of 2 minutes at 92 ° C, followed by 30 seconds at 94 ° C, 30 seconds at 58 ° C, and 3 minutes at 72 ° C for 30 cycles and a final extension. 10 minutes at 72 ° C. The IPCR / Up-PCR products were purified (PCR Purification Kit, Qiagen, Germany) and sequenced (ABI 377 sequencer, Amersham Biosciences Inc.).

The primers for CT_2 were as follows (UP-PCR):

External primers:

sWP28- 5'-TTTTTTTTTTGTTTGTTGTGGGGGTGT-3 '(SEQ ID NO. 78)

SP (External) -5'-CTGGGGTTACTTGCTAATGG-3 '(SEQ ID NO: 79)

Internal (nested) primers:

sWP- 5'-TTTTTGTTTGTTGTGGG-3 '(SEQ ID NO: 80)

SP (Internal) - 5'-GCTCCGGGCTTTGGTTAACG-3 '(SEQ ID NO: 81)

The internal genomic sequence of CT_2 resulting from the above procedure is provided in SEQ ID NO: 14. The primers for CT_6 were as follows (UP-PCR):

External primers:

sWP28- 5'-TTTTTTTTTTGTTTGTTGTGGGGGTGT-3 '(SEQ ID NO. 78)

SP (External) - 5'-GGCTTTGGGATGTTTGAGGTGG-3 '(SEQ ID NO. 82)

Internal (nested) primers:

sWP- 5'-TTTTTGTTTGTTGTGGG-3 '(SEQ ID NO: 83)

SP (Internal) -5'-GGTGGTGGGCTCTTGCAACAG-3 '(SEQ ID NO: 84)

The internal genomic sequence of CT_2 resulting from the above procedure is provided in SEQ ID NO: 85.

To clone the promoters and putative 5 'UTRs, PCR amplification was carried out using a new set of primers (below) in which there was an 8-12 bp extension that included a restriction site (HindIII, SalI, XbaI, BamHI, or SmaI) at the 5 'end. For each promoter, restriction sites that did not exist in the promoter sequence were selected. Furthermore, the restriction sites in the primer sequences were designed so that the resulting PCR products were cloned into the binary vector pPI in the correct orientation, upstream of the GUS marker gene.

The plasmid pPI was constructed by inserting a synthetic poly (A) signal sequence, derived from the basic plasmid vector pGL3 (Promega, Acc. No. U47295; 4658-4811 bp) into the HindIII restriction site of the binary vector pBI101.3 ( Clontech, Accession No. U12640).

Below are the primers used for the amplification and cloning of the promoter and the 5 'UTR (P + U) within pPI, and the amplified and cloned sequence. Restriction sites within each primer are shown in bold letters:

CT_2:

Direct P + U (HindIII): 5'- ATTCAAGCTTTTTTTGTTTGTTGTGGGGG-3 '(SEQ ID NO: 86)

Reverse P + U (BamHI): 5'- TTGGATCCTTGGGCATTGAGCTTCTGTAC-3 '(SEQ ID NO: 87)

The P + U sequence of CT_2 is as set forth in SEQ ID NO: 88.

CT6:

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Direct P + U (HindIII): 5'- TTAAAGCTTTGGGCTCTTGCAACAGAGGC-3 '(SEQ ID NO: 89)

Reverse P + U (BamHI): 5'-AAGGATCCGACGACGACAACAACAACAAC-3 '(SEQ ID NO: 90)

The P + U sequence of CT_6 is as set forth in SEQ ID NO: 91.

Genomic DNA or the IPCR / UP-PCR product was used as template DNA for PCR amplification, using the newly designed oligonucleotide primers. The PCR products were purified (PCR Purification Kit, Qiagen, Germany) and digested with the restriction sites existing in the primers (Roche, Switzerland). The digested PCR products were re-purified and cloned into the binary vector pPI, which was digested with the same restriction enzymes. The PCR product and the open plasmid vector were ligated using the enzyme T4 DNA ligase (Roche, Switzerland).

Example 10

Transforming Agobacterium tumefaciens cells with binary vectors harboring cotton fiber promoters

The binary vector pPI, which includes either the CT2 or CT6 promoter, upstream of the GUS marker gene was used to transform Agrobacterium cells.

The binary vectors were introduced into competent Agrobacterium tumefaciens GV301 or LB4404 cells (approximately 10⁹ cells / ml) by electroporation. Electroporation was performed using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and the EC-2 electroporation program (Biorad). The treated cells were cultured in LB liquid medium at 28 ° C for 3 h, then plated on LB agar supplemented with gentamicin (50 mg / l; for Agrobacterium GV301 strains) or streptomycin (300 mg / l; for the strain of Agrobacterium LB4404) and kanamycin (50 mg / l) at 28 ° C for 48 h. Agrobacterium colonies that grew on the selective medium were analyzed by PCR using the primers set forth in SEQ ID NOs: 70-71, which were designed to span the sequence inserted into the plasmid pPI. The resulting PCR products were isolated and sequenced as described above in Example 4, to verify that the correct sequences were properly introduced into the Agrobacterium cells.

Example 11

Cotton fiber specific promoters are expressed in tomato leaves and tomato fruits

GUS staining was performed to illustrate specific expression in trichomes and tomato fruits.

Experimental procedures

Transformation of Micro-Tom tomato plants with the putative cotton promoter.- As described above.

Transformation of Arabidopsis thaliana plants with the putative cotton promoter.- As described above.

GUS staining of Arabidopsis.- GUS staining of Arabidopsis plants was carried out as previously described (Jefferson RA. Et. Al. 1987, Meissner et. Al. 2000).

GUS staining of tomato leaves. GUS staining of tomato plants was carried out as previously described (Jefferson RA. Et. Al. 1987, Meissnet et. Al. 2000).

Tissue fixation was effected in the following manner. Tomato leaves were immersed in ice cold 90% acetone, then incubated on ice for 15-20 minutes followed by removal of acetone. Thereafter the tissue was rinsed twice with the Working Solution [100 mM Sodium Phosphate buffer (Sigma. USA) pH = 7, Ferricianide (Sigma. USA) 5 mM, Ferrocyanide (Sigma. USA) 5 mM, EDTA ( BioLab) pH = 8 1 mM, Triton X-100 (Sigma. USA) 1%] for 15-20 minutes in the dark. The wash solution was then removed and replaced with X-gluc [Working Solution + 5-bromo-4-chloro-3-indolyl-p-D-glucuronic acid (X-GlcA, Duchefa) staining solution solubilized in N , N-dimethylformamide (BioLab) 0.75 mg / ml, Dithiothreitol (BioLab) 100 mM] and incubated overnight at 37 ° C in the dark (tubes wrapped with aluminum foil). Destinations were carried out by dipping the plant tissue in 70% ethanol and heating at 50 ° C ~ 120 minutes. The destining step was repeated until the plant tissue became transparent excluding the blue-stained regions. The bleached plants were stored in 70% ethanol (BioLab) at room temperature.

GAS staining of tomato fruits.- Gus staining of tomato fruits was carried out as previously described (Jefferson RA. Et. Al. 1987, Meissner et. Al. 2000). Briefly: Tomato fruit thin slices were dipped in staining solution [Sodium Phosphate buffer (Sigma. USA) 100 mM pH = 8, Ferricianide (Sigma. USA) 5 mM, Ferrocyanide (Sigma. USA) 5 mM, EDTA (BioLab) pH = 8 15 mM, Methanol (BioLab) 20%, 5-bromo-4-chloro-3-indolyl-pD-glucuronic acid (X-GlcA, Duchefa) solubilized in N, N-dimethylformamide (BioLab) 0.75

ES 2 635 546 T3 mg / ml] in the dark (tubes wrapped with aluminum foil) and incubated overnight at 37 ° C. Destinations were carried out by dipping the plant tissue in 70% ethanol and heating at 50 ° C ~ 20 minutes. The destining step was repeated until the fruit sheet became transparent except for the blue-stained regions. The bleached fruits were stored in 70% ethanol (BioLab) at room temperature.

Results

GUS staining was performed on T1 tomato plant seeds.

GUS was expressed under the regulation of CT2 and CT6, promoters in genetically transformed tomato plants (Figures 7a-b).

The results for the T1 tomato generation are summarized in Table 10, below.

Table 10

<td>Promoter</td><td>No. of floors T1 independent</td><td>Leaf</td><td>Leaf trichome</td><td>Young fruit seed cover</td><td>Ripe green seed cover</td><td>Ripe fruit seed cover</td>

<td>CT2</td><td>four</td><td> 0</td><td> 2</td><td> 3</td><td> 5</td><td> 3</td>

<td>CT6</td><td>one</td><td> 0</td><td> 1</td><td> 1</td><td> 2,5</td><td> 1</td>

Numbers represent average grade, 0-not expressed, 5-high expression

Example 12

Tomato seed hair as a model system for cotton fibers

Genetic modification of cotton is long and time consuming. Therefore to find genes that are capable of improving cotton fiber yield and quality, there is a need for a model system for the development of cotton fiber in other plants.

Trichome cells and root hair share common characteristics with cotton fiber cells, and are widely accepted as model systems for cotton fiber development [Reviewed in Wagner. GJ et. to the. (2004) and Wang et al. 2004].

However, measuring changes in growth rate, length and thickness as well as other structural parameters is not an easy task due to the small size, remote accessibility and lack of uniformity in size of the trichome cells.

To overcome these limitations, tomato seed hairs were analyzed for possible use as a model tissue for the development of cotton fiber. For this purpose, the GUS marker gene was overexpressed under the regulation of the cotton fiber specific promoter element derived from CT2, as described above.

Transformation of the primary construct, plant regeneration and GUS staining were carried out as described above.

Tomato seed hairs (Figure 8a) are maternal epidermal cells, which cover the surface of the seed ovule. In anatomical aspects, tomato seed hair is closer to cotton fibers than either trichome or root hair cells.

4 independent transgenic tomato fruits were produced that overexpress the GUS gene under the cotton-specific CT_2 promoter. GUS staining of fruits in the ripe-green stage (fruit in its size just before the ripening process) was observed only in the seed coat, where the seed hair is developing (Figures 7a and b).

Five independent transgenic tomato fruits were produced that overexpress 35S-expansin (AF043284), and the length of the seed hair was measured and compared with the wt. The seed hair of transgenic plants was significantly longer than that of the wt (Figures 8a and b).

Table 11

<td>Plant</td><td>Number of independent plants</td><td>Seed hair length (mm)</td>

<td>WT</td><td> 3</td><td> 0,300±0,019</td>

<td>35S: expansin</td><td> 5</td><td>0.357 ± 0.017 (Figure 8b)</td>

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