COTTON FIBERS WITH INCREASED GLUCOSAMINE CONTENT

Abstract

An isolated nucleic acid molecule is provided comprising a nucleotide sequence which encodes a glutamine:fructose-6-phosphate amidotransferase from E. coli which is particularly suitable for expression in cotton plant cells. The invention also relates to plant cells or plants, in particular to cotton plant cells or cotton plants which produce an increased amount of positively charged polysaccharides in their cell walls. Furthermore, methods and means are provided to increase the content of positively charged polysaccharides in the cell walls of cotton cells, in particular in cotton fibers. Fibers obtained from such cotton plants have an altered chemical reactivity which can be used to attach reactive dyes or other textile finish reagents to these fibers.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the modification of the chemical reactivity of cotton fibers. In particular, the present invention provides cotton fibers comprising positively charged oligosaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines. Due to the amino groups these fibers have a modified reactivity which can be exploited for attaching other substances to the fibers to alter their characteristics. Such substances can e.g. be reactive dyes or other reactants such as flame retardants, water, oil and soil repellents, anticrease agents, softeners, antistatic agents, fluorescent whitening agents etc.

[0002] The current invention provides methods and means to increase the efficiency of production of glucosamine oligomers in cotton plant cells such as fiber cells.

BACKGROUND OF THE INVENTION

[0003] Cotton fiber is the single most important textile worldwide. About 80 million acres of cotton are harvested annually across the globe. Cotton is the fifth largest crop in the U.S. in terms of acreage production, with an average of 10,3 million acres planted in the years 2006 to 2008. About 90% of cotton grown worldwide is Gossypium hirsutum L., whereas Gossypium barbadense accounts for about 8%.

[0004] However, like other natural cellulose containing fibers, cotton fibers do not possess the chemical versatility of synthetic fibers, due to the relative inert nature of the .beta.-1-4 linked glucose monomers in cellulose. This relative inert nature is e.g. apparent during the dyeing process of cotton fibers and fabrics.

[0005] Generally two types of dyes are used to color cotton: direct dyes and fiber-reactive dyes, which are both anionic molecules. Cotton itself develops an anionic charge in water, so that without special treatment, the uptake of dye by the fiber or fabric is quite elaborate. Direct dyes create a relatively weak hydrogen bond with the cellulose polymer forming a semi-permanent attachment. Direct dyes are easier to use and less expensive than fiber-reactive dyes, but do not withstand well washing. Fiber-reactive dyes are molecules that combine chromophores with a reactive group that forms strong covalent bonds with the fiber via reaction with hydroxyl groups. The covalent bonds provide a good resistance of the dyed fiber against laundring. Colorfastness can be improved using cationic fixatives.

[0006] During the dyeing process using reactive dyes, large amounts of electrolytes are needed to shield the anionic dyes from the anionic fiber charges. Unreacted dyes (up to 40%) need to be removed by a washing step, generating large volumes of wastewater, also containing the above mentioned electrolytes.

[0007] Providing the cellulose fiber with a positive electric charge, e.g. by incorporation of positively charged chemical compounds such as positively charged polysaccharides, could therefore improve the dyeability of natural cellulose fibers, as well as improve any chemical reaction of the modified cellulose fiber with negatively charged chemical compounds. It would also make the use of acidic dyes possible.

[0008] Several publications have described the incorporation into or coating of chitosan oligomers into cellulose fibers to make chitosan/cellulose blends, yarns or fabrics. Chitosan is a positively charged polymer of glucosamine, which can be obtained by deacetylation of chitin, e.g. by alkalic treatments. Chitin itself is a polymer of .beta.-1-4 linked N-acetylglucosamine (GlcNAc). Based on the physiological function of chitosan in inhibiting e.g. dermatophytes, many functional clothes, fabrics and fibers employ cellulose-chitosan blend fibers, cellulose fiber-chitosan conjugates and fabrics coated with chitosan-containing resins.

[0009] US patent application US2003/0134120 describes the coating of natural fibers with chitosan.

[0010] Liu et al. (Carbohydrate Polymers 44(2003) 233-238) describe a method for coating cotton fibers with chitosan, by oxidation of the cotton thread with potassium periodate at 60.degree. C. in water and subsequent treatment with a solution of chitosan in aqueous acetic acid. With the chitosan coating, the cotton fiber surface became physiologically and biologically active. Since the chemical reactivity of the amino group is greater than the hydroxyl group of cellulose monomers, the fiber has more potential for further chemical modification. Moreover, the smooth surface of the cotton fiber became coarse, suggesting a greater potential for drug absorption and controlled release thereof.

[0011] WO2006/136351 provides methods and means for the modification of the reactivity of plant cell walls, particularly as they can be found in natural fibers of fiber producing plants by inclusion of positively charged oligosaccharides or polysaccharides into the cell wall. This can be conveniently achieved by expressing a chimeric gene encoding an N-acetylglucosamine transferase, particularly an N-acetylglucosamine transferase capable of being targeted to the membranes of the Golgi apparatus in cells of a plant. One of the applications is increased dyeability.

[0012] WO2011/089021 provides methods and means for the modification of the reactivity of plant secondary cell walls, particularly in cotton cell walls found in cotton fibers. This can be conveniently achieved by expressing a chimeric gene encoding a Saprolegnia monoica chitin synthase in cotton plants.

[0013] WO2012/048807 provides alternative methods and means to produce positively charged oligosaccharides in the plant cell wall by introducing into said plant cell a Nodulation C (NOD C) protein fused to a heterologous Golgi signal anchor sequence.

[0014] The polysaccharide chitin is built from N-acetylglucosamine residues. It is synthesized from UDP-N-acetylglucosamine which is the end-product of the hexosamine biosynthesis pathway also active in plants (Mayer et al. 1968, Plant Physiol. 43, 1097-1107). The first and rate limiting step of this pathway is the conversion of glutamine to glucosamine-6-phosphate which is catalyzed by the enzyme glutamine:fructose-6-phosphate-amidotransferase (GFAT).

[0015] WO 2007/039314 describes transgenic plants having the activity of a hyaluronan synthase and additionally an increased glutamine:fructose-6-phosphate amidotransferase (GFAT) activity. These plants synthesize an increased amount of hyaluronan compared to plants having only the activity of a hyaluronan synthase. Like chitin, hyaluronan is synthesized from UDP-N-acetylglucosamine.

[0016] WO 2011/089021 discloses transgenic cotton plants comprising a chimeric chitin synthase gene and a chimeric glutamine:fructose-6-phosphate-amidotransferase gene under the control of a cotton selective promotor. Fibers from these transgenic cotton plants have an increased amount of N-acetylglucosamine polymers which are evenly distributed throughout the cell wall.

[0017] Yet there remains a need for improved methods and means to produce cotton fibers which comprise an increased level of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines. These and other problems are solved as described hereinafter in the summary, detailed embodiments, examples, drawings and claims.

SUMMARY OF THE INVENTION

[0018] The invention shows that the expression of a chimeric gene comprising

[0019] (a) a nucleotide sequence according to SEQ ID 1, or

[0020] (b) a variant thereof which differs from SEQ ID 1 in one or more nucleotides provided that in total it differs from SEQ ID 1 in no more than 20 nucleotides, which encodes a glutamine:fructose-6-phosphate-amidotransferase (GFAT) polypeptide according to SEQ ID 2, in plant cells such as cotton plant cells unexpectedly leads to an increase in the glucosamine content of the cells.

[0021] In a second embodiment the invention provides a chimeric gene comprising the following operably linked DNA regions:

[0022] (a) a plant-expressible promotor such as a fiber-preferential promotor,

[0023] (b) a DNA region coding for a GFAT polypeptide wherein said GFAT is encoded by a nucleotide sequence according to SEQ ID 1 or said variant thereof and

[0024] (c) optionally a DNA region involved in transcription termination and polyadenylation.

[0025] In another embodiment the invention provides a cotton plant cell comprising a chimeric gene comprising the following operably linked DNA regions:

[0026] (a) a plant-expressible promotor such as a fiber-preferential promotor,

[0027] (b) a DNA region coding for a GFAT polypeptide wherein said GFAT is encoded by a nucleotide sequence according to SEQ ID 1 or said variant thereof and

[0028] (c) optionally a DNA region involved in transcription termination and polyadenylation.

[0029] In some embodiments the invention provides a plant cell which in addition to said first chimeric gene comprises a second chimeric gene comprising the following operably linked DNA regions:

[0030] (a) a plant-expressible promotor such as a fiber-preferential promotor,

[0031] (b) a DNA sequence coding for a chitin synthase polypeptide and

[0032] (c) optionally a DNA region involved in transcription termination and polyadenylation.

[0033] In yet another embodiment the invention provides a cotton plant consisting of the plant cells as herein described.

[0034] The invention also provides fibers such as cotton fibers which can be obtained from the plant as herein described. Furthermore a yarn or a fabric made from the fibers is provided.

[0035] In another embodiment of the invention a method is provided to produce cotton fibers with positively charged polysaccharides, such as oligo-N-acetylglucosamines or oligo-glucosamines comprising the steps of

[0036] i) expressing a chimeric gene comprising a GFAT encoding nucleotide sequence according to the invention in a cotton plant cell,

[0037] ii) regenerating a cotton plant from cotton plant cells of step i) and

[0038] iii) optionally isolating fibers from said cotton plant.

[0039] In yet another embodiment of the invention, said method to produce cotton fibers with positively charged polysaccharides comprises the steps of

[0040] i) expressing a first chimeric gene comprising a GFAT-encoding nucleotide sequence as described herein before and a second chimeric gene comprising a nucleotide sequence which encodes a chitin synthase,

[0041] ii) regenerating a cotton plant from cotton plant cells of step i) and

[0042] iii) optionally isolating fibers from said cotton plant.

[0043] The invention further relates to the use of a nucleic acid molecule as herein described to produce a cotton plant with positively charged polysaccharides in the fibers.

[0044] The invention also relates to the use of a nucleic acid molecule as herein described to increase the amount of positively charged polysaccharides in cotton fibers.

DESCRIPTION OF FIGURES

[0045] FIG. 1: Nucleotide sequence of the synthetic nucleic acid molecule which encodes the glutamine:fructose-6-phosphate amidotransferase (GFAT) of E. coli according to SEQ ID 2 (SEQ ID 1).

[0046] FIG. 2: Amino acid sequence of the glutamine:fructose-6-phosphate amidotransferase (SEQ ID 2) of E. coli.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The current invention is based upon the unexpected finding that expression of a nucleotide sequence according to SEQ ID 1 which encodes a glutamine:fructose-6-phosphate-amidotransferase (GFAT) in plant cells, particularly in cotton plant cells of cotton plants leads to the production of an increased amount of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines in plant cells or fibers of such plants such as cotton fibers, compared to plant cells or fibers not comprising a GFAT protein or compared to plant cells expressing a GFAT encoding gene known in the art which is not optimized for expression in cotton plant cells.

[0048] This unexpected finding can also be achieved by expression of a variant of SEQ ID 1 in a plant cell, particularly in a cotton plant cell, which encodes a glutamine:fructose-6-phosphate-amidotransferase according to SEQ ID 2, wherein said variant differs from SEQ ID 1 in one or more nucleotides provided that in total it differs in no more than 20 nucleotides from SEQ ID 1.

[0049] Thus, in a first embodiment, the invention provides an isolated nucleic acid molecule comprising

[0050] i) a nucleotide sequence according to SEQ ID 1,

[0051] ii) or a variant thereof, wherein one or more nucleotides differ from the nucleotide sequence of SEQ ID 1, provided that said variant differs in no more than 20 nucleotides from SEQ ID 1, which encodes a glutamine:fructose-6-phosphate-amidotransferase (GFAT) according to SEQ ID 2

[0052] iii) or a complementary sequence of i) or ii).

[0053] SEQ ID 1 encodes a glutamine:fructose-6-phosphate-amidotransferase from E. coli. The corresponding amino acid sequence of the protein is described in SEQ ID 2. This enzyme catalyzes the conversion of fructose-6-phosphate and glutamine into glucosamine-6-phosphate and glutamate as a side product. It has been described in WO2007/039314 for the production of hyaluronan in plants. During the hexosamine pathway, glucosamine-6-phosphate is further converted to UDP-N-acetylglucosamine which in turn serves as starting material for the synthesis of glycosaminoglycans such as hyaluronan or chitin if the appropriate enzymes are present.

[0054] WO2007/039314 discloses a GFAT nucleotide sequence which was derived from the E. coli gene coding for GFAT but was adapted to the use of codons in plant cells. The nucleotide sequence disclosed as SEQ ID 1 in the current application varies from the nucleotide sequence described in WO2007/039314 by about 25%. While the sequence disclosed in WO2007/039314 was optimized for expression in plant cells in general, the expression of a chimeric gene comprising a nucleotide sequence according to SEQ ID 1 leads to particularly good results in cotton cells. Cotton cells comprising a plant-expressible nucleotide sequence according to SEQ ID 1 or a variant thereof which encodes a GFAT protein from E. coli according to SEQ ID 2 and which differs from SEQ ID 1 in one or more nucleotides provided that in total it does not differ in more than 20 nucleotides from SEQ ID 1, or cotton plants made up by such cotton plant cells, produce an increased amount of glucosamine compared to cotton cells expressing a nucleotide sequence as disclosed in WO2007/039314 or plants made up by such cotton cells (see experimental data).

[0055] As used herein "no more than 20 nucleotides difference from SEQ ID 1", means e.g. 20 nt, 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, 5 nt, 4 nt, 3 nt, 2 nt or 1 nt different from SEQ ID 1, while still encoding the glutamine:fructose-6-phosphate-amidotransferase (GFAT) according to SEQ ID 2.

[0056] Nucleic acids can be DNA or RNA, single- or double-stranded. Nucleic acids can be synthesized chemically or produced by biological expression in vitro or even in vivo. Nucleic acids can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. In connection with the chimeric gene of the present disclosure, DNA includes cDNA and genomic DNA.

[0057] In another embodiment of the invention, a chimeric gene is provided comprising as operably linked DNA regions

[0058] (a) a plant-expressible promotor such as a fiber-preferential promotor,

[0059] (b) a DNA region coding for a GFAT polypeptide wherein said GFAT is encoded by a nucleotide sequence as described herein above and

[0060] (c) optionally a DNA region involved in transcription termination and polyadenylation.

[0061] As used herein, the term "plant-expressible promoter" means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i. e. certain promoters of viral or bacterial origin such as the CaMV35S, the subterranean clover virus promoter No 4 or No 7 (WO9606932) or T-DNA gene promoters and the like.

[0062] In one embodiment of the invention, the promoter may be a heterologous promoter not naturally associated with the DNA region operably linked to it.

[0063] It will be clear that constitutive plant-expressible promoters may be suitable for the invention. Examples of constitutive promoters include the promoter from the actin gene (McElroy et al. (1990) Plant Cell 2: 163-171), the CaMV35S promoter (Odell et al. (1985) Nature 313: 810-812), the CaMV19S promoter (Nilsson et al. (1997) Physiol. Plant. 100: 456-462), the GOS2 promoter (de Pater et al. (1992) Plant. J. 2(6): 837-44), the promoter from ubiquitin gene (Christensen et al. (1992) Plant Mol. Biol. 18: 675-689), the promoter from rice cyclophilin gene (Buchholz et al. (1994) Plant. Mol. Biol. 25(5): 837-43), the promoter from the maize H3 histone gene (Lepetit et al. (1992) Mol. Gen. Genet. 231: 276-285) or the promoter from the actin 2 gene (An et al. (1996) Plant J. 10(1): 107-121).

[0064] It is also clear that inducible promoters, such as a temperature inducible or a chemically inducible promoter or a promoter which is responsive to developmental cues, may be used in accordance with the invention. Tissue selective promoters may also be used.

[0065] In a preferred embodiment of the invention, the chimeric gene comprises a fiber-preferential or fiber-selective promoter. The term "fiber-preferential" or "fiber-selective", with respect to the expression of a gene or with respect to a promoter, refers to, for practical purposes, the highly specific expression of a gene or expression directed by a promoter, in fiber cells of plants, such as cotton plants. In other words, transcript levels of a DNA in tissues different of fiber cells is either below the detection limit or very low (less than about 0.2 picogram per microgram total RNA).

[0066] The term "fiber-preferential" or "fiber-cell preferential" with respect to the expression of a DNA in accordance with this invention, refers to an expression pattern whereby the DNA is expressed predominantly in fiber cells or fibers, but expression can be identified in other tissues of the plant. Preferably, the expression in fiber cells is about 2 to about 10 times higher in the fiber cells than in other tissues.

[0067] Such promoters (all herein incorporated by reference) include the promoter from cotton from a fiber-specific .beta.-tubulin gene (as described in WO0210377), the promoter from cotton from a fiber-specific actin gene (as described in WO0210413), the promoter from a fiber-specific lipid transfer protein gene from cotton (as described in U.S. Pat. No. 5,792,933), a promoter from an expansin gene from cotton (WO9830698) or a promoter from a chitinase gene in cotton (US2003106097) or the promoters of the fiber-specific genes described in U.S. Pat. No. 6,259,003 or U.S. Pat. No. 6,166,294 or the promotors derived from the E6 family as disclosed in U.S. Pat. No. 6,096,950. Fiber selective promoters as described in WO08/083969 (from cotton glucanase genes), WO12/093032 (from cotton FS18 or SCW-PRP gene) or US 2013/0081154 (from cotton FB8-like genes) are also suitable plant-expressible promoters. Also suitable for the invention is the promoter disclosed in EP13172094 comprising the nucleotide sequence of SEQ ID No. 5 as described therein from nucleotide position 4208 to nucleotide position 5615 or having the nucleotide sequence of SEQ ID No. 5 from nucleotide position 75 to 1482.

[0068] The chimeric genes as herein described optionally comprise a DNA region involved in transcription termination and polyadenylation. A variety of DNA regions involved in transcription termination and polyadenylation functional in plants are known in the art and those skilled in the art will be aware of terminator and polyadenylation sequences that may be suitable in performing the methods herein described. The polyadenylation region may be derived from a natural gene, from a variety of other plant genes, from T-DNA genes or even from plant viral genomes. The 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or from any other eukaryotic gene.

[0069] In a particular embodiment of the invention a cotton plant cell is provided comprising a chimeric gene comprising as operably linked DNA regions

[0070] (a) a plant-expressible promotor such as a fiber-preferential promotor,

[0071] (b) a DNA region coding for a GFAT polypeptide wherein said GFAT is encoded by a nucleotide sequence as herein described and

[0072] (c) optionally a DNA region involved in transcription termination and polyadenylation.

[0073] The chimeric gene may be introduced into a plant cell by methods well-known in the art. "Introducing" in connection with the present application relates to the placing of genetic information in a plant cell or plant by artificial means. This can be effected by any method known in the art for introducing RNA or DNA into plant cells, tissues, protoplasts or whole plants.

[0074] The term "introducing" may refer to introduction of an exogenous DNA molecule to a plant cell by transformation, optionally followed by regeneration of a plant from the transformed plant cell. The term may also refer to introduction of the recombinant DNA molecule by crossing of a transgenic plant comprising the recombinant DNA molecule with another plant and selecting progeny plants which have inherited the recombinant DNA molecule or transgene. Yet another alternative meaning of providing refers to introduction of the recombinant DNA molecule by techniques such as protoplast fusion, optionally followed by regeneration of a plant from the fused protoplasts.

[0075] It will be clear that the methods of transformation used are of minor relevance to the current invention. Transformation of plants is now a routine technique. Advantageously, any of several transformation methods may be used to introduce the nucleic acid/gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296: 72-74; Negrutiu et al. (1987) Plant. Mol. Biol. 8: 363-373); electroporation of protoplasts (Shillito et al. (1985) Bio/Technol. 3: 1099-1102); microinjection into plant material (Crossway et al. (1986) Mol. Gen. Genet. 202: 179-185); DNA or RNA-coated particle bombardment (Klein et al. (1987) Nature 327: 70) infection with (non-integrative) viruses and the like.

[0076] Methods to transform cotton plants are also well known in the art. Agrobacterium-mediated transformation of cotton has been described e.g. in U.S. Pat. No. 5,004,863 or in U.S. Pat. No. 6,483,013 and cotton transformation by particle bombardment is reported e.g. in WO 92/15675. Other suitable cotton transformation methods are disclosed e.g. in WO 00071733 and U.S. Pat. No. 5,159,135, which disclosures are incorporated by reference herein as if fully set forth.

[0077] The recombinant DNA molecules according to the invention may be introduced into plants in a stable manner or in a transient manner using methods well known in the art. The chimeric genes may be introduced into plants, or may be generated inside the plant cell as described e.g. in EP 1339859.

[0078] In yet another embodiment, the invention provides a cotton plant cell as described herein above wherein said cotton plant cell additionally comprises a second chimeric gene comprising the following operably linked DNA regions:

[0079] (a) a plant-expressible promotor such as a fiber-preferential promotor,

[0080] (b) a DNA sequence coding for a chitin synthase polypeptide and

[0081] (c) optionally a DNA region involved in transcription termination and polyadenylation.

[0082] Several embodiments and specifications on what is meant by the term "plant-expressible promotor" are given above and equally apply for the second chimeric gene comprising a DNA region encoding a chitin synthase. The same is true for the specifications given above on the DNA region involved in transcription termination and polyadenylation and also for methods and means to provide a plant cell with a chimeric gene.

[0083] The first chimeric gene and the second chimeric gene can be introduced into a plant cell individually in any order or simultaneously. They can be introduced on the same vector or on separate vectors.

[0084] The chitin synthase can be any protein having the enzymatic activity of a chitin synthase (EC 2.4.1.16), i. e. that converts UDP-N-acetyl-D-glucosamine into chitin and UDP. A chitin synthase catalyzes the reaction: UDP-N-acetyl-alpha-D-glucosamine+(1,4-(N-acetyl-beta-D-glucosaminyI))(n).- revreaction.UDP+(1,4-(N-acetyl-beta-D-glucosaminyl))(n+1). Suitable for the present invention is any chitin synthase derived from any organism. Examples for suitable chitin synthases are chitin synthase from Saprolegnia monoica (WO 2011/089021) or chitin synthases of the NOD C type as described in WO 2006/136351 or in WO 2012/048807 for example.

[0085] In a particular embodiment of the invention, the chitin synthase in said cotton plant cell as described before is an N-acetylglucosamine transferase of the NOD C type. Particular good results are achieved if said chitin synthase polypeptide comprises a Golgi localization signal.

[0086] Although good results have been achieved with plant cells comprising a chitin synthase activity in addition to the GFAT activity, the GFAT activity as obtained by means described in the invention can also beneficially be combined with any enzymatic activity that leads to the production of glycosaminoglycans from the GFAT product glucosamine-6-phosphate or from UDP-N-acetylglucosamine. As described in the introduction, glucosamine-6-phosphate is further converted to UDP-N-acetylglucosamine via the hexosamine pathway in plants. One such enzymatic activity that converts UDP-N-acetylglucosamine into glycosaminoglycans other than chitin is that of a hyaluronan synthase. Thus a hyaluronan synthase can also be used instead of a chitin synthase.

[0087] In another particular embodiment the invention provides a plant consisting essentially of plant cells comprising a chimeric gene herein described before. The chimeric gene can be a first chimeric gene comprising a GFAT encoding region or a first and a second chimeric gene as described before. In a particular embodiment the plant is a cotton plant.

[0088] "Cotton" or "cotton plant" as used herein can be any variety useful for growing cotton. The most commonly used cotton varieties are Gossypium barbadense, G. hirsutum, G. arboreum and G. herbaceum. Further varieties include G. africanum and G. raimondii. Also included are progeny from crosses of any of the above species with other species or crosses between such species.

[0089] A cotton plant cell may be any cell comprising essentially the genetic information necessary to define a cotton plant, which may, apart from the chimeric gene disclosed herein, be supplemented by one or more further transgenes. Cells may be derived from the various organs and/or tissues forming a cotton plant, including but not limited to fruits, seeds, embryos, reproductive tissue, meristematic regions, callus tissue, leaves, roots, shoots, flowers, vascular tissue, gametophytes, sporophytes, pollen, and microspores.

[0090] Whereas certain plant cells according to the invention may be able to regenerate into complete plants, in some embodiments said plant cells cannot further develop or regenerate into a complete plant. In one embodiment of the invention, fiber cells are committed. Mature fiber cells are dead cells.

[0091] The invention is also directed towards fiber-producing plants comprising one or more recombinant construct according to the invention. Although the nucleotide sequence encoding the GFAT protein has been optimized for expression in cotton plants, it is thought that the coding region could also be beneficially used in other fiber producing plants such as hemp, jute, flax and woody plants including but not limited to Pinus spp., Populus spp., Picea spp., Eucalyptus spp. etc. The plant cell may be derived from any trichome-producing plant.

[0092] The plants according to the invention can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the chimeric gene according to the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from the transformed plants contain the chimeric genes of the invention as a stable genomic insert and are also encompassed by the invention.

[0093] The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, fibers and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.

[0094] In a specific embodiment the invention provides cotton fibers obtainable from a cotton plant according to the invention.

[0095] The cotton fibers according to the invention can be distinguished from naturally occurring cotton fibers, i. e. cotton fibers obtained from an isogenic line which does not comprise a nucleic acid sequence according to the invention, by the increased content of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines. The GlcNAc polymers or oligo-glucosamines can be detected directly. Alternatively, positively charged polysaccharides in the cotton fibers can be detected by measuring the glucosamine content after treatment with trifluoro-acetic acid (TFA) to hydrolyze the polysaccharides. The cotton fibers according to the invention may also be distinguished by their increased nitrogen content. Due to the reactivity of the nitrogen-containing groups within the glucosamine-polymers, cotton fibers according to the invention are characterized by an altered chemical reactivity compared to fibers obtained from cotton plants which do not comprise a nucleic acid region encoding a GFAT polypeptide as herein described. Fibers according to the invention have an increased capacity to react with dyes or other suitable chemicals via the nitrogen-containing groups.

[0096] Cotton fibers according to the invention are characterized by an increased content of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines. "Increased content" means that the amount of positively charged polysaccharides present in the plant cells or fibers is higher than in plant cells or fibers not comprising a GFAT protein or compared to plant cells or fibers expressing a GFAT encoding gene known in the art which is not optimized for expression in cotton plant cells. In one embodiment, the content of glucosamine (GlcN) is at least twice that of cells or fibers from plants not expressing an artificially introduced gene construct. This background level was observed to be approximately 0.010 to 0.015% GlcN of total fiber weight. Preferably, fibers according to the invention contain more than 0.03% GlcN of total fiber weight. More preferably the GlcN content of fibers according to the invention is more than 0.06%, even more preferably more than 0.08%, most preferably more than 0.10% GlcN of total fiber weight. In another embodiment, the GlcN content of plant cells or cotton fibers according to the invention is at least four times that of cells or fibers from plants not expressing an artificially introduced gene construct. In particularly suitable embodiments of the invention, plant cells or fibers have a GlcN content which is at least five times, preferably at least seven times and most preferably ten times that of cells or fibers from plants not expressing an artificially introduced gene construct.

[0097] A "fiber" is botanically defined as a long narrow tapering cell, dead and hollow at maturity with a rigid thick cell wall composed mostly of cellulose and usually lignin. Soft or bast fibers are found in the phloem (inner bark) of dicotyledonous stems (flax, jute, hemp, ramie). Hard or leaf fibers are found in monocot leaf vascular bundles (sisal, manilla hemp, pineapple). Surface fibers grown from the surface of seeds (cotton), leaves or fruits (coconut coir).

[0098] "Cotton fiber", as used herein, refers to a seed trichome, more specifically a single cell of a fiber-producing plant, such as cotton, that initiates from the epidermis of the outer integument of the ovules, at or just prior to anthesis. The morphological development of cotton fibers has been well documented (Basra and Malik, 1984, Int Rev of Cytology 89: 65-113; Graves and Stewart, 1988, J. Exp. Bot. 39 (1): 59-69; Ramsey and Berlin, 1976, American Journal of Botany 63 (6): 868-876; Ruan and Chourey, 1998, Plant Physiology 118: 399-406; Ruan et al. 2000, Aust. J. Plant Physiol. 27:795-800; Stewart, 1975, Am. J. Bot. 62, 723-730).

[0099] Another embodiment of the invention are therefore plant cell walls such as cell walls from cotton cells, comprising an increased level of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines compared to cell walls from unmodified plant cells or from plant cells not expressing a GFAT encoding nucleotide sequence as herein described.

[0100] The invention also relates to yarns made from fibers according to the invention as well as fabrics made from these yarns.

[0101] In another embodiment, the invention provides a method to produce cotton fibers with positively charged polysaccharides, such as oligo-N-acetylglucosamines or oligo-glucosamines, comprising the steps of

[0102] i) expressing a chimeric gene comprising a GFAT encoding region as described above in a cotton plant cell,

[0103] ii) regenerating a cotton plant from cotton plant cells of step i) and

[0104] iii) optionally isolating fibers from said cotton plant.

[0105] In a particular embodiment, a method is provided to produce cotton fibers with positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines comprising i) expression of a first chimeric gene comprising a GFAT encoding region according to the invention and a second chimeric gene comprising a chitin synthase encoding region in a cotton plant cell, ii) regenerating a cotton plant from said cotton plant cells and iii) optionally isolating fibers from said cotton plant. Said first and second chimeric gene can be introduced into the plant cell simultaneously or separately in any order as described above.

[0106] In another embodiment, a method is provided to produce cotton fibers with an increased content of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines comprising the steps of i) expressing said first chimeric gene or expressing said first and second chimeric gene in a cotton plant cell, ii) regenerating a cotton plant from said cotton plant cells and iii) optionally isolating fibers from said cotton plant. The term "increased content" is to be understood as described above.

[0107] Further, a method is provided for producing cotton fibers with altered chemical reactivity of the fibers comprising the steps of i) expressing a chimeric gene comprising a GFAT encoding region according to the invention in a cotton plant cell, ii) regenerating a cotton plant from said cotton plant cells and iii) optionally isolating fibers from said cotton plant.

[0108] In yet another embodiment, a method is provided for producing cotton fibers with altered chemical reactivity of the fibers comprising the steps of i) expressing a first chimeric gene comprising a GFAT encoding region as described above and a second chimeric gene comprising a chitin synthase encoding region in a cotton plant, ii) regenerating a cotton plant from said cotton plant cells and iii) optionally isolating fibers from said cotton plant.

[0109] The nucleic acid molecule according to the invention can be used to produce a cotton plant with positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines in the fibers. In particular it can be used to increase the amount of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines in fibers. It can also be used for the production of cotton fibers with altered chemical reactivity. This might allow the convenient, easy and/or efficient further finish of the fibers. Fibers obtained from a cotton plant according to the invention can e. g. be stained with reactive dyes that bind to the fibers via covalent bonds to the amino groups of the glucosamine residues in the polysaccharides. Alternatively, other substances can be attached via chemical reactions to the amino groups of the glucosamine residues. Substances can also be attached to fibers according to the invention via electrostatic or ionic bonding to the N-containing groups of the polysaccharides. The attachment of other substances to cotton fibers can be beneficial to transfer special properties to the fibers. Such finishes can be but are not limited to dying, attachment of flame retardants, water, oil and soil repellents, anticrease agents, softeners, antistatic agents, fluorescent whitening agents or any other textile finish.

[0110] As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids may comprise more nucleotides or amino acids than the cited ones, i. e. may be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.

[0111] The following non-limiting examples describe the generation of cotton fibers with an increased content of positively charged polysaccharides such as oligo-N-acetylglucosamines or oligo-glucosamines.

[0112] Unless stated otherwise in the examples, all recombinant techniques are carried out according to standard protocols as described in "Sambrook J and Russell DW (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York" and in "Ausubel F A, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A and Struhl K (eds.) (2006) Current Protocols in Molecular Biology. John Wiley & Sons, New York".

[0113] Standard materials and references are described in "Croy RDD (ed.) (1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd., Oxford and Blackwell Scientific Publications, Oxford" and in "Brown T A, (1998) Molecular Biology LabFax, 2nd Edition, Academic Press, San Diego". Standard materials and methods for polymerase chain reactions (PCR) can be found in "McPherson M J and Moller S G (2000) PCR (The Basics), BIOS Scientific Publishers Ltd., Oxford" and in "PCR Applications Manual, 3rd Edition (2006), Roche Diagnostics GmbH, Mannheim or www.roche-applied-science.com".

Description of Sequences

[0114] Reference is made throughout the application to the following sequences represented in the sequence listing named "BCS14-2002_ST25", which is 42 kB (size as measured in Microsoft Windows.RTM.), contains 4 sequences SEQ ID NO: 1 through SEQ ID NO: 4, which is filed herewith by electronic submission and is incorporated by reference herein:

[0115] SEQ ID 1: Synthetic nucleotide sequence coding for a protein having the activity of a glutamine:fructose-6-phosphate-amidotransferase (GFAT) from E. coli. The sequence is optimized for expression in cotton plant cells. The nucleotide sequence shown codes for a polypeptide having the amino acid sequence of SEQ ID 2.

[0116] SEQ ID 2: Amino acid sequence of a polypeptide having the activity of a glutamine:fructose-6-phosphate-amidotransferase (GFAT) from E. coli. The amino acid sequence shown can be derived from SEQ ID 1.

[0117] SEQ ID 3: T-DNA of pTDBI 252. It comprises a nucleotide sequence according to SEQ ID 1 encoding a GFAT polypeptide from E. coli under control of a fiber-selective SCW-PRP promotor, a DNA region encoding a NOD C chitin synthase under control of a fiber-selective SCW-PRP promotor and the epsps gene as a selectable marker.

[0118] SEQ ID 4: T-DNA of pTDBI 250. It comprises a nucleotide sequence according to SEQ ID 1 encoding a GFAT polypeptide from E. coli under control of a fiber-selective Fb8-like-1 promotor, a DNA region encoding a NOD C chitin synthase under control of a fiber-selective Fb8-like-1 promotor and the epsps gene as a selectable marker.

EXAMPLES

Example 1

Construction of a Chimeric Gene Encoding a Glutamine:Fructose-6-Phosphate-Amidotransferase (GFAT) Protein for Expression in Cotton Cells

[0119] A DNA molecule having the nucleic acid sequence according to SEQ ID 1 was synthesized by Entelechon GmbH. The nucleotide sequence was designed i) to encode a polypeptide according to SEQ ID 2 and ii) to optimize the nucleotide sequence for expression in cotton plant cells. For this purpose, factors such as codon usage, mRNA secondary structure, the AT content, cryptic splice sites or restriction sites were taken into account.

[0120] The resulting nucleotide sequence as disclosed in SEQ ID 1 is 75% identical (1390 matching bases out of 1830) to the published nucleotide sequence encoding a GFAT protein from E. coli which was adapted to the codon usage in plants (WO 2007/039314).

[0121] Using standard recombinant DNA techniques, the following chimeric GFAT gene was constructed: A chimeric glutamine-6-phosphate-amidotransferase gene comprising the following operably linked DNA regions:

[0122] i. the fiber-selective SCW-PRP promoter region according to the sequence from nucleotide position 61 to 1499 of SEQ ID 3,

[0123] ii. a DNA fragment coding for the 35 N-terminal amino acids of a DNA xylosyltransferase from Arabidopsis thaliana which function as a Golgi localisation signal peptide,

[0124] iii. a DNA fragment coding for NOD C of Azorhizobium caulinodans cloned in frame with the previous DNA fragment,

[0125] iv. the 3' untranslated sequence of the 35S transcript of the Cauliflower Mosaic Virus,

[0126] v. the fiber-selective SCW-PRP promoter region according to the sequence from nucleotide position 61 to 1499 of SEQ ID 3,

[0127] vi. a DNA region having the nucleotide sequence according to SEQ ID 1 encoding a glutamine: fructose-6-phosphate amidotransferase from E. coli according to SEQ ID 2,

[0128] vii. the 3' untranslated sequence of histone H4 gene of Arabidopsis thaliana.

[0129] This chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric double mutated 5-enol-pyruvylshikimate-3-phosphate synthase (epsps) gene from Zea mays (corn) providing resistance to N-(phosphonomethyl)glycin as a selectable marker. The resulting T-DNA vector was named pTDBI 252. The sequence of the T-DNA of this vector is provided in SEQ ID 3. The genetic elements of the T-DNA of this vector are represented in Table 1.

[0130] Another chimeric GFAT gene was constructed containing the following operably linked DNA regions:

[0131] i. the fiber-selective Fb8-like-1 promoter region according to the sequence from nucleotide position 60 to 1495 of SEQ ID 4,

[0132] ii. a DNA fragment coding for the 35 N-terminal amino acids of a DNA xylosyltransferase from Arabidopsis thaliana which serves as a Golgi localization peptide,

[0133] iii. a DNA fragment coding for NOD C of Azorhizobium caulinodans cloned in frame with the previous DNA fragment,

[0134] iv. the 3'' untranslated sequence of the 35S transcript of the Cauliflower Mosaic Virus,

[0135] v. the fiber-selective Fb8-like-1 promoter region according to the sequence from nucleotide position 60 to 1495 of SEQ ID 4,

[0136] vi. a DNA region having the nucleotide sequence according to SEQ ID 1 encoding a glutamine: fructose-6-phosphate amidotransferase from E. coli according to SEQ ID 2,

[0137] vii. the 3' untranslated sequence of histone H4 gene of Arabidopsis thaliana.

[0138] This chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric epsps gene as a selectable marker. The resulting T-DNA vector was named pTDBI 250, The sequence of the T-DNA of this vector is provided in SEQ ID 4. The genetic elements of the T-DNA are represented in Table 2.

TABLE-US-00001 TABLE 1 Elements of the T-DNA of pTDBI 252 Start End Name Description 1 25 RB Right border repeat from the T-DNA of Agrobacterium tumefaciens 61 1499 PSCW-PRP sequence including the promoter region of a proline-rich cell wall protein gene of Gossypium hirsutum 1503 1607 RPXylTAt coding sequence for the Golgi retention peptide of the beta-1,2- xylosyltransferase gene of A. thaliana 1608 2798 NodC coding sequence of the N-acetylglucosaminyltransferase gene NodC of Azorhizobium caulinodans 2810 3030 3'35S sequence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus 3068 4506 PSCW-PRP sequence including the promoter region of a proline-rich cell wall protein gene of Gossypium hirsutum 4510 6339 GFAT coding region of the glutamine:fructose-6-phosphate amidotransferase gene of Escherichia coli optimized for expression in cotton plant cells 6357 7017 3'H4 At sequence including the 3' untranslated region of the histone H4 gene of Arabidopsis thaliana 7067 7983 PH4 sequence including the promoter region of the histone H4 gene of Arabidopsis thaliana 8017 8497 intron1 H3At + sequence including the first intron of gene II of the histone H3.III flanking region variant of Arabidopsis thaliana 8502 8873 TP_opt coding sequence of the optimized transit peptide, containing sequence of the RuBisCO small subunit genes of Zea mays (corn) and Helianthus annuus 8874 10211 2mepsps coding sequence of the double-mutant 5-enol-pyruvylshikimate-3- phosphate synthase gene of Zea mays (corn) 10235 10895 3'H4 At sequence including the 3' untranslated region of the histone H4 gene of Arabidopsis thaliana 11008 11032 LB Left border repeat from the T-DNA of Agrobacterium tumefaciens

TABLE-US-00002 TABLE 2 Elements of pTDBI 250 Start End Name Description 1 25 RB Right border repeat from the T-DNA of Agrobacterium tumefaciens 60 1495 Pfb8-like-1 sequence including the promoter region of the fb8-like gene of Gossypium hirsutum (cotton) 1497 1601 RPxylTAt coding sequence for the Golgi retention peptide of the beta-1,2- xylosyltransferase gene of Arabidopsis thaliana 1602 2792 NodC coding sequence of the N-acetylglucosaminyl-transferase gene nodC of Azorhizobium caulinodans 2804 3026 3'35S sequence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus 3061 4496 Pfb8-like-1 sequence including the promoter region of the fb8-like gene of Gossypium hirsutum (cotton) 4498 6327 GFAT coding region of the glutamine:fructose-6-phosphate amidotransferase gene of Escherichia coli optimized for expression in cotton plant cells 6345 7005 3'H4 At sequence including the 3' untranslated region of the histone H4 gene of Arabidopsis thaliana 7056 7970 PH4 AT sequence including the promoter region of the histone H4 gene of Arabidopsis thaliana 8005 8486 intron1 H3At + sequence including the first intron of gene II of the histone H3.III flanking region variant of Arabidopsis thaliana 8490 8861 TP_opt coding sequence of the optimized transit peptide, containing sequence of the RuBisCO small subunit genes of Zea mays (corn) and Helianthus annuus 8862 10199 2mepsps coding sequence of the double-mutant 5-enol-pyruvylshikimate-3- phosphate synthase gene of Zea mays (corn) 10223 10883 3'H4 At sequence including the 3' untranslated region of the histone H4 gene of Arabidopsis thaliana 10996 11020 LB Left border repeat from the T-DNA of Agrobacterium tumefaciens

[0139] As a control a chimeric gene was used containing the following operably linked DNA regions:

[0140] i. the fiber-selective SCW-PRP promoter region according to the sequence from nucleotide position 61 to 1499 of SEQ ID 3,

[0141] ii. a DNA fragment coding for the 35 N-terminal amino acids of a DNA xylosyltransferase from Arabidopsis thaliana,

[0142] iii. a DNA fragment coding for NOD C of Azorhizobium caulinodans cloned in frame with the previous DNA fragment,

[0143] iv. the 3' untranslated sequence of the 35S transcript of the Cauliflower Mosaic Virus,

[0144] v. the fiber-selective SCW-PRP promoter region according to the sequence from nucleotide position 61 to 1499 of SEQ ID 3,

[0145] vi. a DNA region encoding a glutamine:fructose-6-phosphate amidotransferase from E. coli which was optimized for codon usage in plants as described in WO 2007/039314 under SEQ ID 10 therein,

[0146] vii. the 3' untranslated sequence of histone H4 gene of Arabidopsis thaliana.

[0147] This chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric epsps gene as a selectable marker. The resulting T-DNA vector was named pTGK 110, This vector is identical to pTDBI252 except for the GFAT encoding sequence.

Example 2

Generation of Transgenic Cotton Plants Expressing a Glutamine: Fructose-6-Phosphate Amidotransferase

[0148] The T-DNA vectors were introduced into Agrobacterium tumefaciens strains containing a helper Ti-plasmid and used in cotton transformation essentially as described in WO00/71733. T0 plants were further analyzed as described in Example 3.

Example 3

Determination of the Glucosamine Content of Cotton Fibers

[0149] Fibers from transgenic cotton T0 plants were isolated, treated with trifluoroacetic acid (TFA) to hydrolyze the glucosamine polymers and analyzed for the glucosamine content by HPLC. All steps were carried out following standard protocols.

[0150] Fibers of untransformed lines contained about 0,01% of GlcN. The results for the measured glucosamine content of cotton fibers from different T0 plants expressing the GFAT gene according to the invention under the control of the SCW-PRP promotor (transformed with pTDBI 252) are depicted in Table 3.

TABLE-US-00003 TABLE 3 GlcN content of cotton fibers from individual T0 plants GFAT optimized (pTDBI 252) GFAT control (pTGK 110) pl1 0.0115 * cpl1 0.0101 * pl2 0.0118 * cpl2 0.0112 * pl3 0.0136 * cpl3 0.0120 * pl4 0.0141 * cpl4 0.0121 * pl5 0.0318 cpl5 0.0125 * pl6 0.0340 cpl6 0.0316 pl7 0.0371 cpl7 0.0330 pl8 0.0389 cpl8 0.0334 pl9 0.0401 cpl9 0.0349 pl10 0.0405 cpl10 0.0425 pl11 0.0431 cpl11 0.0446 pl12 0.0448 cpl12 0.0536 pl13 0.0472 cpl13 0.0546 pl14 0.0502 cpl14 0.0566 pl15 0.0530 cpl15 0.0589 pl16 0.0538 cpl16 0.0597 pl17 0.0558 cpl17 0.0599 pl18 0.0630 cpl18 0.0714 pl19 0.0674 Average: 0.0385 pl20 0.0762 pl21 0.0783 pl22 0.0811 pl23 0.0817 pl24 0.0910 pl25 0.0929 pl26 0.0965 Pl27 0.1016 pl28 0.1152 pl29 0.1243 pl30 0.1319 Average: 0.0607 Values represent % GlcN of total fiber weight; * considered as background

[0151] The numbers given represent % GlcN of total fiber weight. Values below 0.015 were considered as background. Table 3 also shows the GlcN content found in fibers from individual T0 cotton plants that were transformed with the control vector pTGK 110 which comprises a GFAT encoding DNA region which is optimized for codon usage in plants and is known in the art.

[0152] Table 4 shows the average and maximum GlcN content (measured in % of total fiber weight) of cotton fibers derived from T0 plants expressing either the GFAT gene according to the invention under control of the SCW-PRP promotor or under the control of the Fb8-like-1 promotor. As a control values are given for plants expressing the plant-optimized GFAT gene described in WO 2007/039314. The mean GlcN content of fibers expressing the GFAT gene sequence according to SEQ ID 1 under control of the SCW-PRP promotor was about four times above background level (0.061% vs. 0.015%) and nearly twice that of control plants expressing a plant-optimized GFAT gene sequence published in WO 2007/039314 (0.061% vs. 0.039%). The maximum GlcN content that was measured in a T0 plant expressing the GFAT gene according to the invention under control of the SCW-PRP promotor was nearly 10-fold above the background level of fibers from plants not expressing an artificially introduced gene construct (0.132% vs. 0.015%). Moreover, it was nearly twice that of control plants expressing a plant-optimized GFAT gene sequence published in WO 2007/039314 (0.132% vs. 0.071%). Likewise, plants expressing a GFAT gene according to SEQ ID 1 under the control of the Fb8-like-1 promotor had a maximum increase in the GlcN content of the fibers by more than 10-fold (0.178% vs. 0.015%) and a mean 2-fold increase in the GlcN content of the fibers (0.039% vs. 0.015%) compared to plants not expressing an artificially introduced gene construct.

TABLE-US-00004 TABLE 4 Mean and average GlcN content of fibers from T0 cotton plants expressing a GFAT gene according to SEQ ID 1 under the control of different fiber-selective promotors) GFAT ctrl. GFAT ctrl. GFAT opt. GFAT opt. Promoter average max. average maximum SCW-PRP 0.039 0.071 0.061 0.132 Fb8-like-1 0.039 0.178 Values represent % GlcN of total fiber weight

Example 4

Cotton Fibers with Increased Reactivity

[0153] Transgenic cotton plants comprising a chimeric GFA gene and a chimeric NOD C gene operably linked to a fiber-specific promoter as outlined in Example 1 are generated as described in Example 2. Mature cotton fibers are harvested from these plants and can be stained with anionic dyes such as Congo Red or can be reacted with wheat germ agglutinin (WGA) coupled Alexa fluor 555. WGA specifically binds to N-acetylglucosamine in plant cells and therefore can be used as a detection reagent for N-acetylglucosamine. In addition, the resulting mature cotton fibers can be stained with commercial dyes including cotton reactive dyes (e.g. Reactive Red 120, Levafix Blue CA), acid dyes (Acid Orange 7, Acid Blue 281) and wool reactive dyes (e.g. Reactive Red 116, Realan Amber EHF).

Sequence CWU 1

1

411830DNAartificialartificial sequence encoding the glutaminefructose-6-phosphate-amidotransferase (GFAT) of E. coliCDS(1)..(1830) 1atg tgc gga att gtt ggc gca ata gca caa agg gac gta gca gaa atc 48Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Glu Ile1 5 10 15ctt ctt gaa gga ctc cgt cgt ctg gaa tac aga gga tat gat tct gcc 96Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30ggt cta gcc gtt gta gat gcc gaa ggt cac atg aca cgt cta aga cgt 144Gly Leu Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45ctg ggt aag gtt caa atg ctg gct caa gca gcc gaa gaa cat cct tta 192Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60cat ggt ggc aca ggt att gct cac act aga tgg gct act cac ggt gaa 240His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu65 70 75 80cct tca gag gta aat gct cat cca cat gtc tct gag cac att gtg gtc 288Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val 85 90 95gtt cac aac ggg atc atc gaa aac cat gaa cca ctt cga gaa gag ctg 336Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110aaa gct cgt ggc tat act ttc gtt tca gag aca gac act gag gtg att 384Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile 115 120 125gct cat ctc gtg aac tgg gaa ctg aaa caa ggg gga act ctg aga gag 432Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140gct gtt cta cgt gct atc cct caa tta cgt ggt gct tac ggg aca gtg 480Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val145 150 155 160atc atg gat tca aga cac cca gat aca ctg ctg gca gca agg tct ggt 528Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly 165 170 175agt cca ctg gtg att gga ctg ggg atg gga gaa aac ttt atc gct tcg 576Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser 180 185 190gat caa ctg gct ctg tta cct gtg aca cgg aga ttt atc ttc ctt gaa 624Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu 195 200 205gag ggc gat atc gcg gaa ata act cga cgt agc gta aac atc ttc gat 672Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp 210 215 220aaa acc gga gca gaa gta aaa cgc cag gat atc gaa tcc aat ctt caa 720Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln225 230 235 240tac gac gcc ggc gat aaa ggc ata tac cga cac tac atg cag aaa gag 768Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu 245 250 255atc tac gag caa ccg aac gct atc aag aat acc ctt act ggg cgt atc 816Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile 260 265 270tca cat ggt cag gtt gac tta tct gaa ctg gga cca aac gca gac gaa 864Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285cta ctg tcg aag gta gaa cat att cag atc ctc gcg tgt ggt act tct 912Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser 290 295 300tat aac tct ggt atg gtc agt cgc tat tgg ttt gaa tca ctg gca gga 960Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly305 310 315 320att cct tgc gac gtc gaa att gcc tcg gaa ttc aga tat cgc aag tct 1008Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335gca gta aga cgc aac agc ctg atg ata acg tta tct cag tct gga gaa 1056Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu 340 345 350acg gct gat aca ctg gct gga tta cgt ctg tca aaa gag ctt ggc tac 1104Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365ctt ggt tct cta gca atc tgt aac gtt cct ggt agc tct ctt gtg cga 1152Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380gaa tct gat ctt gct ctt atg act aac gct ggt aca gaa atc ggg gtg 1200Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val385 390 395 400gca tcc aca aaa gca ttt aca act caa ctt acg gtg ctg cta atg ctt 1248Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415gtg gca aag ctg tct aga ctc aaa ggt cta gat gcc tcc atc gag cat 1296Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His 420 425 430gat atc gtt cat ggt ctg caa gct ctt cct agc cga att gag cag atg 1344Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met 435 440 445ctg tca caa gac aaa agg att gaa gcc ctg gca gaa gat ttc tca gac 1392Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460aag cat cac gct ttg ttt ctc ggt cgt ggc gat cag tat cct atc gct 1440Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala465 470 475 480ctc gaa ggc gca ttg aag ctc aaa gag atc tcc tat ata cac gct gaa 1488Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu 485 490 495gct tac gct gca ggc gaa ctg aaa cac gga cct cta gct ctt att gac 1536Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510gca gat atg ccc gtt atc gtc gtt gca cca aac aac gaa ttg ctg gag 1584Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu Glu 515 520 525aag ctg aaa tca aat att gaa gag gta cgt gca aga ggc gga caa ctt 1632Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu 530 535 540tat gtc ttc gct gag caa gat gcc ggt ttt gta agt agc gat aac atg 1680Tyr Val Phe Ala Glu Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met545 550 555 560cac atc atc gag atg cct cac gtg gaa gag gtg att gct ccg atc ttc 1728His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe 565 570 575tac aca gtt ccc ctg cag ctt ctg gct tat cac gtt gcc ctt atc aaa 1776Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys 580 585 590gga act gac gtt gac cag cca agg aat ctc gca aag tca gta acg gtt 1824Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605gag taa 1830Glu2609PRTartificialSynthetic Construct 2Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Glu Ile1 5 10 15Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30Gly Leu Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu65 70 75 80Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val 85 90 95Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile 115 120 125Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val145 150 155 160Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly 165 170 175Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser 180 185 190Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu 195 200 205Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp 210 215 220Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln225 230 235 240Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu 245 250 255Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile 260 265 270Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser 290 295 300Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly305 310 315 320Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu 340 345 350Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val385 390 395 400Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His 420 425 430Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met 435 440 445Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala465 470 475 480Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu 485 490 495Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu Glu 515 520 525Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu 530 535 540Tyr Val Phe Ala Glu Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met545 550 555 560His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe 565 570 575Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys 580 585 590Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605Glu311032DNAArtificialT-DNA of pTDBI 252 3aattacaacg gtatatatcc tgccagtact gggccccctc gagggcgatc gcgcggccgc 60ttcacggaaa gttgttatat ataagttcag taaataataa tgaaatataa attttaatta 120tatctagtac tcaataagaa gatggagaaa gttatgttaa ttatagttat aaattattta 180taaatttaat atatatatat aaagaaaata gttgtataac taataattat ttttacaata 240ctttatatag ttatatttaa aaaaatttta aaattaaaat actattattt tgttcaatat 300attaatattt atattattta atttattatt gaatatgaat aaattttttt tgaaaattat 360atttttaatt tttagaaatt ttatataact ttccatatat atatttctga tttgtcaatt 420tcttttgaga tttatctaaa ttgatttgaa ttttttttat ttttaaaaaa taaaataatt 480ttaaaatttc ttggaatttt atataaattt ttggattttt caaaaaaaat tgagattttt 540ttcttttttt tcgatttttt aaatttattt caggaaaata taaactaact tttctttgct 600ttgggtataa ttaatattag ataacccaca aattagatca ataggagctt catgtcctaa 660tcccatttaa ttacttttgt tgtatcatta atttagtcga ccttacatag tagctctatg 720gggcaaatag ttataaatgt taaattagta tttaaatctt gaagttttta atttaaagtt 780cagactatta gtattatatc aaatatttaa gggtaaatat atattctaat atctaagctt 840gggtcaaggt ttaaattaag tacttaaact tggttttata gttcaaattg atttaaataa 900ctaagtatta atttgaatta agaagcaaag ttcaagtacc taattagact ataaaaaaaa 960cttttgctag taaattgaac cttaaagtcg agtttagtta tctaattgga caaaaaaatc 1020ttaaatacca atttaaaccc taaagtcaag tttaggtacc aaagtgtata tttatctaat 1080atttaaattt gatccaccta atttaaattt ttttggtcca atgcaataag agaattaatt 1140aatacttaca cacatgatag agatataccc acaacagata cacactacaa aaaacattaa 1200aaaatagaaa gatatatttc ctacaaaatt taaaagcatt taatttttta actaacatta 1260gacaaatgga aatggaaaga cttattttta agtttatgga tgaatctaat ttatctaaac 1320attgggtttt ttttttttgt gacgaaatat gggtgagaga aggtagtaag ctaagtaggg 1380gagtaatatc tcaaacaaat aattaaaaaa ctcctttaaa tgtggctata aatacctgaa 1440accaatcctt ctttcctcaa ctcaaatctt caatctttag atcatctctc caaaaaaata 1500ccatgagtaa acggaatccg aagattctga agatttttct gtatatgtta cttctcaact 1560ctctctttct catcatctac ttcgtttttc actcatcgtc gttttcaagt gtcgtagatg 1620tgatcggttt gcttgcgact gcagcctacg tgacgttggc gagcgcatac aaggtggtcc 1680agttcattaa cgtgtcgagc gtaacggatg tcgctggtct cgaaagtgat gctttgccgc 1740tcactccaag ggttgacgtt atcgtgccga cattcaatga gaactccagc acattgctcg 1800agtgcgtcgc ttctatatgc gcacaagact accgcggacc aataacgatt gtcgtggtag 1860acgatgggtc gaccaacaaa acatcatttc acgcagtatg cgacaagtac gcgagcgacg 1920aaaggttcat atttgtcgaa cttgatcaaa acaaggggaa gcgcgccgcg caaatggagg 1980ccatcaggag aacagacgga gacctgatac taaacgtaga ctcggacacg gttatagata 2040aggatgttgt tacaaagctt gcgtcgtcca tgagagcccc gaatgtcggt ggtgtcatgg 2100ggcagctcgt tgcaaagaat cgagaaagat cttggcttac cagattaatc gatatggagt 2160actggcttgc gtgtaacgag gagcgcattg cgcagtcgag gtttggctcc gtgatgtgtt 2220gttgtgggcc gtgcgccatg tatagaagat ctgcaattac gccactattg gcagaatatg 2280agcaccagac attcctaggg cgtccgagca actttggtga ggatcgccat ctcacaatcc 2340tgatgctgaa ggcgggattt cggaccgggt acgtcccagg tgccgtagcg aggacgttgg 2400ttccggatgg gctggcgccg tacctgcgcc agcaactccg ctgggcccgc agcacttatc 2460gcgacaccgc cctcgcctta cgtataaaga aaaatctaag caaatatatc acctttgaga 2520tatgcgcaca gaatttgggt acggctctct tacttgtgat gaccatgatt tcgctttcgc 2580tgactacatc agggtcgcaa acgcccgtta tcattctggg tgtcgttgtg gggatgtcta 2640taataagatg ttgttctgtc gcccttatag cgaaagattt tcggtttcta tacttcatcg 2700ttcactcagc gttgaatgtt ctaattttaa cgccgttaaa actctatgcc ctgttaacca 2760ttcgggatag tcggtggcta tcacgcgaga gttcctaagc tagcaagctt ggacacgctg 2820aaatcaccag tctctctcta caaatctatc tctctctatt ttctccataa taatgtgtga 2880gtagttccca gataagggaa ttagggttcc tatagggttt cgctcatgtg ttgagcatat 2940aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt 3000cctaaaacca aaatccagta ctaaaatcca gacgcgtcct gcaggcccgg gttaattaag 3060cggccgcttc acggaaagtt gttatatata agttcagtaa ataataatga aatataaatt 3120ttaattatat ctagtactca ataagaagat ggagaaagtt atgttaatta tagttataaa 3180ttatttataa atttaatata tatatataaa gaaaatagtt gtataactaa taattatttt 3240tacaatactt tatatagtta tatttaaaaa aattttaaaa ttaaaatact attattttgt 3300tcaatatatt aatatttata ttatttaatt tattattgaa tatgaataaa ttttttttga 3360aaattatatt tttaattttt agaaatttta tataactttc catatatata tttctgattt 3420gtcaatttct tttgagattt atctaaattg atttgaattt tttttatttt taaaaaataa 3480aataatttta aaatttcttg gaattttata taaatttttg gatttttcaa aaaaaattga 3540gatttttttc ttttttttcg attttttaaa tttatttcag gaaaatataa actaactttt 3600ctttgctttg ggtataatta atattagata acccacaaat tagatcaata ggagcttcat 3660gtcctaatcc catttaatta cttttgttgt atcattaatt tagtcgacct tacatagtag 3720ctctatgggg caaatagtta taaatgttaa attagtattt aaatcttgaa gtttttaatt 3780taaagttcag actattagta ttatatcaaa tatttaaggg taaatatata ttctaatatc 3840taagcttggg tcaaggttta aattaagtac ttaaacttgg ttttatagtt caaattgatt 3900taaataacta agtattaatt tgaattaaga agcaaagttc aagtacctaa ttagactata 3960aaaaaaactt ttgctagtaa attgaacctt aaagtcgagt ttagttatct aattggacaa 4020aaaaatctta aataccaatt taaaccctaa agtcaagttt aggtaccaaa gtgtatattt 4080atctaatatt taaatttgat ccacctaatt taaatttttt tggtccaatg caataagaga 4140attaattaat acttacacac atgatagaga tatacccaca acagatacac actacaaaaa 4200acattaaaaa atagaaagat atatttccta caaaatttaa aagcatttaa ttttttaact 4260aacattagac aaatggaaat ggaaagactt atttttaagt ttatggatga atctaattta 4320tctaaacatt gggttttttt tttttgtgac gaaatatggg tgagagaagg tagtaagcta 4380agtaggggag taatatctca aacaaataat taaaaaactc ctttaaatgt ggctataaat 4440acctgaaacc aatccttctt tcctcaactc aaatcttcaa tctttagatc atctctccaa 4500aaaaatacca tgtgcggaat tgttggcgca atagcacaaa gggacgtagc agaaatcctt 4560cttgaaggac tccgtcgtct ggaatacaga ggatatgatt ctgccggtct agccgttgta 4620gatgccgaag gtcacatgac acgtctaaga cgtctgggta aggttcaaat gctggctcaa 4680gcagccgaag aacatccttt acatggtggc acaggtattg ctcacactag atgggctact 4740cacggtgaac cttcagaggt aaatgctcat ccacatgtct ctgagcacat tgtggtcgtt 4800cacaacggga tcatcgaaaa ccatgaacca cttcgagaag agctgaaagc tcgtggctat 4860actttcgttt cagagacaga cactgaggtg attgctcatc tcgtgaactg ggaactgaaa 4920caagggggaa ctctgagaga ggctgttcta cgtgctatcc ctcaattacg tggtgcttac 4980gggacagtga tcatggattc aagacaccca gatacactgc tggcagcaag gtctggtagt 5040ccactggtga ttggactggg gatgggagaa aactttatcg cttcggatca actggctctg 5100ttacctgtga cacggagatt tatcttcctt gaagagggcg atatcgcgga aataactcga

5160cgtagcgtaa acatcttcga taaaaccgga gcagaagtaa aacgccagga tatcgaatcc 5220aatcttcaat acgacgccgg cgataaaggc atataccgac actacatgca gaaagagatc 5280tacgagcaac cgaacgctat caagaatacc cttactgggc gtatctcaca tggtcaggtt 5340gacttatctg aactgggacc aaacgcagac gaactactgt cgaaggtaga acatattcag 5400atcctcgcgt gtggtacttc ttataactct ggtatggtca gtcgctattg gtttgaatca 5460ctggcaggaa ttccttgcga cgtcgaaatt gcctcggaat tcagatatcg caagtctgca 5520gtaagacgca acagcctgat gataacgtta tctcagtctg gagaaacggc tgatacactg 5580gctggattac gtctgtcaaa agagcttggc taccttggtt ctctagcaat ctgtaacgtt 5640cctggtagct ctcttgtgcg agaatctgat cttgctctta tgactaacgc tggtacagaa 5700atcggggtgg catccacaaa agcatttaca actcaactta cggtgctgct aatgcttgtg 5760gcaaagctgt ctagactcaa aggtctagat gcctccatcg agcatgatat cgttcatggt 5820ctgcaagctc ttcctagccg aattgagcag atgctgtcac aagacaaaag gattgaagcc 5880ctggcagaag atttctcaga caagcatcac gctttgtttc tcggtcgtgg cgatcagtat 5940cctatcgctc tcgaaggcgc attgaagctc aaagagatct cctatataca cgctgaagct 6000tacgctgcag gcgaactgaa acacggacct ctagctctta ttgacgcaga tatgcccgtt 6060atcgtcgttg caccaaacaa cgaattgctg gagaagctga aatcaaatat tgaagaggta 6120cgtgcaagag gcggacaact ttatgtcttc gctgagcaag atgccggttt tgtaagtagc 6180gataacatgc acatcatcga gatgcctcac gtggaagagg tgattgctcc gatcttctac 6240acagttcccc tgcagcttct ggcttatcac gttgccctta tcaaaggaac tgacgttgac 6300cagccaagga atctcgcaaa gtcagtaacg gttgagtaaa cgcgtggcgc gcccccgatc 6360cgcgtttgtg ttttctgggt ttctcactta agcgtctgcg ttttactttt gtattgggtt 6420tggcgtttag tagtttgcgg tagcgttctt gttatgtgta attacgcttt ttcttcttgc 6480ttcagcagtt tcggttgaaa tataaatcga atcaagtttc actttatcag cgttgtttta 6540aattttggca ttaaattggt gaaaattgct tcaattttgt atctaaatag aagagacaac 6600atgaaattcg acttttgacc tcaaatcttc gaacatttat ttcctgattt cacgatggat 6660gaggataacg aaagggcggt tcctatgtcc gggaaagttc ccgtagaaga caatgagcaa 6720agctactgaa acgcggacac gacgtcgcat tggtacggat atgagttaaa ccgactcaat 6780tcctttatta agacataaac cgattttggt taaagtgtaa cagtgagctg atataaaacc 6840gaaacaaacc ggtacaagtt tgattgagca acttgatgac aaacttcaga attttggtta 6900ttgaatgaaa atcatagtct aatcgtaaaa aatgtacaga agaaaagcta gagcagaaca 6960aagattctat attctggttc caatttatca tcgctttaac gtccctcaga tttgatcggg 7020gaattcgata tcattaccct gttatcccta aagcttatta atgtttgtcg aggagaaata 7080tgagtcgagg catggataca ctaagttccc ctgaagtgag catgatcttt gatgctgaga 7140tgattcccag agcaagatag tttgtgctgc aagtgacaca attgtaatga aaccaccact 7200caacgaattt acttgtggct ttgacatgtc gtgtgctctg tttgtatttg tgagtgccgg 7260ttggtaatta tttttgttaa tgtgatttta aaacctctta tgtaaatagt tactttatct 7320attgaagtgt gttcttgtgg tctatagttt ctcaaaggga aattaaaatg ttgacatccc 7380atttacaatt gataacttgg tatacacaaa ctttgtaaat ttggtgatat ttatggtcga 7440aagaaggcaa tacccattgt atgttccaat atcaatatca atacgataac ttgataatac 7500taacatatga ttgtcattgt ttttccagta tcaatataca ttaagctact acaaaattag 7560tataaatcac tatattataa atctttttcg gttgtaactt gtaattcgtg ggtttttaaa 7620ataaaagcat gtgaaaattt tcaaataatg tgatggcgca attttatttt ccgagttcca 7680aaatattgcc gcttcattac cctaatttgt ggcgccacat gtaaaacaaa agacgattct 7740tagtggctat cactgccatc acgcggatca ctaatatgaa ccgtcgatta aaacagatcg 7800acggtttata catcatttta ttgtacacac ggatcgatat ctcagccgtt agatttaata 7860tgcgatctga ttgctcaaaa aatagactct ccgtctttgc ctataaaaac aatttcacat 7920ctttctcacc caaatctact cttaaccgtt cttcttcttc tacagacatc aatttctctc 7980gactctagag gatccaagct tatcgatttc gaacccctca ggcgaagaac aggtatgatt 8040tgtttgtaat tagatcaggg gtttaggtct ttccattact ttttaatgtt ttttctgtta 8100ctgtctccgc gatctgattt tacgacaata gagtttcggg ttttgtccca ttccagtttg 8160aaaataaagg tccgtctttt aagtttgctg gatcgataaa cctgtgaaga ttgagtctag 8220tcgatttatt ggatgatcca ttcttcatcg tttttttctt gcttcgaagt tctgtataac 8280cagatttgtc tgtgtgcgat tgtcattacc tagccgtgta tcgagaacta gggttttcga 8340gtcaattttg ccccttttgg ttatatctgg ttcgataacg attcatctgg attagggttt 8400taagtggtga cgtttagtat tccaatttct tcaaaattta gttatggata atgaaaatcc 8460ccaattgact gttcaatttc ttgttaaatg cgcagatcac aatggcttcg atctcctcct 8520cagtcgcgac cgttagccgg accgcccctg ctcaggccaa catggtggct ccgttcaccg 8580gccttaagtc caacgccgcc ttccccacca ccaagaaggc taacgacttc tccacccttc 8640ccagcaacgg tggaagagtt caatgtatgc aggtgtggcc ggcctacggc aacaagaagt 8700tcgagacgct gtcgtacctg ccgccgctgt ctatggcgcc caccgtgatg atggcctcgt 8760cggccaccgc cgtcgctccg ttccaggggc tcaagtccac cgccagcctc cccgtcgccc 8820gccgctcctc cagaagcctc ggcaacgtca gcaacggcgg aaggatccgg tgcatggccg 8880gcgccgagga gatcgtgctg cagcccatca aggagatctc cggcaccgtc aagctgccgg 8940ggtccaagtc gctttccaac cggatcctcc tactcgccgc cctgtccgag gggacaacag 9000tggttgataa cctgctgaac agtgaggatg tccactacat gctcggggcc ttgaggactc 9060ttggtctctc tgtcgaagcg gacaaagctg ccaaaagagc tgtagttgtt ggctgtggtg 9120gaaagttccc agttgaggat gctaaagagg aagtgcagct cttcttgggg aatgctggaa 9180tcgcaatgcg gtccttgaca gcagctgtta ctgctgctgg tggaaatgca acttacgtgc 9240ttgatggagt accaagaatg agggagagac ccattggcga cttggttgtc ggattgaagc 9300agcttggtgc agatgttgat tgtttccttg gcactgactg cccacctgtt cgtgtcaatg 9360gaatcggagg gctacctggt ggcaaggtca agctgtctgg ctccatcagc agtcagtact 9420tgagtgcctt gctgatggct gctcctttgg ctcttgggga tgtggagatt gaaatcattg 9480ataaattaat ctccattccg tacgtcgaaa tgacattgag attgatggag cgttttggtg 9540tgaaagcaga gcattctgat agctgggaca gattctacat taagggaggt caaaaataca 9600agtcccctaa aaatgcctat gttgaaggtg atgcctcaag cgcaagctat ttcttggctg 9660gtgctgcaat tactggaggg actgtgactg tggaaggttg tggcaccacc agtttgcagg 9720gtgatgtgaa gtttgctgag gtactggaga tgatgggagc gaaggttaca tggaccgaga 9780ctagcgtaac tgttactggc ccaccgcggg agccatttgg gaggaaacac ctcaaggcga 9840ttgatgtcaa catgaacaag atgcctgatg tcgccatgac tcttgctgtg gttgccctct 9900ttgccgatgg cccgacagcc atcagagacg tggcttcctg gagagtaaag gagaccgaga 9960ggatggttgc gatccggacg gagctaacca agctgggagc atctgttgag gaagggccgg 10020actactgcat catcacgccg ccggagaagc tgaacgtgac ggcgatcgac acgtacgacg 10080accacaggat ggcgatggct ttctcccttg ccgcctgtgc cgaggtcccc gtcaccatcc 10140gggaccctgg gtgcacccgg aagaccttcc ccgactactt cgatgtgctg agcactttcg 10200tcaagaatta agctctagaa ctagtggatc ccccgatccg cgtttgtgtt ttctgggttt 10260ctcacttaag cgtctgcgtt ttacttttgt attgggtttg gcgtttagta gtttgcggta 10320gcgttcttgt tatgtgtaat tacgcttttt cttcttgctt cagcagtttc ggttgaaata 10380taaatcgaat caagtttcac tttatcagcg ttgttttaaa ttttggcatt aaattggtga 10440aaattgcttc aattttgtat ctaaatagaa gagacaacat gaaattcgac ttttgacctc 10500aaatcttcga acatttattt cctgatttca cgatggatga ggataacgaa agggcggttc 10560ctatgtccgg gaaagttccc gtagaagaca atgagcaaag ctactgaaac gcggacacga 10620cgtcgcattg gtacggatat gagttaaacc gactcaattc ctttattaag acataaaccg 10680attttggtta aagtgtaaca gtgagctgat ataaaaccga aacaaaccgg tacaagtttg 10740attgagcaac ttgatgacaa acttcagaat tttggttatt gaatgaaaat catagtctaa 10800tcgtaaaaaa tgtacagaag aaaagctaga gcagaacaaa gattctatat tctggttcca 10860atttatcatc gctttaacgt ccctcagatt tgatcgggaa accaaaacgt cgtgagacag 10920tttggttaac tataacggtc ctaaggtagc gatcgaggca ttacggcatt acggcactcg 10980cgagggtccg aattcgagca tggagccatt tacaattgaa tatatcctgc cg 11032411020DNAArtificialT-DNA of pTDBI 250 4aattacaacg gtatatatcc tgccagtact gggccccctc gagggcgatc gcgcggccgc 60atgattagtt agatcaagct tttgagtctt caaaaacata aaaattacaa aaaaaaaaca 120aacttaaaat catttatcaa tttgaacaac aaagcttggc cgaatgctaa gagcttaaaa 180atggcttctt ttgtttcttt ttgttgcaaa cggtggagag aagagggaaa tgaagattga 240ccatattttt ttattatgtt ttaacatata atattaataa tttaatcata attatacttt 300ggtgaatgtg acagtgggga gatacgtaaa gtatataaca ttatactttt tgcaagcagt 360tggctggtct acccaagagt gatcaaagtt tgagctgcct tcaatgagcc aatttttgcc 420cataatggat aaaggcaatt tgtttagttc aactgctcac agaataatgt taaaatgaaa 480ttaaaataag gtggcctggt cacacacaca aaaaaaaact aatgttggtt ggttgaattt 540tatattacgg aatgtaatat tatattttaa aataaaatta tgttatttag attcttaata 600ttttgagcat tccatactat aatttcgtat acataatatt aaaatatagt aatataaagt 660gtaattaact ttaaattaca agcataatat taaattttga atcaattaat ttttatttct 720attattttaa ttaatttagt ctattttttc aaaataaaat ttaaatctaa ataaaaataa 780tttttcctta atgttgaaac aactcatgtt atacttcaaa attataagta ttatatttac 840cttgatgatt tatttattag tatattaatt ctgattataa ttatggtggg atacaatcgc 900tttccactaa atattttaac tatgatttat aaatttattt caacatcgta tatttactta 960ttaatacata atttatcata attttatgga aattgagacc aagaaacatt aagagaacaa 1020attctataac aaagacaatt tagaaaaaaa tgtactttta ggtaatttta agtactctta 1080accaaacaca aaaattcaaa tcaaatgaac taaataagat aatataacat acggaacatc 1140ttacttgtaa tcttacattc ccataatttt attatgaaaa ataatcttat attactcgaa 1200ctaaatgttg tcacaaatta ttatctaaat aaagaaaaac acttaatttt tataacattt 1260tttcatatat ttgaaagatt atattttgta tatttacgta aaaatatttg acatagattg 1320agcaccttct taacataatc ccaccataag tcaagtatgt agatgagaaa ttggtacaaa 1380caacgtgggg ccaaatccca ccaaaccatc tctcattctc tcctataaaa ggcttgctac 1440acatagacaa caatccacac acaaatacac gttcttttct ttctatttga ttaaccatga 1500gtaaacggaa tccgaagatt ctgaagattt ttctgtatat gttacttctc aactctctct 1560ttctcatcat ctacttcgtt tttcactcat cgtcgttttc aagtgtcgta gatgtgatcg 1620gtttgcttgc gactgcagcc tacgtgacgt tggcgagcgc atacaaggtg gtccagttca 1680ttaacgtgtc gagcgtaacg gatgtcgctg gtctcgaaag tgatgctttg ccgctcactc 1740caagggttga cgttatcgtg ccgacattca atgagaactc cagcacattg ctcgagtgcg 1800tcgcttctat atgcgcacaa gactaccgcg gaccaataac gattgtcgtg gtagacgatg 1860ggtcgaccaa caaaacatca tttcacgcag tatgcgacaa gtacgcgagc gacgaaaggt 1920tcatatttgt cgaacttgat caaaacaagg ggaagcgcgc cgcgcaaatg gaggccatca 1980ggagaacaga cggagacctg atactaaacg tagactcgga cacggttata gataaggatg 2040ttgttacaaa gcttgcgtcg tccatgagag ccccgaatgt cggtggtgtc atggggcagc 2100tcgttgcaaa gaatcgagaa agatcttggc ttaccagatt aatcgatatg gagtactggc 2160ttgcgtgtaa cgaggagcgc attgcgcagt cgaggtttgg ctccgtgatg tgttgttgtg 2220ggccgtgcgc catgtataga agatctgcaa ttacgccact attggcagaa tatgagcacc 2280agacattcct agggcgtccg agcaactttg gtgaggatcg ccatctcaca atcctgatgc 2340tgaaggcggg atttcggacc gggtacgtcc caggtgccgt agcgaggacg ttggttccgg 2400atgggctggc gccgtacctg cgccagcaac tccgctgggc ccgcagcact tatcgcgaca 2460ccgccctcgc cttacgtata aagaaaaatc taagcaaata tatcaccttt gagatatgcg 2520cacagaattt gggtacggct ctcttacttg tgatgaccat gatttcgctt tcgctgacta 2580catcagggtc gcaaacgccc gttatcattc tgggtgtcgt tgtggggatg tctataataa 2640gatgttgttc tgtcgccctt atagcgaaag attttcggtt tctatacttc atcgttcact 2700cagcgttgaa tgttctaatt ttaacgccgt taaaactcta tgccctgtta accattcggg 2760atagtcggtg gctatcacgc gagagttcct aagctagcaa gcttggacac gctgaaatca 2820ccagtctctc tctacaaatc tatctctctc tattttctcc ataataatgt gtgagtagtt 2880cccagataag ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa 2940cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 3000accaaaatcc agtactaaaa tccagacgcg tcctgcaggc ccgggttaat taagcggccg 3060catgattagt tagatcaagc ttttgagtct tcaaaaacat aaaaattaca aaaaaaaaac 3120aaacttaaaa tcatttatca atttgaacaa caaagcttgg ccgaatgcta agagcttaaa 3180aatggcttct tttgtttctt tttgttgcaa acggtggaga gaagagggaa atgaagattg 3240accatatttt tttattatgt tttaacatat aatattaata atttaatcat aattatactt 3300tggtgaatgt gacagtgggg agatacgtaa agtatataac attatacttt ttgcaagcag 3360ttggctggtc tacccaagag tgatcaaagt ttgagctgcc ttcaatgagc caatttttgc 3420ccataatgga taaaggcaat ttgtttagtt caactgctca cagaataatg ttaaaatgaa 3480attaaaataa ggtggcctgg tcacacacac aaaaaaaaac taatgttggt tggttgaatt 3540ttatattacg gaatgtaata ttatatttta aaataaaatt atgttattta gattcttaat 3600attttgagca ttccatacta taatttcgta tacataatat taaaatatag taatataaag 3660tgtaattaac tttaaattac aagcataata ttaaattttg aatcaattaa tttttatttc 3720tattatttta attaatttag tctatttttt caaaataaaa tttaaatcta aataaaaata 3780atttttcctt aatgttgaaa caactcatgt tatacttcaa aattataagt attatattta 3840ccttgatgat ttatttatta gtatattaat tctgattata attatggtgg gatacaatcg 3900ctttccacta aatattttaa ctatgattta taaatttatt tcaacatcgt atatttactt 3960attaatacat aatttatcat aattttatgg aaattgagac caagaaacat taagagaaca 4020aattctataa caaagacaat ttagaaaaaa atgtactttt aggtaatttt aagtactctt 4080aaccaaacac aaaaattcaa atcaaatgaa ctaaataaga taatataaca tacggaacat 4140cttacttgta atcttacatt cccataattt tattatgaaa aataatctta tattactcga 4200actaaatgtt gtcacaaatt attatctaaa taaagaaaaa cacttaattt ttataacatt 4260ttttcatata tttgaaagat tatattttgt atatttacgt aaaaatattt gacatagatt 4320gagcaccttc ttaacataat cccaccataa gtcaagtatg tagatgagaa attggtacaa 4380acaacgtggg gccaaatccc accaaaccat ctctcattct ctcctataaa aggcttgcta 4440cacatagaca acaatccaca cacaaataca cgttcttttc tttctatttg attaaccatg 4500tgcggaattg ttggcgcaat agcacaaagg gacgtagcag aaatccttct tgaaggactc 4560cgtcgtctgg aatacagagg atatgattct gccggtctag ccgttgtaga tgccgaaggt 4620cacatgacac gtctaagacg tctgggtaag gttcaaatgc tggctcaagc agccgaagaa 4680catcctttac atggtggcac aggtattgct cacactagat gggctactca cggtgaacct 4740tcagaggtaa atgctcatcc acatgtctct gagcacattg tggtcgttca caacgggatc 4800atcgaaaacc atgaaccact tcgagaagag ctgaaagctc gtggctatac tttcgtttca 4860gagacagaca ctgaggtgat tgctcatctc gtgaactggg aactgaaaca agggggaact 4920ctgagagagg ctgttctacg tgctatccct caattacgtg gtgcttacgg gacagtgatc 4980atggattcaa gacacccaga tacactgctg gcagcaaggt ctggtagtcc actggtgatt 5040ggactgggga tgggagaaaa ctttatcgct tcggatcaac tggctctgtt acctgtgaca 5100cggagattta tcttccttga agagggcgat atcgcggaaa taactcgacg tagcgtaaac 5160atcttcgata aaaccggagc agaagtaaaa cgccaggata tcgaatccaa tcttcaatac 5220gacgccggcg ataaaggcat ataccgacac tacatgcaga aagagatcta cgagcaaccg 5280aacgctatca agaataccct tactgggcgt atctcacatg gtcaggttga cttatctgaa 5340ctgggaccaa acgcagacga actactgtcg aaggtagaac atattcagat cctcgcgtgt 5400ggtacttctt ataactctgg tatggtcagt cgctattggt ttgaatcact ggcaggaatt 5460ccttgcgacg tcgaaattgc ctcggaattc agatatcgca agtctgcagt aagacgcaac 5520agcctgatga taacgttatc tcagtctgga gaaacggctg atacactggc tggattacgt 5580ctgtcaaaag agcttggcta ccttggttct ctagcaatct gtaacgttcc tggtagctct 5640cttgtgcgag aatctgatct tgctcttatg actaacgctg gtacagaaat cggggtggca 5700tccacaaaag catttacaac tcaacttacg gtgctgctaa tgcttgtggc aaagctgtct 5760agactcaaag gtctagatgc ctccatcgag catgatatcg ttcatggtct gcaagctctt 5820cctagccgaa ttgagcagat gctgtcacaa gacaaaagga ttgaagccct ggcagaagat 5880ttctcagaca agcatcacgc tttgtttctc ggtcgtggcg atcagtatcc tatcgctctc 5940gaaggcgcat tgaagctcaa agagatctcc tatatacacg ctgaagctta cgctgcaggc 6000gaactgaaac acggacctct agctcttatt gacgcagata tgcccgttat cgtcgttgca 6060ccaaacaacg aattgctgga gaagctgaaa tcaaatattg aagaggtacg tgcaagaggc 6120ggacaacttt atgtcttcgc tgagcaagat gccggttttg taagtagcga taacatgcac 6180atcatcgaga tgcctcacgt ggaagaggtg attgctccga tcttctacac agttcccctg 6240cagcttctgg cttatcacgt tgcccttatc aaaggaactg acgttgacca gccaaggaat 6300ctcgcaaagt cagtaacggt tgagtaaacg cgtggcgcgc ccccgatccg cgtttgtgtt 6360ttctgggttt ctcacttaag cgtctgcgtt ttacttttgt attgggtttg gcgtttagta 6420gtttgcggta gcgttcttgt tatgtgtaat tacgcttttt cttcttgctt cagcagtttc 6480ggttgaaata taaatcgaat caagtttcac tttatcagcg ttgttttaaa ttttggcatt 6540aaattggtga aaattgcttc aattttgtat ctaaatagaa gagacaacat gaaattcgac 6600ttttgacctc aaatcttcga acatttattt cctgatttca cgatggatga ggataacgaa 6660agggcggttc ctatgtccgg gaaagttccc gtagaagaca atgagcaaag ctactgaaac 6720gcggacacga cgtcgcattg gtacggatat gagttaaacc gactcaattc ctttattaag 6780acataaaccg attttggtta aagtgtaaca gtgagctgat ataaaaccga aacaaaccgg 6840tacaagtttg attgagcaac ttgatgacaa acttcagaat tttggttatt gaatgaaaat 6900catagtctaa tcgtaaaaaa tgtacagaag aaaagctaga gcagaacaaa gattctatat 6960tctggttcca atttatcatc gctttaacgt ccctcagatt tgatcgggga attcgatatc 7020attaccctgt tatccctaaa gcttattaat gtttgtcgag gagaaatatg agtcgaggca 7080tggatacact aagttcccct gaagtgagca tgatctttga tgctgagatg attcccagag 7140caagatagtt tgtgctgcaa gtgacacaat tgtaatgaaa ccaccactca acgaatttac 7200ttgtggcttt gacatgtcgt gtgctctgtt tgtatttgtg agtgccggtt ggtaattatt 7260tttgttaatg tgattttaaa acctcttatg taaatagtta ctttatctat tgaagtgtgt 7320tcttgtggtc tatagtttct caaagggaaa ttaaaatgtt gacatcccat ttacaattga 7380taacttggta tacacaaact ttgtaaattt ggtgatattt atggtcgaaa gaaggcaata 7440cccattgtat gttccaatat caatatcaat acgataactt gataatacta acatatgatt 7500gtcattgttt ttccagtatc aatatacatt aagctactac aaaattagta taaatcacta 7560tattataaat ctttttcggt tgtaacttgt aattcgtggg tttttaaaat aaaagcatgt 7620gaaaattttc aaataatgtg atggcgcaat tttattttcc gagttccaaa atattgccgc 7680ttcattaccc taatttgtgg cgccacatgt aaaacaaaag acgattctta gtggctatca 7740ctgccatcac gcggatcact aatatgaacc gtcgattaaa acagatcgac ggtttataca 7800tcattttatt gtacacacgg atcgatatct cagccgttag atttaatatg cgatctgatt 7860gctcaaaaaa tagactctcc gtctttgcct ataaaaacaa tttcacatct ttctcaccca 7920aatctactct taaccgttct tcttcttcta cagacatcaa tttctctcga ctctagagga 7980tccaagctta tcgatttcga acccctcagg cgaagaacag gtatgatttg tttgtaatta 8040gatcaggggt ttaggtcttt ccattacttt ttaatgtttt ttctgttact gtctccgcga 8100tctgatttta cgacaataga gtttcgggtt ttgtcccatt ccagtttgaa aataaaggtc 8160cgtcttttaa gtttgctgga tcgataaacc tgtgaagatt gagtctagtc gatttattgg 8220atgatccatt cttcatcgtt tttttcttgc ttcgaagttc tgtataacca gatttgtctg 8280tgtgcgattg tcattaccta gccgtgtatc gagaactagg gttttcgagt caattttgcc 8340ccttttggtt atatctggtt cgataacgat tcatctggat tagggtttta agtggtgacg 8400tttagtattc caatttcttc aaaatttagt tatggataat gaaaatcccc aattgactgt 8460tcaatttctt gttaaatgcg cagatcacaa tggcttcgat ctcctcctca gtcgcgaccg 8520ttagccggac cgcccctgct caggccaaca tggtggctcc gttcaccggc cttaagtcca 8580acgccgcctt ccccaccacc aagaaggcta acgacttctc cacccttccc agcaacggtg 8640gaagagttca atgtatgcag gtgtggccgg cctacggcaa caagaagttc gagacgctgt 8700cgtacctgcc gccgctgtct atggcgccca ccgtgatgat ggcctcgtcg gccaccgccg 8760tcgctccgtt ccaggggctc aagtccaccg ccagcctccc cgtcgcccgc cgctcctcca 8820gaagcctcgg caacgtcagc aacggcggaa ggatccggtg catggccggc gccgaggaga 8880tcgtgctgca gcccatcaag gagatctccg gcaccgtcaa gctgccgggg tccaagtcgc 8940tttccaaccg gatcctccta ctcgccgccc tgtccgaggg gacaacagtg gttgataacc 9000tgctgaacag tgaggatgtc cactacatgc tcggggcctt gaggactctt ggtctctctg 9060tcgaagcgga caaagctgcc aaaagagctg tagttgttgg ctgtggtgga aagttcccag 9120ttgaggatgc taaagaggaa

gtgcagctct tcttggggaa tgctggaatc gcaatgcggt 9180ccttgacagc agctgttact gctgctggtg gaaatgcaac ttacgtgctt gatggagtac 9240caagaatgag ggagagaccc attggcgact tggttgtcgg attgaagcag cttggtgcag 9300atgttgattg tttccttggc actgactgcc cacctgttcg tgtcaatgga atcggagggc 9360tacctggtgg caaggtcaag ctgtctggct ccatcagcag tcagtacttg agtgccttgc 9420tgatggctgc tcctttggct cttggggatg tggagattga aatcattgat aaattaatct 9480ccattccgta cgtcgaaatg acattgagat tgatggagcg ttttggtgtg aaagcagagc 9540attctgatag ctgggacaga ttctacatta agggaggtca aaaatacaag tcccctaaaa 9600atgcctatgt tgaaggtgat gcctcaagcg caagctattt cttggctggt gctgcaatta 9660ctggagggac tgtgactgtg gaaggttgtg gcaccaccag tttgcagggt gatgtgaagt 9720ttgctgaggt actggagatg atgggagcga aggttacatg gaccgagact agcgtaactg 9780ttactggccc accgcgggag ccatttggga ggaaacacct caaggcgatt gatgtcaaca 9840tgaacaagat gcctgatgtc gccatgactc ttgctgtggt tgccctcttt gccgatggcc 9900cgacagccat cagagacgtg gcttcctgga gagtaaagga gaccgagagg atggttgcga 9960tccggacgga gctaaccaag ctgggagcat ctgttgagga agggccggac tactgcatca 10020tcacgccgcc ggagaagctg aacgtgacgg cgatcgacac gtacgacgac cacaggatgg 10080cgatggcttt ctcccttgcc gcctgtgccg aggtccccgt caccatccgg gaccctgggt 10140gcacccggaa gaccttcccc gactacttcg atgtgctgag cactttcgtc aagaattaag 10200ctctagaact agtggatccc ccgatccgcg tttgtgtttt ctgggtttct cacttaagcg 10260tctgcgtttt acttttgtat tgggtttggc gtttagtagt ttgcggtagc gttcttgtta 10320tgtgtaatta cgctttttct tcttgcttca gcagtttcgg ttgaaatata aatcgaatca 10380agtttcactt tatcagcgtt gttttaaatt ttggcattaa attggtgaaa attgcttcaa 10440ttttgtatct aaatagaaga gacaacatga aattcgactt ttgacctcaa atcttcgaac 10500atttatttcc tgatttcacg atggatgagg ataacgaaag ggcggttcct atgtccggga 10560aagttcccgt agaagacaat gagcaaagct actgaaacgc ggacacgacg tcgcattggt 10620acggatatga gttaaaccga ctcaattcct ttattaagac ataaaccgat tttggttaaa 10680gtgtaacagt gagctgatat aaaaccgaaa caaaccggta caagtttgat tgagcaactt 10740gatgacaaac ttcagaattt tggttattga atgaaaatca tagtctaatc gtaaaaaatg 10800tacagaagaa aagctagagc agaacaaaga ttctatattc tggttccaat ttatcatcgc 10860tttaacgtcc ctcagatttg atcgggaaac caaaacgtcg tgagacagtt tggttaacta 10920taacggtcct aaggtagcga tcgaggcatt acggcattac ggcactcgcg agggtccgaa 10980ttcgagcatg gagccattta caattgaata tatcctgccg 11020

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. Fibers obtainable from a cotton plant consisting essentially of plant cells comprising a chimeric gene comprising the following operably linked DNA regions: a) a plant-expressible promotor, b) a DNA region coding for a GFAT polypeptide wherein said GFAT is encoded by a nucleotide sequence comprising i) a nucleotide sequence according to SEQ ID NO: 1, ii) or a variant thereof, wherein one or more nucleotides differ from the nucleotide sequence according to SEQ ID NO: 1, provided that said variant differs in no more than 20 nucleotides from SEQ ID NO: 1, which encodes a glutamine:fructose-6-phosphate-amidotransferase (GFAT) according to SEQ ID NO: 2 iii) or a complementary sequence of i) or ii)), and c) optionally a DNA region involved in transcription termination and polyadenylation wherein said cotton plant cells additionally comprise a second chimeric gene comprising the following operably linked DNA regions: a) a plant-expressible promotor, b) a DNA sequence coding for a chitin synthase polypeptide and c) optionally a DNA region involved in transcription termination and polyadenylation.

10. A yarn or fabric made from fibers according to claim 9.

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. Fibers according to claim 9, wherein said chitin synthase is an N-acetylglucosamine transferase of the Nod C type.

16. Fibers according to claim 15, wherein said chitin synthase polypeptide comprises a Golgi localization signal.