Patent application title: COTTON FIBERS WITH INCREASED GLUCOSAMINE CONTENT
Inventors:
IPC8 Class: AC12N1582FI
USPC Class:
1 1
Class name:
Publication date: 2019-03-21
Patent application number: 20190085349
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.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.
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
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