METHODS FOR ALTERING THE REACTIVITY OF PLANT CELL WALLS

Abstract

Methods and means are provided to produce positively charged oligosaccharides in the plant cell wall by introducing into said plant cell a Nodulation C protein fused to a heterologous Golgi signal anchor sequence.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 13/878,873 filed Apr. 24, 2013 which claims the benefit of PCT International Application Serial No. PCT/EP2011/004929 filed Sep. 30, 2011 which claims the benefit of U.S. Provisional Application Ser. No. 61/394,109 filed Oct. 18, 2010 and European Patent Application Serial No. 10013693.6, filed Oct. 15, 2010, the contents of which are herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "BCS10-2016.text," created on Apr. 11, 2013, and having a size of 115 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to the modification of the reactivity of plant cell walls, including secondary plant cell walls, particularly as they can be found in natural fibers of fiber producing plants. In particular, the present invention is related to cotton fibers with altered reactivity. The modified reactivity could be applied in methods for dyeing cell wall containing plant derived material such as natural fibers, using fiber-reactive dyes, to improve e.g. colorfastness, or to decrease the volumes of waste-water used during the dyeing process. The modified reactivity could also be applied to improve the reactivity of the natural fibers with reactants such as flame retardants, water, oil and soil repellents, anticrease agents, softeners, antistatic agents, fluorescent whitening agents etc.

[0004] The current invention provides methods to increase the efficiency of production of N-acetylglucosamine oligomers in plant cell walls, with the further advantage that the plants produced by methods of the invention do not show retardation in root growth.

BACKGROUND ART

[0005] Natural fibers, including cellulose containing natural fibers from plants, such as cotton and linen, have been used by mankind for more than 5000 years. Natural cellulose containing fibers, however, do not possess the chemical versatility of synthetic fibers, due to the relative inert nature of the cellulose consisting of .beta.-1-4 linked glucose monomers.

[0006] This relatively inert nature is e.g. apparent during the dyeing process of cotton fibers and fabrics. Several types of dyes are used to color cotton, such as direct dyes and, most importantly, 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.

[0007] 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.

[0008] During the dyeing process, large amounts of electrolytes are needed to shield the anionic dyes from the anionic fiber charges. Unreacted hydrolyzed dyes (up to 40%) need to be removed by multiple washing steps, generating large volumes of wastewater, also containing the above mentioned electrolytes.

[0009] Providing the cellulose fiber with a positive electric charge, e.g. by incorporation of positively charged chemical compounds, 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.

[0010] 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).

[0011] US patent application US2003/0134120 describes the coating of natural fibers with chitosan.

[0012] 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.

[0013] 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.

[0014] WO 00/09729 describes the expression of chitin synthase and chitin deacetylase genes in plants to alter the cell wall for industrial uses and improved disease resistance. Specifically cited uses are: to provide a single plant source of cellulose, chitin and chitosan, to increase tensile strength and to increase brittle snap. Specifically suggested chitin synthase genes are derived from fungal organisms. No experimental data are provided on the production of chitin or chitosan in plants, nor on the incorporation thereof in plant cell walls.

[0015] WO2006/136351 showed that the strategy as proposed in WO00/09729 does not lead to the functional incorporation of chitin into the plant cell wall. Instead, WO 2006/136351 discloses that chitin is effectively produced in the secondary cell wall of cotton fibers only when the N-acetylglucosamine transferase is relocated to the Golgi apparatus. For the fungal chitin synthase from Neurospora crassa, relocation to the Golgi apparatus is achieved by operable fusion of this fungal chitin synthase with a heterologous signal anchor sequence specific for the Golgi apparatus, and by expressing the resulting chimeric gene in plants. For the NODC type of N-acetylglucosamine transferase however, addition of a signal anchor sequence is not required for localization of the NodC protein to the Golgi apparatus, and for incoporation of chito-oligosaccharides into the plant cell wall without external GlcNAc feeding. Although chitin could be efficiently produced in the plant cell walls, it was also observed that transgenic plants comprising NODC had shorter roots as compared to wild-type plants.

[0016] Thus there remains a need for alternative methods to produce plant cell walls such as secondary cell walls which comprise positively charged polysaccharides. In particular a need exists for providing methods to produce plants with positively charged oligosaccharides in their cell walls, but without root growth retardation. These and other problems are solved as described hereinafter in the different embodiments, examples and claims.

SUMMARY OF THE INVENTION

[0017] In one embodiment, the invention provides a method for production of a plant cell, or a plant, such as a cotton plant, comprising positively charged oligosaccharides in the cell wall, particularly the secondary cell wall of a plant cell, comprising introducing a chimeric gene into the plant cell, whereby the chimeric gene comprises a plant-expressible promoter operably linked to a DNA region coding for a NODC-type N-acetylglucosamine transferase fused to a Golgi signal anchor sequence; and a transcription termination and polyadenylation region. In another embodiment, a method is provided for production of a plant, such as a cotton plant, comprising positively charged oligosaccharides in the cell using methods according to the invention, characterized in that the root length of said plant is essentially the same as that of a wild-type plant not comprising the NODC gene.

[0018] The invention further provides a method to produce plants comprising positively charged oligosaccharides in the cell wall, further comprising the step of deacetylating said oligosaccharides consisting of N-acetylglucosamine monomers by treating the cell wall from said plant with an alkali solution or through the enzymatic action of chitin deacetylases.

[0019] The invention also provides chimeric genes comprising a plant-expressible promoter; a DNA region coding for a Nodulation C protein fused to a signal anchor sequence for targeting to the membranes of the Golgi-apparatus; and a transcription termination and polyadenylation region, and plant cells, plants, such as cotton and cotton fibers comprising such a chimeric gene. In another embodiment, the invention provides plants consisting essentially of plant cells comprising a chimeric gene comprising a NODC fused to a Golgi signal anchor sequence, characterised in that the root length of said plant is essentially the same as that of a wild-type plant not comprising NODC.

[0020] The invention further provides plant cells, plants, such as cotton plants, cotton fibers and yarns generated from cotton fibers comprising the chimeric gene comprising NODC fused to a Golgi signal anchor sequence.

[0021] The invention also provides plant cell walls, comprising an increased amount of oligosaccharides, which can be positively charged oligosaccharides, such as oligo-N acetylglucosamines, with a polymerization degree of 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 3 to 5. Such plant cell walls are obtainable by the methods of the invention. These plant cell walls may be subjected to further chemical modification.

[0022] In a specific embodiment, the invention provides cotton fibers comprising an increased amount of the positively charged oligosaccharides mentioned herein, and yarns, textiles which comprise such cotton fibers. The cotton fibers may be used as such or may have been subjected to further chemical modification, including dying. These cotton fibers can be recognized e.g. through detection of the NODC comprising chimeric genes, through their increased binding of anionic dyes, including congo red, through their increased binding of wheat germ agglutinin or through their increased reactivity with amine-reactive dyes when compared to cotton fibers obtained from cotton plants of a an isogenic line which does not contain a chimeric NODC gene operably linked to a Golgi signal anchor sequence as described herein. The presence and/or the amount of oligosaccharides in the cotton fibers can also be determined directly through e.g. high performance thin layer chromatography (HPTLC) or high-performance liquid chromatography and mass spectrometry (HPLC-MS).

[0023] In another embodiment, the invention is directed towards the use of a DNA region coding for an N-acetylglucosamine transferase capable of being targeted to the Golgi apparatus of a plant cell to increase the amount of positively charged oligosacccharides in the cell wall of a plant cell or to increase the reactivity of plant cell walls for chemical modifications of such plant cell walls.

[0024] In one embodiment, the invention is directed to a method of dying cotton fibers, yarn or fabric comprising providing the fiber described herein or the yarn or fabric described herein and applying a dye reactive to said fibers, yarn or fabric.

[0025] The invention also provides chimeric genes comprising the following operably linked DNA regions: a plant-expressible promoter; a DNA region coding for a NODC-type N-acetylglucosamine transferase fused to a Golgi signal anchor sequence; and a transcription termination and polyadenylation region, and the use of these chimeric genes to increase the amount of positively charged oligosaccharides in the plant cell wall and to produce cotton fibers, yarns and fabrics with improved reactivity, such as dyeability.

BRIEF DESCRIPTION OF THE FIGURES

[0026] FIG. 1: Alignment of the amino acid sequence of different NODC proteins. Amino acid residues conserved in all proteins are indicated in bold. ROT_NODC_RHILP: NODC protein from Rhizobium leguminosarum (biovar phaseoli) (SEQ ID No 7); ROT_NODC_BRAJA: NODC protein from Bradyrhizobium japonicum (SEQ ID No 2); ROT_NODC_RHIS3 NODC protein from Rhizobium sp. (strain N33) (SEQ ID No 8); ROT_NODC_RHISN: NODC protein from Rhizobium sp (SEQ ID No 17); ROT_NODC_RHILV: NODC protein from Rhizobium leguminosarum (biovar viciae) (SEQ ID No 4) and ROT_NODC_AZOCA: NODC protein from Azorhizobium caulinodans (SEQ ID No 1).

[0027] FIG. 2: Alignment of the amino acid sequence of different NODC proteins. Amino acid residues conserved in all proteins are indicated in bold. ROT_NODC_BRAJA: NODC protein from Bradyrhizobium japonicum (SEQ ID No 2); ROT_NODC_RHIS3 NODC protein from Rhizobium sp. (strain N33) (SEQ ID No 8); ROT_NODC_RHISN: NODC protein from Rhizobium sp (SEQ ID No 17); ROT_NODC_RHILV: NODC protein from Rhizobium leguminosarum (biovar viciae) (SEQ ID No 4) and ROT_NODC_AZOCA: NODC protein from Azorhizobium caulinodans (SEQ ID No 1).

[0028] FIG. 3: Root length of wild-type (col) and transgenic Arabidopsis plants transformed with pTJN6 and pTGK42. A: comparison between wild-type plants and plants transformed with pTJN6 and with two lines containing pTGK42. Black bars: wild-type; checked bars: pTJN6-23; hatched bars: pTGK42-10; vertically striped bars: pTGK42-28. B: comparison between wild-type plants and different transgenic lines containing pTJN6. Black bars: wild-type; checked bars: pTJN6-4; hatched bars: TJN6-14; vertically striped bars: pTJN6-23; horizontally striped bars: pTJN6-26.

[0029] FIG. 4. Presence of mono-, di-, tri-, tetra- and pentamers of GlcNAc in Arabidopsis plants transformed with pTJN6. The circled values represent the values for the GlcNAc oligomers; the number of hexagons above these values represents the degree of polymerisation of these oligomers. The graphs are shown in pairs, of which the upper graph is wild-type, and the lower graph is the transformant containing pTJN6.

DETAILED DESCRIPTION OF DIFFERENT EMBODIMENTS OF THE INVENTION

[0030] The current invention is based on the finding that, upon expression in plant cells, fusion of a heterologous Golgi signal anchor sequence to a N-acetylglucosamine transferase of the NODC type unexpectedly increased incorporation of specific N-acetylglucosamine oligomers in plant cell walls up to 65-fold when compared to cell walls from plants expressing an N-acetylglucosamine transferase of the NODC type not fused to a Golgi signal anchor sequence. The synthesis of the GlcNAc oligomers did not require the external addition of GlcNAc to the growth medium.

[0031] At the same time, whereas expression of NODC in plants negatively affected root length, fusion of the heterologous Golgi signal anchor sequence to NODC restored the root length to wild-type level.

[0032] Thus, in a first embodiment of the invention, a method is provided for the production of positively charged oligosaccharides in the plant cell wall, particularly the secondary cell wall, wherein the method comprises the step of introducing a chimeric gene into the plant cell, and the chimeric gene comprising the following operably linked DNA fragments:

[0033] a plant-expressible promoter

[0034] a DNA region coding for an N-acetylglucosamine transferase of the NODC type fused to a signal anchor sequence for targeting to the membranes of the Golgi apparatus; and

[0035] a transcription termination and polyadenylation region.

[0036] In another embodiment, a method is provided for the production of a plant comprising positively charged oligosaccharides in the cell wall, particularly the secondary cell wall, wherein the method comprises the steps of

[0037] introducing a chimeric gene into the plant cell, and the chimeric gene comprising the following operably linked DNA fragments:

[0038] a plant-expressible promoter;

[0039] a DNA region coding for an N-acetylglucosamine transferase of the NODC type fused to a signal anchor sequence for targeting to the membranes of the Golgi apparatus; and

[0040] a transcription termination and polyadenylation region;

[0041] regeneration of said plant cell into a plant.

[0042] Suitable for the method of the invention is that the Nodulation C protein is expressed in said plant cell. The Nodulation C protein may be expressed in all cells of said plant. Alternatively, the Nodulation C protein may be expressed in specific tissues of said plant only, but not in others, such as roots or cotton leaves.

[0043] Nodulation proteins and their encoding genes are involved in the synthesis of the lipochitooligosacccharide signals or acetylated chitooligomers (Nod factors) which lead to the nodule formation typical of the symbiosis between Rhizobiaceae and leguminous plants.

[0044] The most crucial nod gene products required for the synthesis of these lipo-chito-oligosaccharides are NODA, NODB and NODC. In the absence of other nod gene products they can form a core signal consisting of oligomers of four or five N-acetylglucosamine residues carrying an N-linked acyl group. The function of each of the three proteins in the synthesis of nodulation factors is well known: NODC is an N-acetylglucosaminyl transferase which produces the chito-oligosacharide chain; the N-acetyl group from the non-reducing N-acetylglucosamine residue of the chito-oligosaccharide chain is removed by NODB, which acts as a chitin oligosaccharide deacetylase; NODA is involved in the attachment of the acyl chain to the free amino group generated by the action of NODB. Other Nod factors, encoded by other nod genes, provide for any of the decorating chemical groups discriminating the different nodulation factors. For the purposes of the present invention, only the NODC proteins and encoding genes are of relevance.

[0045] Nodulation C protein ("NODC protein") is a well characterized protein (for a review see Kamst and Spaink, 1999, Trends in Glycoscience and Glycotechnology, 11, pp 187-199). It belongs to a family of .beta.-polysaccharide synthase proteins that are involved in the synthesis of linear polysaccharides containing .beta.-linked monosaccharide residues. The enzymes that are structurally most closely related to NODC are transferases involved in the synthesis of chitin (.beta.-1-4 linked N-acetylglucosamines); cellulose (the polymer of .beta.-1-4 linked glucose residues); hyaluronic acid (a co-polymer of N-acetylglucosamine and glucuronic acid) and chitin oligosaccharides produced during early development of zebrafish embryos. Six short regions conserved between these proteins can be recognized. For NODC proteins, these short sequences correspond to:

[0046] 1) a K residue at position 23 of SEQ ID No 1 (NODC from Azorhizobium caulinodans)

[0047] 2) the sequence DDG at position 86-88 of SEQ ID No 1

[0048] 3) the sequence VDSDT at position 137-141 of SEQ ID No 1

[0049] 4) the sequence GPCAMYR at position 207-213 of SEQ ID No 1

[0050] 5) the sequence GEDRHL at position 237-242 of SEQ ID No 1; and

[0051] 6) the sequence QQLRW at position 274-278 of SEQ ID No 1

[0052] However, it is important to realize that some NODC proteins or variants thereof may exist wherein one or more of the above mentioned consensus sequences are not absolutely conserved.

[0053] NODC proteins are also frequently characterized by hydrophobic stretches of amino acid residues representing transmembrane domains (Barney et al. 1996, Molecular Microbiology 19, pp 443-453). The N-terminal hydrophobic domain spans the bacterial membrane in a N.sub.out-C.sub.in orientation, with the adjacent large hydrophilic domain being exposed to the bacterial cytoplasm. This orientation appears to be dependent upon the presence of the hydrophobic region(s) near the C-terminus, potentially containing three membrane spans, such that the C-terminus of NODC is normally located in the bacterial periplasm.

[0054] The large hydrophilic loop of NODC also has other structural similarity to similar regions in the other .beta.-glucosyl transferases. This region has been proposed to be made up of an A domain (which extends from about residue 45 to 140 in the sequence of SEQ ID No 4) consisting of alternating .beta.-sheets and .alpha.-helices, and a B-domain (corresponding to residues 215-280 of SEQ ID No 4) thought to be responsible for the processivity of NODC. In the A-domain, two aspartate residues are conserved (residues 88 and 139 of SEQ ID No. 4); in the B-domain one aspartate residue and the motif QXXRW (residue 240 and 276-280 of SEQ ID No 4) are also conserved and thought to be crucial for catalytic activity.

[0055] When different NODC proteins are compared among themselves, amino acid sequences which are more conserved are revealed. FIG. 1 represents an alignment of different NODC proteins from SEQ ID No 1, 2, 8, 4, 7, 17 and indicates a number of conserved residues between the different NODC proteins including (in order):

TABLE-US-00001 the sequence (SEQ ID No 18) PXVDVIXPXXNE the sequence (SEQ ID No 19) VDDGSXN the sequence (SEQ ID No 20) GDXXLDVDSDTXXXXDV the sequence (SEQ ID No 21) GXXMGQ the sequence (SEQ ID No 22) DMEYWLACNEERXXQXRFGXVMXCXGXCXMYR the sequence (SEQ ID No 23) FRTXYXPXAXAXTXVP the sequence (SEQ ID No 24) YLXQQLRWARSTXRXTXL the sequence (SEQ ID No 25) QNXGXXLL the sequence (SEQ ID No 26) RFXFXXXHXXXNXXXLXPLKXYALXT

[0056] FIG. 2 represents an alignment of a subset of different NODC proteins, showing even more conserved residues such as:

TABLE-US-00002 the sequence (SEQ ID No 27) WLTRLIDMEYWLACNEERXXQXRFGXVMCCCGPCAMYRRS the sequence (SEQ ID No 28) LLXXYEXQXFXGXPSXFGEDRHLTILMLXAGFRTXYVPXAXAXTXVP the sequence (SEQ ID No 29) YLRQQLRWARSTXRDTXLA

[0057] The length of the oligosaccharide backbone in lipo-chitin oligosaccharides produced by different Rhizobiaceae varies between two and six residues. It has been shown that the nodulation protein NODC is an important determinant of the chitin oligosaccharide chain length in the synthesis of the chito-oligosaccharide chain (Kamst et al., 1997, Journal of Bacteriology 179, p 2103-2108).

[0058] Coding regions coding for an N-acetylglucosamine transferase of the NODC type may be obtained directly from bacteria belonging to the genera Rhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Ralstonia, Cupriavidus, Streptomyces, Burkholderia, Sinorhizobium, Desulfobacterium, Dokdonia, Methylobacterium, Phyllobacterium or Psychroflexus. However, it will be immediately clear that such coding regions may also be made synthetically, even with a codon usage adapted to the plant, particularly the fiber producing plant into which the chimeric gene overexpresing the NODC type protein is introduced.

[0059] Different sequences for NODC proteins are available from databases such as the protein sequences identified by the following accession numbers: 1615305C, 1615305D, 1615305E, AAA26226, AAA63602, AAB16897, AAB24745, AAB34509, AAB47353, AAB51164, AAB71694, AAB91695, AAB95329, AAC80567, AAD11313, AAD11315, AAD11317, AAD11319, AAD11321, AAD11323, AAD11325, AAD11327, AAD11329, AAD11331, AAD11333, AAD11335, AAD11337, AAD11339, AAD11341, AAD11343, AAD11345, AAD11347, AAD11349, AAD11351, AAD11353, AAD11355, AAD11357, AAD11359, AAD11361, AAD11363, AAD11365, AAD11367, AAD11369, AAD11371, AAD11373, AAD11375, AAD11377, AAD11379, AAD11381, AAD11383, AAD11385, AAD11387, AAD11389, AAD11391, AAD11393, AAD11395, AAD11397, AAD11399, AAD11401, AAD11403, AAD11405, AAG60998, AAK00157, AAK39956, AAK39957, AAK39958, AAK39959, AAK39960, AAK39961, AAK39962, AAK39963, AAK39964, AAK39965, AAK39966, AAK39967, AAK50872, AAK65131, AAL88670, AAN62903, AAS91748, AAU11338, AAU11339, AAU11340, AAU11341, AAU11342, AAU11343, AAU11344, AAU11345, AAU11346, AAU11347, AAU11348, AAU11349, AAU11350, AAU11351, AAU11352, AAU11353, AAU11354, AAU11355, AAU11356, AAU11357, AAU11358, AAU11359, AAU11360, AAU11361, AAU11362, AAU11363, AAU11364, AAU11365, AAX30049, AAX30050, AAY44091, AAY44092, AAY44093, AAY89044, AAZ81541, ABC40958, ABC67303, ABD39006, ABD39007, ABD39008, ABD39009, ABD39010, ABD39011, ABD39012, ABD39013, ABD39014, ABD39015, ABD39016, ABD39017, ABD39018, ABD39019, ABD39020, ABD39021, ABD39022, ABD39023, ABD39024, ABD39025, ABD39026, ABD39027, ABD39028, ABD39029, ABD39030, ABD39031, ABD39032, ABD39033, ABD39034, ABD39035, ABD39036, ABD39037, ABD39038, ABD67413, ABD67416, ABD67419, ABD67422, ABD67425, ABD67428, ABD67431, ABD67434, ABD73319, ABD73320, ABD73321, ABD73322, ABD73323, ABD73324, ABD73325, ABD73326, ABD73327, ABD73328, ABD73329, ABD73330, ABD94161, ABD94162, ABD94163, ABD94164, ABD94165, ABF93198, ABF93199, ABF93200, ABF93201, ABF93202, ABM69186, ABM69187, ABM69188, ABM69189, ABM69190, ABN09217, ABN09218, ABN09219, ABN11177, ABN11178, ABN11179, ABP93834, ABS85176, ABS85177, ABS85178, ABS85179, ABS85180, ABS85181, ABS85182, ABU69044, ABU69045, ABU69046, ABU69047, ABU69048, ABU69049, ABU69050, ABU69051, ABU69052, ABU69053, ABU69054, ABU69055, ABU69056, ABU69057, ABU69058, ABU69059, ABU69060, ABU69061, ABU89879, ABV25689, ABV25690, ABV25691, ABV25692, ABV25693, ABV25694, ABW96196, ABW96197, ABW96198, ABW96199, ABW96200, ABW96201, ABW96202, ABW96203, ABW96204, ABW96205, ABW96206, ABW96207, ABW96208, ABW96209, ABW96210, ABW96211, ABY59633, ABY59634, ABY59635, ABY59636, ABY59637, ACA80309, ACA80310, ACA80311, ACA80312, ACA80313, ACC77565, ACD39337, ACD39338, ACD39339, ACD39340, ACD39341, ACD39342, ACD39343, ACD39344, ACD39345, ACD39346, ACD39347, ACD62595, ACD63093, ACD63094, ACD63095, ACD63096, ACD63097, ACD63098, ACD63099, ACD63100, ACD63101, ACD63102, ACD63103, ACD63104, ACF19762, ACF19763, ACF19764, ACF19765, ACF19766, ACF19767, ACF19768, ACF19769, ACF19770, ACH91221, ACH91222, ACH91223, ACH91224, ACH91225, ACH91226, ACH91227, ACH91228, ACH91229, ACH91230, ACH91231, ACH91232, ACH91233, ACH91242, ACH91243, ACH91244, ACH91245, ACH91246, ACH91247, ACH91248, ACH91249, ACI47333, ACI47334, ACI47335, ACI47336, ACI47337, ACI47338, ACI47339, ACI47340, ACI47341, ACI47342, ACI47343, ACI47344, ACI47345, ACL12058, ACL12059, ACL50517, ACL50518, ACL50519, ACL50520, ACL50521, ACL50522, ACL50523, ACM69382, ACM79634, ACM79635, ACM79636, ACM79637, ACM79638, ACM79639, ACM79640, ACM79641, ACM79642, ACM79643, ACM79644, ACM79645, ACM79646, ACN17701, ACN69201, ACN69202, ACN69203, ACN69204, ACN69205, ACN69206, ACN69207, ACN69208, ACN69209, ACN69210, ACN69211, ACN69212, ACN69213, AC058664, AC058665, AC058666, AC058667, AC058668, AC058669, AC058670, AC058671, AC058672, AC058673, AC058674, AC058675, ACP40990, ACS35430, ACS35434, ACT34091, ACT34094, ACT34097, ACT34100, ACT34101, ACT34104, ACT34107, ACT34110, ACT34113, ACT34116, ACT34119, ACT34122, ACT34125, ACT34128, ACT34131, ACT34134, ACT34137, ACT34140, ACT34143, ACV52950, ACV52951, ACV52952, ACV52953, ACV52954, ACV52955, ACX47326, ACX47327, ACX47328, ACX47329, ACX47330, ACX47331, ACX47332, ACX47333, ACX47334, ACY02884, ACY78518, ACZ52692, ACZ52693, ACZ52694, ACZ52695, ACZ52696, ACZ52697, ADD20957, ADD20958, ADD20959, ADD20960, ADD20961, ADD20962, ADD20963, ADD20964, ADD20965, ADD20966, ADD20967, ADD20968, ADD20969, ADD20970, ADD20971, ADG63645, ADG63646, ADG63647, ADG63648, ADG63649, ADG63650, ADG63651, ADG63652, ADJ18191, ADJ18192, BAA06082, BAA06083, BAA06084, BAA06085, BAA06086, BAA06087, BAA06088, BAA06089, BAA06090, BAA24092, BAB52500, C26813, CAA25810, CAA25811, CAA25814, CAA26310, CAA26311, CAA51773, CAA51774, CAA608779, CAA67139, CAB56055, CAC42489, CAD29949, CAD29950, CAD29951, CAD29952, CAD29953, CAD29954, CAD29955, CAD29956, CAD29957, CAD31533, CAD43933, CAD90257, CAD90583, CAD90584, CAD90585, CAD90586, CAD90587, CAD90588, CAH04369, CAN84684, CAP64017, EAQ38847, EAS72439, NP_106714, NP_435719, NP_443883, P04340, P04341, P04677, P04678, P04679, P06234, P06235, P17862, P24151, P26024, P50357, P53417, P72334, Q07755, Q53513, YP_001796208, YP_002605865, ZP_01050448 or ZP_01252570 (incorporated herein by reference).

[0060] Other entries in the UNIPROT databases referring to full length NODC proteins are summarized in the table below. All mentioned amino acid sequences referenced by the accession number are herein incorporated by reference.

TABLE-US-00003 TABLE full length NODC proteins UniProt UniProt/UniParc ID Accessions Species Name Length NODC_BRAJA P26024 Bradyrhizobium japonicum 485 NODC_AZOCA Q07755 Azorhizobium caulinodans 395 Q6PTX8_9RHIZ Q6PTX8 Rhizobium sp. SIN-1 408 Q70YC2_9BURK Q70YC2 Cupriavidus taiwanensis 450 Q6EX51_SINSB Q6EX51 Sinorhizobium sp. 452 NODC_RHIS3 P72334 Rhizobium sp. 450 NODC_RHILP P24151 Rhizobium leguminosarum 428 Q8GNH5_RHIME Q8GNH5 Rhizobium meliloti 421 Q53254_RHITR Q53254 Rhizobium tropici 452 Q9AQ23_BRASW Q9AQ23 Bradyrhizobium sp. 452 NODC_RHISN P50357 Rhizobium sp. 413 Q8KLG3_RHIET Q8KLG3 Rhizobium etli 443 Q9RAN5_MESS7 Q9RAN5 Mesorhizobium sp. 416 Q9Z3I6_BRASS Q9Z3I6 Bradyrhizobium sp. 481 NODC_RHILO P17862 Rhizobium loti 424 Q8KJI5_RHILO Q8KJI5 Rhizobium loti 424 NODC_RHIGA P50356 Rhizobium galegae 433 NODC_RHIME P04341 Rhizobium meliloti 426 Q9R614_RHIME Q9R614 Rhizobium meliloti 424 O52478_RHIME O52478 Rhizobium meliloti 402 Q52971_RHIME Q52971 Rhizobium meliloti 402 NODC_RHILV P04340 Rhizobium leguminosarum 424

[0061] However, it will be clear that variants of NODC proteins, wherein one or more amino acid residues have been deleted, substituted or inserted, which can be deduced from the above mentioned amino acid sequences, can also be used to the same effect in the methods according to the invention, provided that the enzymatic activity has not changed. These variant NODC proteins may have about 95% sequence identity to any one of the herein mentioned NODC proteins. A method for determining enzymatic activity of NODC proteins in vitro has been described e.g. by Kamst et al., 1997 Journal of Bacteriology, 179, p 2103-2108.

[0062] Thus, as used herein, an "N-acetylglucosamine transferase that is of the NODC type" is an N-acetylglucosamine transferase that catalyzes the transfer of the GlcNAc moiety from UDP-GlcNAc to a nascent chitin oligosaccharide. Preferably the protein contains the conserved regions which can be found by comparing the different NODC proteins.

[0063] Suitable for the methods of the invention are the proteins listed in SEQ ID No 1 to SEQ ID No 9, particularly the protein listed in SEQ ID No 1, and the DNA fragments encoding such a protein.

[0064] NODC should be equipped with heterologous signal anchor sequences targeting the NODC to the membranes of the Golgi apparatus. Such sequences are known in the art, including the sequences within and adjacent to the transmembrane segment of .alpha.-2,6-sialyltransferase (particularly the first 44 or 52 amino acids thereof; Munro et al. 1991, EMBO Journal, 10: 3577-3588); the signal anchor sequence from human galactosyl transferase (particularly the first 60 amino acids thereof) or the signal anchor sequence from the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) (Saint-Jore et al., 2002, The Plant Journal, 29: 661-678), the signal anchor sequence from .beta.1,2-xylosyltransferase protein (particularly the first 36 amino acids thereof; Pagny et al., 2003, The Plant Journal 33: 189-203), the signal anchor sequences of N-acetyl-glucosaminyl transferase I (particularly the first 77 amino acids thereof; Essl et al. 1999, FEBS Lett. 453:169-173) or a 20 amino acid fragment of the human lysosomal protein LAMP1 (Brandizzi et al., 2002, Plant Cell 14: 1077-1092) (all publication incorporated herein by reference). Other Golgi targeting signals to be employed by fusion at the C-terminus of the N-acetylglucosamine transferase include the amino acid sequence "YYHDL" (SEQ ID No 30) as can be found in Arabidopsis DAGAT1 protein or "LKLEI" (SEQ ID No 31) as can be found in Arabidopsis DAGAT2. Fusion of such Golgi signal anchor sequences to NODC by linking DNA fragments encoding the respective polypeptides can be achieved using standard recombinant DNA techniques.

[0065] A heterologous signal anchor sequence as used herein means a signal anchor sequence that is not naturally part of the protein to which it is fused. The heterologous signal anchor sequence can thus be derived from another protein from the same species, or can be derived from a protein from another species.

[0066] The chimeric genes according to the invention comprise a plant-expressible promoter. As used herein, the term "promoter" denotes any DNA which is recognized and bound (directly or indirectly) by a DNA-dependent RNA-polymerase during initiation of transcription. A promoter includes the transcription initiation site, and binding sites for transcription initiation factors and RNA polymerase, and can comprise various other sites (e.g., enhancers), at which gene expression regulatory proteins may bind.

[0067] 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, T-DNA gene promoters such as Pmas, Pnos, Ptr1, Ptr2, Cassava vein mosaic virus and the like.

[0068] A transcription termination and polyadenylation region as used herein is a sequence that drives the cleavage of the nascent RNA, whereafter a poly(A) tail is added at the resulting RNA 3' end. Transcription termination and polyadenylation signals functional in plants include, but are not limited to, 3'nos, 3'35S, 3'his and 3'g7.

[0069] A plant-expressible promoter that controls initiation and maintenance of transcription preferentially in fiber cells is a promoter that drives transcription of the operably linked DNA region to a higher level in fiber cells and the underlying epidermis cells than in other cells or tissues of the plant. Such promoters 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), the promoter from the seed coat and fiber-specific protease from cotton (Hou et al., 2008, Chinese Science Bulletin 53, pp 2639-2645), the promoter from fiber-specific R2R3 MYB gene from cotton (Pu et al., 2008, Genetics 180, pp 811-820), a promoter from an expansin gene from cotton (WO9830698), a promoter from a chitinase gene in cotton (US2003106097), the promoter of CesA1 (U.S. Pat. No. 6,271,443), or the promoters of the fiber specific genes described in U.S. Pat. No. 6,259,003 or 6,166,294 or WO96040924.

[0070] Positively charged oligosaccharides according to the invention can consist of N-acetylglucosamine oligomers such as .beta.1-4 linked N-acetylglucosamine oligomers. Said oligosaccharides can comprise 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 3 to 5 N-acetylglucosamine monomers.

[0071] Degree of polymerisation as used herein is the number of monomers present in an oligomer or in a polymer. The degree of polymerisation of an N-acetylglucosamine oligomer is the number of N-acetylglucosamine monomers present in said oligomer.

[0072] In another embodiment, the invention provides a method to produce plants comprising positively charged oligosaccharides in the cell wall using methods according to the invention, characterized in that said plants have a root length which is essentially the same as that of plants not comprising a NODC gene.

[0073] The root length is essentially the same when it has a length of at least 70%, or 80% or 90%, or 95%, or between 70% and 120%, or between 80% and 120%, or between 80% and 110%, or between 90% and 110%, or between 95% and 110% of, or which is identical to the length of the wild-type root not comprising a NODC gene.

[0074] In a further embodiment, the plants according to the invention are selected from cotton, hemp or flax. In a further embodiment, said plants are cotton plants comprising fibers.

[0075] The invention further provides plant cell walls, such as secondary cell walls or fibers including such cell walls obtained from plant cells using the methods according to the invention. Such plant cell walls comprise positively charged oligosaccharides, such as N-acetylglucosamine oligomers, .beta.1-4 linked N-acetylglucosamines, or chitin, embedded into the cellulose. The invention also provides the step of isolating plant cell walls and fibers from the plants obtained by the method of the invention.

[0076] The positively charged polysaccharides according to the invention may be further modified, e.g. partly or completely deacetylated such that oligomers comprising glucosamine residues are obtained. The amino-group of the resulting glucosamines is chemically more reactive than the aminoacetyl group of N-acetylglucosamine or the hydroxyl group of cellulose.

[0077] Deacetylation of N-acetylglucosamine can be performed chemically using methods including alkali hydrolysis, through application of thermo-mechano-chemical technology (Pelletier et al, 1990, Biotechnol Bioeng. 36, pp 310-315), using alkali impregnation technique (Rao et al, 1987, Indian Journal of Technology, 25, pp 194-196), using water-miscible organic solvents as diluents (Batista and Roberts, 1990, Makromolekulare Chemie-Macromolecular Chemistry and Physics, 191, pp 429-434.), using thiophenol to trap oxygen during deacetylation processes (Domard and Rinaudo, 1983, International Journal of Biological Macromolecules, 5, pp 49-52.), or using autoclaving conditions (No et al., 2000, Journal of Agricultural and Food Chemistry, 48, pp 2625-2627). Deacetylation of chitin can also be performed enzymatically using chitin deacetylases. Such chitin deacetylases include those from Mucor rouxii, Absidia coerulea, Aspergillus nidulans, Colletotrichum lindemuthianum and Saccharomyces cerevisiae.

[0078] The plant cell wall obtained according to the invention, particularly those which have been subjected to a deacetylation step, can be further chemically modified. Products containing such plant cell walls, such as fibers, yarns or fabrics have qualities resembling those of the cellulose-chitosan blends described in the art, including improved dyeability, improved inhibition of e.g. dermatophytes, controlled drug release etc.

[0079] The invention also provides the chimeric genes as herein described, and plant cells or plants containing such chimeric genes, and the use of said chimeric genes to increase the amount of positively charged oligosaccharides in the cell wall, or to increase the reactivity of plant cell walls, cotton fibers or yarns or fabrics for chemical modifications such as dyeability. The invention further provides plants, such as cotton plants, containing such chimeric genes characterized in that the root length of said plant is essentially the same as that of isogenic plants not containing such chimeric genes. The invention further provides the fibers from such cotton plant, and a yarn or fabric made from said fibers.

[0080] An increase in the efficiency of production of N-acetylglucosamine oligomers, or an increase in the amount of positively charged oligosaccharides in plant cell walls as used herein means an increase of positively charged oligosaccharides or N-acetylglucosamine oligomers in the plant cell walls which is at least 2-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or 2 to 100-fold, or 5 to 100-fold, or 10 to 100-fold or 20 to 100-fold.

[0081] In a specific embodiment, the invention provides cotton fibers obtained from or which can be obtained from cotton plants according to the methods of the invention, or comprising the chimeric gene according to the invention. In other words, cotton fibers are provided from cotton plants comprising in the genome, such as the nuclear genome, of their cells a chimeric gene comprising a plant-expressible promoter operably linked to a DNA region coding for a NODC-type N-acetylglucosamine transferase fused to a Golgi signal anchor sequence. Particular embodiments of DNA coding regions or promoters comprised in the chimeric genes transferred into cotton plants are as described elsewhere in this document.

[0082] 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 chimeric gene according to the invention, by the capacity of such fibers for increased staining with anionic dyes (including e.g. Congo Red), by the capacity of such fibers for increased staining with amine-reactive dyes (including e.g. tetrafluorophenyl ester). The cotton fibers according to the invention also have the capacity of binding of Wheat germ agglutinin which binds chito-oligmers. The cotton fibers according to the invention can also be distinguished from naturally occurring cotton fibers by direct detection of the N-acetylglucosamine and GlcNAc oligmers, such as chitobiose, chitotriose or chitotetraose, preferably after treatment of the fiber cell wall material with cellulase. The cotton fibers according to the invention may also be distinguished by their increased nitrogen content.

[0083] Cotton fibers according to the invention can also be distinguished from the chitosan coated fibers or from chitosan/cellulose blended yarns, in that the positively charged oligomers are more or less evenly distributed in the secondary plant cell walls making up the fibers. Accordingly, in microscopical sections of cotton fibers, stained e.g. with WGA or with congo red or with tetrafluorophenyl as described hereinafter, the dyes will be distributed more or less evenly throughout the cell walls making up the cotton fibers, whereas in chitosan-coated fibers, the staining will be concentrated at the coat of chitosan located as a sheet at the surface of the treated fibers.

[0084] Cotton fibers according to the invention can also be distinguished from other cotton fibers by detection of the NODC comprising chimeric genes in nucleic acids which remain in the plant material associated with cotton fibers.

[0085] The increased staining of the plant cell wall material according to the invention, by anionic dyes such as congo-red can be quantified e.g. by dying a uniform amount of material under standard conditions, spreading out the material over a standardized area (such as a well in a multiwell plate) digitalizing a picture of the area for the gray scale of the colored layer of material. The less gray, the more stained the plant cell wall material is. In this way, fibers and cell wall material according to the invention may be obtained with an increase of at least 10%, or at least 30%, or at least 50% in staining by congo-red compared to control cell wall material or fibers from isogenic plant lines without a NODC encoding gene.

[0086] The plant cell wall material according to the invention can also be stained with acid dyes such as Acid Orange 7. Fibers and cell wall material according to the invention may be obtained with an increase of at least 50%, or at least 70%, or between 50% and 100% in staining by Acid Orange 7 as compared to control cell wall material or fibers from isogenic plant lines without a NODC encoding gene.

[0087] The capacity of the novel cotton fibers to specifically bind wheat germ agglutin (detectable by the coupled fluorophoric group) is a clear distinguishing feature of the provided novel cotton fibers over the naturally occurring cotton fibers. Except for a very low background fluorescence, naturally occurring cotton fibers do not stain/fluoresce when treated with WGA-alexa fluor 488 or 555. The fluorescence of cotton fibers increases at least 5 times when chito-oligomers are present. Accordingly, the invention provides cotton fibers which are capable of specifically binding wheat germ agglutinin, or WGA coupled to a flurophore, such as WGA Alexa 488 or WGA Alexa 555 or which, when treated with WGA Alexa 488 or WGA Alexa 555 provide a bright fluorescence under UV light. This fluorescence is not restricted to the surface of the cotton fiber but is distributed throughout the cell wall of the fiber cells.

[0088] Plant cell wall material according to the invention, including cotton fibers typically possess chito-oligosaccharides in a concentration of at least 0.1 .mu.g/mg cell wall material, or at least 1 .mu.g/mg cell wall material, or at least 5 .mu.g/mg cell wall material.

[0089] Wherever the methods of the invention are directed to introduction of a chimeric gene in a plant cell, it will be clear that such methods can also be applied in cases whereby the plant cell is incorporated into a mature plant. E.g. transgenic cells may be regenerated into transgenic plants according to established methods.

[0090] Methods to transform plants cells and plants are well known in the art. 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.

[0091] The chimeric genes may be introduced by transformation in cotton plants from which embryogenic callus can be derived, such as Coker 312, Coker310, Coker 5Acala SJ-5, GSC25110, FiberMax 819, Siokra 1-3, T25, GSA75, Acala SJ2, Acala SJ4, Acala SJ5, Acala SJ-C1, Acala B1644, Acala B1654-26, Acala B1654-43, Acala B3991, Acala GC356, Acala GC510, Acala GAM1, Acala C1, Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B1810, Acala B2724, Acala B4894, Acala B5002, non Acala "picker" Siokra, "stripper" variety FC2017, Coker 315, STONEVILLE 506, STONEVILLE 825, DP50, DP61, DP90, DP77, DES119, McN235, HBX87, HBX191, HBX107, FC 3027, CHEMBRED A1, CHEMBRED A2, CHEMBRED A3, CHEMBRED A4, CHEMBRED B1, CHEMBRED B2, CHEMBRED B3, CHEMBRED C1, CHEMBRED C2, CHEMBRED C3, CHEMBRED C4, PAYMASTER 145, HS26, HS46, SICALA, PIMA S6 and ORO BLANCO PIMA, Fibermax.RTM. FM5013, FM5015, FM5017, FM989, FM832, FM966 and FM958, FM989, FM958, FM832, FM991, FM819, FM800, FM960, FM966, FM981, FM5035, FM5044, FM5045, FM5013, FM5015, FM5017 or FM5024 and plants with genotypes derived thereof.

[0092] "Cotton" as used herein includes Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium herbaceum or progeny from crosses between such species.

[0093] The methods and means of the current invention may also be employed for other plant species such as hemp, jute, flax and woody plants, including but not limited to Pinus spp., Populus spp., Picea spp., Eucalyptus spp. etc.

[0094] The obtained transformed plant can be used in a conventional breeding scheme to produce more transformed 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.

[0095] Reactive dyes which can be used in connection with the method of dying cotton fibers, yarn or fabric comprise Reactive Red 120 (RR120), Reactive Yellow 176 (RY176), Levafix Blue CA, Reactive Orange 35, Reactive Black 5, Reactive Red 116. Dyeing can also be done with Acid dyes, such as Acid Orange 7, Acid Blue 62, Acid Blue 281, Acid Red 361, Acid Blue 277, Acid Red 4, Acid Blue 113, Acid Yellow 137, Acid Blue 127:1, and Acid Blue 193. These dyes are applied according to protocols well-known in the art.

[0096] 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 actually cited ones, i.e., 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.

[0097] The transgenic plant cells and plants obtained by the methods disclosed herein may also be further used in subsequent transformation procedures, e.g. to introduce a further chimeric gene.

[0098] The cotton plants or seed comprising the chimeric gene disclosed herein or obtained by the methods disclosed herein may further be treated with cotton herbicides such as Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; cotton insecticides such as Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor; and cotton fungicides such as Azoxystrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fenamidone, Fluazinam, Fluopyram, Fluoxastrobin, Fluxapyroxad, Iprodione, Isopyrazam, Isotianil, Mancozeb, Maneb, Metominostrobin, Penthiopyrad, Picoxystrobin, Propineb, Prothioconazole, Pyraclostrobin, Quintozene, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin. For a treatment with cotton herbicides, said cotton plants or seed preferably further comprise a trait conferring a respective herbicide tolerance or are naturally tolerant to said herbicide.

[0099] The following non-limiting Examples describe the methods for altering plant cell walls. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

[0100] Throughout the description and Examples, reference is made to the following sequences represented in the sequence listing:

SEQ ID No 1: Nodulation protein C of Azorhizobium caulinodans SEQ ID No 2: Nodulation protein C of Bradyrhizobium japonicum SEQ ID No 3: Nodulation protein C of Rhizobium galegae SEQ ID No 4: Nodulation protein C of Rhizobium leguminosarum (biovar viciae) SEQ ID No 5: Nodulation protein C of Rhizobium meliloti SEQ ID No 6: Nodulation protein C of Rhizobium tropici SEQ ID No 7: Nodulation protein C of Rhizobium leguminosarum (biovar phaseoli) SEQ ID No 8: Nodulation protein C of Rhizobium sp. Strain N33 SEQ ID No 9: Nodulation protein C of Rhizobium loti SEQ ID No 10: T-DNA of pTJN6 SEQ ID No 11: Amino acid sequence of NODC linked to Golgi-signal anchor sequence. SEQ ID No 12: nucleic acid sequence of a TDNA comprising a chimeric gene comprising the F286 fiber-selective promoter operably linked to a NODC encoding nucleic acid additionally comprising a Golgi-targeting sequence (=pTDBI146) SEQ ID No 13: nucleic acid sequence of a TDNA comprising a chimeric gene comprising the Gluc1A promoter operably linked to a NODC encoding nucleic acid additionally comprising a Golgi-targeting sequence (=pTDBI158) SEQ ID No 14: nucleic acid sequence of a TDNA comprising a chimeric gene comprising the Gluc1D promoter operably linked to a NODC encoding nucleic acid additionally comprising a Golgi-targeting sequence (=pTDBI159) SEQ ID No 15: nucleic acid sequence of a TDNA comprising a chimeric gene comprising the expansin promoter operably linked to a NODC encoding nucleic acid additionally comprising a Golgi-targeting sequence (=pTDBI165) SEQ ID No 16: nucleic acid sequence of a TDNA comprising a chimeric gene comprising the E6 promoter operably linked to a NODC encoding nucleic acid additionally comprising a Golgi-targeting sequence (=pTGK96)

EXAMPLES

Example 1: Construction of Chimeric Plant-Expressible Genes Encoding a N-Acetylglucosamine Transferase Protein Fused to a Golgi Signal Anchor Sequence

[0101] Using standard recombinant DNA techniques, a plant expressible NODC chimeric gene was constructed containing the following operably linked DNA fragments:

[0102] a 35S promoter region from CaMV

[0103] a DNA fragment coding for an untranslated leader sequence (5'Cab22L)

[0104] a DNA fragment coding for the 35 N-terminal amino acids of .beta.-1,2-xylosyltransferase from Arabidopsis thaliana

[0105] a DNA fragment coding for NODC of Azorhizobium caulinodans cloned in frame with the previous DNA fragment

[0106] a transcription termination and polyadenylation signal from the 35S transcript of CaMV (3' 35S)

[0107] The chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric bar gene providing resistance to phosphinotricin. The resulting T-DNA vector was named pTJN 6. The sequence of the T-DNA of this vector is provided in SEQ ID No 10.

[0108] The T-DNA vector pTJN6 was introduced into the Agrobacterium strain C58C1RIF(pEHA101) (Hood et al (1986) J. Bact. 168: 1291) which was used to transform Arabidopsis thaliana by means of the floral dip method (Clough S J and Bent A F (1998) Plant J. 16: 735-743).

Example 2: Analysis of Root Length of Transgenic Arabidopsis Plants

[0109] Wild-type Arabidopsis and transgenic Arabidopsis plants transformed with pTJN6 (NODC with heterologous Golgi signal anchor sequence) and pTGK42 (NODC without heterologous Golgi signal anchor sequence; see WO2006/136351) were gas sterilized and plated on 0.5.times.Murashige and Skoog (MS) basal salt medium including modified vitamins (Sigma) and 20 g/l glucose. After imbibing for 2 days at 4.degree. C., plates were placed vertically in a growth chamber with a day/night regime of 16 h of light at 21.degree. C. for a period of 9 days. Plates were scanned on a bench-top scanner and measurements were done in ImageJ. Data were exported to Microsoft Excel for analysis. The root length was measured and compared to that of wild-type plants. FIG. 3A shows that, whereas the root length of plants transformed with pTGK42 is about 25% shorter than that of wild-type, there is no significant difference in root length between wild-type and transgenic plants comprising pTJN6. Figure B shows that, for different transgenic lines comprising pTJN6, there is no significant difference in root length between wild-type and transgenic plants.

Example 3: Characterization of GlcNAc Oligomers in Cell Walls of Transgenic Arabidopsis Plants

[0110] The GlcNAc oligomers in transgenic Arabidopsis plants transformed with pTJN6 were analyzed using a combination of derivatization, high-performance liquid chromatography and mass spectrometry as described in Rozaklis et al. (2002, Clinical Chemistry 48:131-139). Briefly, leaf samples (20-150 mg) were snap frozen in liquid nitrogen and grinded in 2 ml eppendorf tubes using a rich mill grinder. 0.5 ml 80% MeOH was added and the tubes were vortexed and centrifuged in a precooled table top centrifuge (5 min 14000 rpm). Supernatant was transferred to a fresh 2 ml tube and freeze dried in a speed vac. The pellet was resuspended in 100 .mu.l MeOH containing 0.5M PMP and 100 .mu.l 800 mM NH3. The reaction mixture was incubated at 70.degree. C. in a thermomixer (Eppendorf) for 9 min at 850 rpm. After this incubation step, the reaction mixture was neutralized by adding 200 .mu.l 800 mM formic acid and further made up to 500 .mu.l with water. After 3 subsequent chloroform extractions using 0.5 ml chloroform, samples were freeze dried and resuspended in 200 ul water. Samples were analyzed on an Acquity UPLC BEH C18 column (Waters Corp., Milford, Mass., USA) (1.7 .mu.m, 150 mm.times.2.1 mm) using a Finnigan Mat LCQ Mass Spectrometer. Mobile phases were composed of (A) water containing 1% ACN and 0.1% ammonium acetate and (B) ACN containing 1% water and 0.1% ammonium acetate. Column temperature was maintained at 55.degree. C. and the autosampler temperature at 10.degree. C. A flow rate of 300 .mu.l/min was applied during the gradient elution initializing. Conditions where as follows: at time 0 min 20% (B), time 10 min 25% (B) and time 14 min 100% (B). Full MS and MS/MS spectra of the eluting compounds were obtained with electrospray ionization (ESI) operated in positive mode. Derivatized GlcNAC has a m/z value of 552 in positive mode, and the total peak area was considered the best approach for quantification. GlcNAC-oligmers were detected by MS/MS where only specific compounds with specific m/z value were selected for further fragmentation. The 552-peak or 755-peak, corresponding to the derivatized monomer or dimer respectively, were used for quantification. These levels were compared to those of Arabidopsis plants transformed with pTGK42 (WO2006/136351) and wild-type Arabidopsis plants. Table 1 shows that, in the presence of the Golgi signal anchor sequence, the amount of GlcNAc2 was up to 65-fold higher, and that of GlcNAc3 was up to 35-fold higher than in the absence of the Golgi signal anchor sequence.

TABLE-US-00004 TABLE 1 Relative amounts of the N-acetylglucosamine oligomers GlcNAc2 and GlcNAc3 in leaves of Arabidopsis plants transformed with pTJN6 (NODC with heterologous Golgi signal anchor sequence) and with pTGK42 (NODC without heterologous Golgi signal anchor sequence; see WO2006/136351). GlcNAc2 GlcNAc3 area 552 area 552 Construct peak average peak average WT 3.78E+04 5.41E+04 .+-. -- -- WT 7.04E+04 2.31E+04 -- pTGK4210 3.07E+06 2.39E+06 .+-. 9.22E+05 6.70E+05 .+-. pTGK4210 1.70E+06 0.97E+06 4.18E+05 3.56E+05 pTGK4228 2.83E+06 1.82E+06 .+-. 1.04E+06 6.16E+05 .+-. pTGK4228 8.00E+05 1.44E+06 1.91E+05 6.00E+05 pTJN6-4 5.20E+04 3.28E+05 .+-. 1.51E+04 5.13E+04 .+-. pTJN6-4 6.04E+05 3.90E+05 8.75E+04 5.12E+04 pTJN6-14 1.67E+05 1.42E+05 .+-. 2.99E+04 2.64E+04 .+-. pTJN6-14 1.17E+05 0.35E+05 2.28E+04 0.50E+04 pTJN6-23 1.23E+08 1.36E+08 .+-. 2.17E+07 2.24E+07 .+-. pTJN6-23 1.49E+08 0.18E+07 2.30E+07 0.09E+07 pTJN6-26 1.35E+05 1.18E+05 .+-. -- -- pTJN6-26 1.00E+05 0.25E+05 -- WT 1.77E+05 1.77E+05 -- -- pTGK4210 4.57E+06 4.57E+06 8.78E+05 8.78E+05 pTJN6-23 (1) 1.43E+07 3.00E+07 .+-. 3.63E+06 8.15E+06 .+-. pTJN6-23 (1) 4.57E+07 0.81E+07 1.27E+07 6.39E+06 pTJN6-23 (2) 5.39E+07 4.87E+07 .+-. 1.40E+07 1.30E+07 .+-. pTJN6-23 (2) 4.35E+07 0.74E+07 1.21E+07 0.14E+07

[0111] As the roots of the plants containing NODC Golgi signal anchor sequence have the same length as those of wild-type plants, whereas those of plants containing NODC without Golgi signal anchor sequence were significantly shorter than wild-type, it was investigated whether oligomers of GlcNAc2 and GlcNAc3 were present in roots of Arabidopsis plants comprising NODC without Golgi signal anchor sequence (pTGK42) and with Golgi signal anchor sequence (pTJN6) using methods as described above for the leaves.

[0112] Table 2 shows that the roots from the pTJN6 plants contain GlcNAc2 and GlcNAc3 oligonucleotides at levels that are higher than in roots from the pTGK42 plants. The levels of GlcNAc2 and GlcNAc3 in roots are significantly higher than in leaves. The presence of GlcNAc oligos in roots of wild-type plants is presumably due to contamination with root material from the pTGK42 plants and pTJN6 plants.

[0113] These results show that the restoration of root length to wild-type levels by adding the Golgi signal anchor sequence to NODC is not due to lack of accumulation of GlcNAc oligos in roots.

TABLE-US-00005 TABLE 2 Roots Leaves area 552 area 552 Construct peak average peak average a. Relative amounts of the N-acetylglucosamine oligomer GlcNAc2 in roots and leaves of Arabidopsis plants transformed with pTJN6 (NODC with heterologous Golgi signal anchor sequence) and with pTGK42 (NODC without heterologous Golgi signal anchor sequence; see WO2006/136351) expressed per mg of tissue. WT 1.72E+06 1.48E+06 .+-. 7.53E+04 8.41E+04 .+-. WT 1.58E+06 0.31E+06 5.26E+04 2.63E+04 WT 1.12E+06 9.40E+04 WT -- 1.14E+05 pTGK4210 5.63E+06 5.27E+06 .+-. 3.81E+05 3.90E+05 .+-. pTGK4210 4.92E+06 0.51E+06 4.00E+05 0.13E+05 pTGK4228 8.61E+06 6.80E+06 .+-. 8.69E+05 8.11E+05 .+-. pTGK4228 4.98E+06 2.56E+06 7.52E+05 0.83E+05 pTJN6-23 2.71E+07 1.74E+07 .+-. 3.00E+06 2.97E+06 .+-. pTJN6-23 1.49E+07 0.66E+07 2.70E+06 0.18E+06 pTJN6-23 1.26E+07 3.08E+06 pTJN6-23 1.50E+07 3.09E+06 b. Relative amounts of the N-acetylglucosamine oligomer GlcNAc3 in roots and leaves of Arabidopsis plants transformed with pTJN6 (NODC with heterologous Golgi signal anchor sequence) and with pTGK42 (NODC without heterologous Golgi signal anchor sequence; see WO2006/136351) expressed per mg of tissue. WT 3.62E+03 2.33E+03 .+-. -- -- WT 1.03E+03 1.83E+03 -- WT -- -- -- WT -- -- -- pTGK4210 3.13E+05 2.59E+05 .+-. 1.69E+04 2.21E+04 .+-. pTGK4210 2.06E+05 0.75E+05 2.73E+04 0.73E+04 pTGK4228 4.80E+05 3.47E+05 .+-. 5.34E+04 4.31E+04 .+-. pTGK4228 2.14E+05 1.88E+05 3.29E+04 1.44E+04 pTJN6-23 1.45E+06 9.57E+05 .+-. 2.09E+05 2.20E+05 .+-. pTJN6-23 8.69E+05 3.36E+05 2.12E+05 0.16E+05 pTJN6-23 7.06E+05 2.43E+05 pTJN6-23 8.01E+05 2.16E+05

Example 4: Characterization of GlcNAc Oligomers with a Degree of Polymerisation of Up to 5 in Cell Walls of Transgenic Arabidopsis Plants

[0114] To detect GlcNAc oligomers with a degree of polymerisation of up to 5 in Arabidopsis plants transformed with pTJN6 as compared to wild-type Arabidopsis plants, PMP-Derivatisation was performed. LC-MS analysis was performed on a Waters Acquity UPLC system (Waters Corp., Milford, Mass., USA) connected to a Synapt HDMS Q-Tof mass spectrometer (Micromass, Manchester, UK). Chromatographic separation was done on an Acquity BEH C18 column (2.1 mm.times.150 mm, 1.7 .mu.m) (Waters Corp., Milford, Mass., USA) using a gradient elution. Mobile phases were composed of (A) water containing 1% ACN and 0.1% formic acid and (B) ACN containing 1% water and 0.1% formic acid. Column temperature was maintained at 40.degree. C. and the autosampler temperature at 10.degree. C. A flow rate of 350 .mu.l/min was applied during the gradient elution initializing at time 0 min 5% (B), time 30 min 50% (B), time 33 min 100% (B). The eluant was directed to the mass spectrometer equipped with an electrospray ionization source and lockspray interface for accurate mass measurement. MS source parameters were as following: capillary voltage 1.5 kV, sampling cone 40V, extraction cone 4V, source temperature 120.degree. C., desolvation temperature 350.degree. C., cone gas flow 50 L/h, desolvation gas 550 L/h. The collision energy for the trap and transfer cell were set at 6 and 4 V, respectively. For data acquisition the dynamic range enhancement mode was activated. Full scan data was recorded in negative centroid V-mode, the mass range was set between m/z 100-1600 with a scan speed of 0.2 s/scan using Masslynx software (Waters Corp., Milford, Mass., USA). Leu-enkephalin (400 pg/.mu.l solubilised in water/ACN, (1:1, v/v) acidified with 0.1% formic acid) was used for the lock mass calibration by scanning every 10 seconds with a scan time of 0.5 seconds, 3 scans were averaged. For MS/MS purposes, the same settings were applied, except the trap collision energy was ramped from 10 to 45 V. All solvents used were ULC/MS grade (Biosolve, Valkenswaard, The Netherlands), water was produced by a DirectQ-UV water purification system (Millipore S.A.S, Molsheim, France). FIG. 4 shows that plants expressing pTJN6-23 contain monomers, dimers, trimers, tetramers and pentamers of GlcNAc.

Example 5: Fiber Specific Expression of NODC Fused to a Golgi Signal Anchor Sequence in Cotton

[0115] Transgenic cotton plants comprising a chimeric NODC gene fused to a Golgi signal anchor sequence as outlined in example 1, under control of the F286 fiber-selective promoter (which is disclosed in US2003/106097) (=pTDBI146), the GluclA (=pTDBI158) and Gluc1D (=pTDBI159) promoter (WO 2008/083969), the E6 promoter (U.S. Pat. No. 6,096,950) (=pTGK96) or with the expansin promoter (U.S. Pat. No. 6,566,586) (=pTDBI165) were generated using the transformation method as described in U.S. Pat. No. 6,483,013. Fibers from these transgenic cotton plants were isolated and analyzed for N-acetylglucosamine polymers by HPLC. These transgenic fibers contained up to 0.5% of glucosamine which could only be detected upon TFA hydrolysis, showing that it is part of a polymer. Fibers of untransformed lines contained less than 0.01% of GlcN. The presence of chitobiose was demonstrated in the fiber from one of the lines transformed with pTDBI158. Chitobiose was not detected in the fiber from untransformed lines. Selected lines with high levels of N-acetyl oligomers were grown in the field and lines that grow normal and form normal bolls could be selected. Fiber from these lines is used to produce yarns and fabrics with improved reactivity, such as improved dyeability. Fibers isolated from cotton bolls of transgenic plants have an increased amount of N-acetylglucosamine polymers which are evenly distributed throughout the cell wall.

Example 6: Cotton Fibers with Increased Reactivity

[0116] Transgenic cotton plants comprising a chimeric NODC gene fused to a Golgi signal anchor sequence operably linked to a fiber-specific promoter were generated as described in Example 5. Mature cotton fibers are harvested from these plants and can be stained with Congo Red or can be reacted with WGA-Alexa fluor 555. 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).

WGA-Alexa 555 Staining

[0117] Cotton fibers do not need to be dehydrated or permeabilized. Instead, lipids and waxes are removed by treating the fibers for 3 times 10 minutes in a chloroform: methanol mixture (1:1), follow by twice a treatment of 10 minutes in acetone and twice 5 minutes in ether. The fibers are allowed to air dry.

[0118] Fibers can be stained with either WGA-Alexa555, WGA-Alexa488 or WGA-tetramethylrhodamine.

[0119] The fibers are placed in blocking solution (150 mM NaCl, 10 mM sodiumphosphate buffer pH 7.4; 0.1% Tween 20 and 1% bovine serum albumin) and are incubated for one hour. Thereafter, the buffer is replaced by the same buffer containing WGA-fluorochrome and incubated for 4 hrs. The WGA-fluorochrome solution is replaced by blocking solution, washed 10 minutes, followed by 3 times 10 min washing with blocking solution without BSA, and 2 times 5 min washing with blocking solution without BSA and without Tween. The stained fibers are mounted on a microscope slide and evaluated by means of fluorescence microscopy (Axioplan 2 (Zeiss, Jena, Germany) using Filterset 38 (exitation: BP470/40; emission: BP525/50) for Alexa fluor 488 conugate or Filterset 20 (exitation: BP546/12; emission: BP575-640) for Alexa fluor 555 or tetramethylrhodamine conjugate. Whereas no specific fluorescence can be detected in cotton fibers from non-transgenic cotton plants, a bright fluorescence is detectable in cotton fibers from transgenic cotton plants comprising a chimeric NODC gene fused to a Golgi signal anchor sequence. Virtual microscopic sections of the cotton fibers show that the WGA-fluor555 is evenly distributed throughout the secondary cell wall of the cotton fiber cells.

[0120] To prepare for dyeing, fiber was treated for 30 min at 80.degree. C. with distilled water containing 1 g/l sodium carbonate and 1 g/l Sandozin NIN (non-ionic detergent) followed by drying. Fiber was dyed using a liquor:fiber ratio of 50:1 with 2% omf Acid Blue 62 for 60 min at 98.degree. C. After dyeing, fibre samples were rinsed with cold water and dried at ambient conditions. Exhaustion was determined by measuring dye concentration of the dye bath before and after dyeing using a spectrophotometer.

[0121] As can be seen on the example of a cotton line transformed with T-DNA vector pTDBI158, fiber dyed with Acid Blue 62 according to the protocol above shows an increased exhaustion level.

TABLE-US-00006 % exhaustion % exhaustion transgene at pH 4 at pH 7 G4GH396-35101 HH gfa-nodc 23.9 18.7 G4GH396-35101 wt none 16.3 13.0

Sequence CWU 1

1

161397PRTAzorhizobium caulinodans 1Met Ser Val Val Asp Val Ile Gly Leu Leu Ala Thr Ala Ala Tyr Val1 5 10 15Thr Leu Ala Ser Ala Tyr Lys Val Val Gln Phe Ile Asn Val Ser Ser 20 25 30Val Thr Asp Val Ala Gly Leu Glu Ser Asp Ala Leu Pro Leu Thr Pro 35 40 45Arg Val Asp Val Ile Val Pro Thr Phe Asn Glu Asn Ser Ser Thr Leu 50 55 60Leu Glu Cys Val Ala Ser Ile Cys Ala Gln Asp Tyr Arg Gly Pro Ile65 70 75 80Thr Ile Val Val Val Asp Asp Gly Ser Thr Asn Lys Thr Ser Phe His 85 90 95Ala Val Cys Asp Lys Tyr Ala Ser Asp Glu Arg Phe Ile Phe Val Glu 100 105 110Leu Asp Gln Asn Lys Gly Lys Arg Ala Ala Gln Met Glu Ala Ile Arg 115 120 125Arg Thr Asp Gly Asp Leu Ile Leu Asn Val Asp Ser Asp Thr Val Ile 130 135 140Asp Lys Asp Val Val Thr Lys Leu Ala Ser Ser Met Arg Ala Pro Asn145 150 155 160Val Gly Gly Val Met Gly Gln Leu Val Ala Lys Asn Arg Glu Arg Ser 165 170 175Trp Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn Glu 180 185 190Glu Arg Ile Ala Gln Ser Arg Phe Gly Ser Val Met Cys Cys Cys Gly 195 200 205Pro Cys Ala Met Tyr Arg Arg Ser Ala Ile Thr Pro Leu Leu Ala Glu 210 215 220Tyr Glu His Gln Thr Phe Leu Gly Arg Pro Ser Asn Phe Gly Glu Asp225 230 235 240Arg His Leu Thr Ile Leu Met Leu Lys Ala Gly Phe Arg Thr Gly Tyr 245 250 255Val Pro Gly Ala Val Ala Arg Thr Leu Val Pro Asp Gly Leu Ala Pro 260 265 270Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Tyr Arg Asp Thr 275 280 285Ala Leu Ala Leu Arg Ile Lys Lys Asn Leu Ser Lys Tyr Ile Thr Phe 290 295 300Glu Ile Cys Ala Gln Asn Leu Gly Thr Ala Leu Leu Leu Val Met Thr305 310 315 320Met Ile Ser Leu Ser Leu Thr Thr Ser Gly Ser Gln Thr Pro Val Ile 325 330 335Ile Leu Gly Val Val Val Gly Met Ser Ile Ile Arg Cys Cys Ser Val 340 345 350Ala Leu Ile Ala Lys Asp Phe Arg Phe Leu Tyr Phe Ile Val His Ser 355 360 365Ala Leu Asn Val Leu Ile Leu Thr Pro Leu Lys Leu Tyr Ala Leu Leu 370 375 380Thr Ile Arg Asp Ser Arg Trp Leu Ser Arg Glu Ser Ser385 390 3952485PRTBradyrhizobium japonicum 2Met Asp Leu Leu Ala Thr Thr Ser Ala Ala Ala Val Ser Ser Tyr Ala1 5 10 15Leu Leu Ser Thr Ile Tyr Lys Ser Val Gln Ala Leu Tyr Ala Gln Pro 20 25 30Ala Ile Asn Ser Ser Leu Asp Asn Leu Gly Gln Ala Glu Val Val Val 35 40 45Pro Ala Val Asp Val Ile Val Pro Cys Phe Asn Glu Asn Pro Asn Thr 50 55 60Leu Ala Glu Cys Leu Glu Ser Ile Ala Ser Gln Asp Tyr Ala Gly Lys65 70 75 80Met Gln Val Tyr Val Val Asp Asp Gly Ser Ala Asn Arg Asp Val Val 85 90 95Ala Pro Val His Arg Ile Tyr Ala Ser Asp Pro Arg Phe Ser Phe Ile 100 105 110Leu Leu Ala Asn Asn Val Gly Lys Arg Lys Ala Gln Ile Ala Ala Ile 115 120 125Arg Ser Ser Ser Gly Asp Leu Val Leu Asn Val Asp Ser Asp Thr Ile 130 135 140Leu Ala Ala Asp Val Val Thr Lys Leu Val Leu Lys Met His Asp Pro145 150 155 160Gly Ile Gly Ala Ala Met Gly Gln Leu Ile Ala Ser Asn Arg Asn Gln 165 170 175Thr Trp Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn 180 185 190Glu Glu Arg Ala Ala Gln Ala Arg Phe Gly Ala Val Met Cys Cys Cys 195 200 205Gly Pro Cys Ala Met Tyr Arg Arg Ser Ala Leu Ala Leu Leu Leu Asp 210 215 220Gln Tyr Glu Ala Gln Phe Phe Arg Gly Lys Pro Ser Asp Phe Gly Glu225 230 235 240Asp Arg His Leu Thr Ile Leu Met Leu Lys Ala Gly Phe Arg Thr Glu 245 250 255Tyr Val Pro Asp Ala Ile Ala Ala Thr Val Val Pro His Ser Leu Arg 260 265 270Pro Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Asp 275 280 285Thr Phe Leu Ala Trp Arg Leu Leu Pro Glu Leu Asp Gly Tyr Leu Thr 290 295 300Leu Asp Val Ile Gly Gln Asn Leu Gly Pro Leu Leu Leu Ala Ile Ser305 310 315 320Ser Leu Ala Ala Leu Ala Gln Leu Leu Ile Asp Gly Ser Ile Pro Trp 325 330 335Trp Thr Gly Leu Thr Ile Ala Ala Met Thr Thr Val Arg Cys Cys Val 340 345 350Ala Ala Leu Arg Ala Arg Glu Leu Arg Phe Ile Gly Phe Ser Leu His 355 360 365Thr Pro Ile Asn Ile Cys Leu Leu Leu Pro Leu Lys Ala Tyr Ala Leu 370 375 380Cys Thr Leu Ser Asn Ser Asp Trp Leu Ser Arg Lys Val Thr Asp Met385 390 395 400Pro Thr Glu Glu Gly Lys Gln Pro Val Ile Leu His Pro Asn Ala Gly 405 410 415Arg Ser Pro Ala Gly Val Gly Gly Arg Leu Leu Leu Phe Val Arg Arg 420 425 430Arg Tyr Arg Ser Leu His Arg Ala Trp Arg Arg Arg Arg Val Phe Pro 435 440 445Val Ala Ile Val Arg Leu Ser Thr Asn Lys Trp Ser Ala Asp Asp Ser 450 455 460Gly Arg Lys Pro Ser Val Ile Arg Ala Arg Val Gly Cys Arg Arg Pro465 470 475 480Val Ala Pro Arg His 4853433PRTRhizobium galegae 3Met Thr Leu Leu Glu Thr Ile Gly Ile Ala Ala Val Thr Leu His Ala1 5 10 15Leu Leu Ser Ala Ile Tyr Lys Ser Met Gln Ala Phe Tyr Ala Arg Lys 20 25 30Ala Ser Gly Ser Gln Pro Arg Ser Lys Asp Ile Asp Pro Ala Ala Leu 35 40 45Pro Ser Val Asp Ile Ile Val Pro Cys Phe Asn Glu Asp Pro Ala Ile 50 55 60Leu Ser Ala Cys Leu Ser Ser Leu Ala Gly Gln Asp Tyr Gly Gly Lys65 70 75 80Leu Arg Ile Tyr Met Val Asp Asp Gly Ser Cys Asn Arg Glu Ala Ile 85 90 95Leu Pro Val His Asp Phe Tyr Thr Ser Asp Pro Arg Phe Glu Phe Leu 100 105 110Leu Leu Ser Lys Asn Val Gly Lys Arg Lys Ala Gln Ile Ala Ala Ile 115 120 125Glu Arg Ser Cys Gly Asp Leu Ile Leu Asn Val Asp Ser Asp Thr Ser 130 135 140Ile Ala Ser Asp Val Val Thr Leu Leu Val Glu Lys Met Arg Asp Ser145 150 155 160Asp Val Gly Ala Ala Met Gly Gln Leu Lys Ala Ser Asn Arg Asp Lys 165 170 175Asn Leu Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn 180 185 190Asp Glu Arg Ala Ala Gln Ala Arg Phe Gly Ala Val Met Cys Cys Cys 195 200 205Gly Pro Cys Ala Met Tyr Arg Arg Ser Ala Leu Leu Leu Leu Leu Asp 210 215 220Gln Tyr Gln Thr Gln Leu Tyr Arg Gly Lys Pro Ser Asp Phe Gly Glu225 230 235 240Asp Arg His Leu Thr Ile Leu Met Leu Ser Ala Gly Phe Arg Thr Glu 245 250 255Tyr Val Pro Glu Ala Ile Ala Lys Thr Val Val Pro Asp Arg Met Gly 260 265 270Ser Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Asp 275 280 285Thr Leu Leu Ala Leu Pro Leu Leu Pro Ser His Asn Arg Phe Leu Thr 290 295 300Leu Asp Ala Ile His Gln Asn Ile Gly Pro Leu Leu Leu Ala Val Ser305 310 315 320Ser Ala Thr Gly Ile Thr Gln Phe Ile Leu Thr Ala Thr Val Pro Gly 325 330 335Trp Thr Ile Ile Ile Ile Ala Ser Met Thr Met Val Arg Cys Ser Val 340 345 350Ala Ala Tyr Arg Ser Arg Gln Ile Arg Phe Leu Ala Phe Ser Leu His 355 360 365Thr Leu Ile Asn Leu Phe Met Leu Ile Pro Leu Lys Gly Phe Ala Leu 370 375 380Leu Thr Leu Ser Asn Ser Asp Trp Leu Ser Arg Gly Ser Thr Thr Asp385 390 395 400Gly Pro Ala Ile Ala Glu Ser Asn Ala Ala Ser Asn Glu Ala Glu Ile 405 410 415Val Ala Ser Ala Ser Pro Phe Gly Gly Gly Thr Ser Trp Arg Phe Arg 420 425 430Arg4424PRTRhizobium leguminosarum 4Met Thr Leu Leu Ala Thr Thr Ser Ile Ala Ala Ile Ser Leu Tyr Ala1 5 10 15Met Leu Ser Thr Val Tyr Lys Ser Ala Gln Val Phe His Ala Arg Arg 20 25 30Thr Thr Ile Ser Thr Thr Pro Ala Lys Asp Ile Glu Thr Asn Pro Val 35 40 45Pro Ser Val Asp Val Ile Val Pro Cys Phe Asn Glu Asp Pro Ile Val 50 55 60Leu Ser Glu Cys Leu Ala Ser Leu Ala Glu Gln Asp Tyr Ala Gly Lys65 70 75 80Leu Arg Ile Tyr Val Val Asp Asp Gly Ser Lys Asn Arg Asp Ala Val 85 90 95Val Ala Gln Arg Ala Ala Tyr Ala Asp Asp Glu Arg Phe Asn Phe Thr 100 105 110Ile Leu Pro Lys Asn Val Gly Lys Arg Lys Ala Ile Ala Ala Ile Thr 115 120 125Gln Ser Ser Gly Asp Leu Ile Leu Asn Val Asp Ser Asp Thr Thr Ile 130 135 140Ala Pro Asp Val Val Ser Lys Leu Ala His Lys Met Arg Asp Pro Ala145 150 155 160Val Gly Ala Ala Met Gly Gln Met Lys Ala Ser Asn Gln Ala Asp Thr 165 170 175Trp Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn Glu 180 185 190Glu Arg Ala Ala Gln Ala Arg Phe Gly Ala Val Met Cys Cys Cys Gly 195 200 205Pro Cys Ala Met Tyr Arg Arg Ser Ala Met Leu Ser Leu Leu Asp Gln 210 215 220Tyr Glu Thr Gln Leu Tyr Arg Gly Lys Pro Ser Asp Phe Gly Glu Asp225 230 235 240Arg His Leu Thr Ile Leu Met Leu Ser Ala Gly Phe Arg Thr Glu Tyr 245 250 255Val Pro Ser Ala Ile Ala Ala Thr Val Val Pro Asp Thr Met Gly Val 260 265 270Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Asp Thr 275 280 285Leu Leu Ala Leu Pro Val Leu Pro Gly Leu Asp Arg Tyr Leu Thr Leu 290 295 300Asp Ala Ile Gly Gln Asn Val Gly Leu Leu Leu Leu Ala Leu Ser Val305 310 315 320Leu Thr Gly Ile Gly Gln Phe Ala Leu Thr Ala Thr Leu Pro Trp Trp 325 330 335Thr Ile Leu Val Ile Gly Ser Met Thr Leu Val Arg Cys Ser Val Ala 340 345 350Ala Tyr Arg Ala Arg Glu Leu Arg Phe Leu Gly Phe Ala Leu His Thr 355 360 365Leu Val Asn Ile Phe Leu Leu Ile Pro Leu Lys Ala Tyr Ala Leu Cys 370 375 380Thr Leu Ser Asn Ser Asp Trp Leu Ser Arg Gly Ser Val Ala Ile Ala385 390 395 400Pro Thr Val Gly Gln Gln Gly Ala Thr Lys Met Pro Gly Arg Ala Thr 405 410 415Ser Glu Ile Ala Tyr Ser Gly Glu 4205426PRTRhizobium meliloti 5Met Tyr Leu Leu Asp Thr Thr Ser Thr Ala Ala Ile Ser Ile Tyr Ala1 5 10 15Leu Leu Leu Thr Ala Tyr Arg Ser Met Gln Val Leu Tyr Ala Arg Pro 20 25 30Ile Asp Gly Pro Ala Val Ala Ala Glu Pro Val Glu Thr Arg Pro Leu 35 40 45Pro Ala Val Asp Val Ile Val Pro Ser Phe Asn Glu Asp Pro Gly Ile 50 55 60Leu Ser Ala Cys Leu Ala Ser Ile Ala Asp Gln Asp Tyr Pro Gly Glu65 70 75 80Leu Arg Val Tyr Val Val Asp Asp Gly Ser Arg Asn Arg Glu Ala Ile 85 90 95Val Arg Val Arg Ala Phe Tyr Ser Arg Asp Pro Arg Phe Ser Phe Ile 100 105 110Leu Leu Pro Glu Asn Val Gly Lys Arg Lys Ala Gln Ile Ala Ala Ile 115 120 125Gly Gln Ser Ser Gly Asp Leu Val Leu Asn Val Asp Ser Asp Ser Thr 130 135 140Ile Ala Phe Asp Val Val Ser Lys Leu Ala Ser Lys Met Arg Asp Pro145 150 155 160Glu Val Gly Ala Val Met Gly Gln Leu Thr Ala Ser Asn Ser Gly Asp 165 170 175Thr Trp Leu Thr Lys Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn 180 185 190Glu Glu Arg Ala Ala Gln Ser Arg Phe Gly Ala Val Met Cys Cys Cys 195 200 205Gly Pro Cys Ala Met Tyr Arg Arg Ser Ala Leu Ala Ser Leu Leu Asp 210 215 220Gln Tyr Glu Thr Gln Leu Phe Arg Gly Lys Pro Ser Asp Phe Gly Glu225 230 235 240Asp Arg His Leu Thr Ile Leu Met Leu Lys Ala Gly Phe Arg Thr Glu 245 250 255Tyr Val Pro Asp Ala Ile Val Ala Thr Val Val Pro Asp Thr Leu Lys 260 265 270Pro Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Asp 275 280 285Thr Phe Leu Ala Leu Pro Leu Leu Arg Gly Leu Ser Pro Phe Leu Ala 290 295 300Phe Asp Ala Val Gly Gln Asn Ile Gly Gln Leu Leu Leu Ala Leu Ser305 310 315 320Val Val Thr Gly Leu Ala His Leu Ile Met Thr Ala Thr Val Pro Trp 325 330 335Trp Thr Ile Leu Ile Ile Ala Cys Met Thr Ile Ile Arg Cys Ser Val 340 345 350Val Ala Leu His Ala Arg Gln Leu Arg Phe Leu Gly Phe Val Leu His 355 360 365Thr Pro Ile Asn Leu Phe Leu Ile Leu Pro Leu Lys Ala Tyr Ala Leu 370 375 380Cys Thr Leu Ser Asn Ser Asp Trp Leu Ser Arg Tyr Ser Ala Pro Glu385 390 395 400Val Pro Val Ser Gly Gly Lys Gln Thr Pro Ile Gln Thr Ser Gly Arg 405 410 415Val Thr Pro Asp Cys Thr Cys Ser Gly Glu 420 4256452PRTRhizobium tropici 6Met Asn Leu Leu Asp Ala Thr Ser Thr Ala Ala Ile Ser Leu Tyr Ala1 5 10 15Met Leu Ser Thr Ala Tyr Lys Ser Met Gln Val Val Tyr Ala Arg Pro 20 25 30Ile Glu Glu Pro Ser Thr Ser Ala Glu Pro Ile Ala Ser Ala Gln Trp 35 40 45Pro Ser Val Asp Val Ile Ile Pro Ser Phe Asn Glu Asp Pro Gly Thr 50 55 60Leu Trp Asp Cys Leu Glu Ser Ile Ala His Glu Glu Tyr Ala Gly Asp65 70 75 80Leu Asn Val Tyr Val Val Asp Asp Gly Ser Ser Asn Arg Asp Ala Ile 85 90 95Thr Pro Val His Thr Ala Phe Ala Arg Asp Pro Arg Phe Thr Phe Ile 100 105 110Leu Leu Arg Lys Asn Val Gly Lys Arg Lys Ala Gln Ile Ala Ala Ile 115 120 125Arg Arg Ser Ser Gly Asp Leu Val Leu Asn Val Asp Ser Asp Thr Ile 130 135 140Leu Ala Pro Asp Val Val Val Lys Leu Ala Leu Lys Met Gln Asp Pro145 150 155 160Ala Ile Gly Ala Ala Met Gly Gln Leu Ala Ala Ser Asn Arg His Glu 165 170 175Thr Trp Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn 180 185 190Glu Glu Arg Ala Ala Gln Ala Arg Phe Gly Ala Val Met Cys Cys Cys 195 200 205Gly Pro Cys Ala Met Tyr Arg Arg Thr Ala Leu Thr Met Leu Leu Asp 210 215 220Gln Tyr Glu Thr Gln Met Phe Arg Gly Lys Arg Ser Asp Phe Gly Glu225 230 235 240Asp Arg His Leu Thr Ile Leu Met Leu Lys Ala Gly Phe Arg Thr Glu 245 250 255Tyr Val Pro Thr Ala Ile Ala Ala Thr Val Val Pro Asn Lys Leu Arg 260 265 270Pro Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Asp 275 280 285Thr Leu

Leu Ala Met Asn Leu Leu Pro Gly Leu Asp Arg Phe Leu Thr 290 295 300Leu Asp Val Ile Gly Gln Asn Leu Gly Pro Leu Leu Leu Ala Leu Ser305 310 315 320Val Leu Thr Gly Leu Ala Gln Phe Ala Leu Thr Gly Thr Val Pro Trp 325 330 335Trp Thr Cys Leu Met Ile Ala Ser Met Thr Met Ile Arg Cys Ser Val 340 345 350Ala Ala Val Arg Ala Arg Gln Phe Arg Phe Ile Gly Phe Ser Leu His 355 360 365Thr Phe Ile Asn Ile Phe Phe Leu Leu Pro Leu Lys Ala Tyr Ala Leu 370 375 380Cys Thr Leu Ser Asn Ser Asp Trp Leu Ser Arg Gly Ser Ala Ala Lys385 390 395 400Ala Thr Gly Lys Gly Gly Lys Leu Asp Ala Ile Gln Asp Pro Val Ala 405 410 415Ala Ser Ser Pro Arg Glu Ser Gln Glu Asn Glu Ala Pro Leu Arg Arg 420 425 430His Asn Leu Ala Arg Asp Ala Thr Arg Ser Met Ala Tyr Asp Gly Ile 435 440 445Cys Thr Asp Gln 4507428PRTRhizobium leguminosarum 7Met Thr Met Leu Asp Thr Thr Ser Thr Val Ala Val Ser Leu Tyr Ala1 5 10 15Leu Leu Ser Thr Ala Tyr Lys Ser Met Gln Ala Val Tyr Ser Leu Pro 20 25 30Thr Asp Val Ser Leu Ala Ser His Gly Leu Gly Gly Phe Asp Glu Leu 35 40 45Pro Ser Val Asp Val Ile Val Pro Ser Phe Asn Glu Asp Pro Arg Thr 50 55 60Leu Ser Glu Cys Leu Ala Ser Ile Ala Gly Gln Glu Tyr Gly Gly Arg65 70 75 80Leu Gln Val Tyr Leu Val Asp Asp Gly Ser Glu Asn Arg Glu Ala Leu 85 90 95Arg Leu Val His Glu Ala Phe Ala Arg Asp Pro Arg Phe Asn Ile Leu 100 105 110Leu Leu Pro Gln Asn Val Gly Lys Arg Lys Ala Gln Asp Arg Cys Asp 115 120 125Gln Arg Ser Ala Gly Asp Met Val Leu Asn Val Asp Ser Asp Thr Ile 130 135 140Leu Ala Ser Asp Val Ile Arg Lys Leu Val Pro Lys Asn Ala Arg Val145 150 155 160Ala Val Gly Arg Met Gly Gln Leu Thr Gly Pro Gln Pro Lys Arg Gln 165 170 175Leu Ala Asp Pro Phe Asp Asp Met Glu Tyr Trp Leu Ala Cys Asn Glu 180 185 190Glu Arg Ser Gln Gln Ala Arg Phe Gly Cys Val Met Phe Cys Ser Gly 195 200 205Ser Cys Val Met Tyr Arg Leu Val Ser Ala Ser Leu Leu Asp Gln Tyr 210 215 220Asp Ala Gln Tyr Phe Arg Lys Gln Arg Phe Gly Glu Ile Asp Ile His225 230 235 240Leu Ser His Ala Glu Gly Ser Phe Arg Thr Glu Tyr Arg Pro Ser Ala 245 250 255His Ala Ala Thr Val Val Pro Asn Lys Leu Gly Pro Tyr Leu Gly Gln 260 265 270Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Thr Thr Leu Leu Gly Ala 275 280 285Pro Leu Pro Asn Leu Asn Arg Phe Leu Met Leu Asp Val Val Gly Gln 290 295 300Asn Leu Gly Pro Leu Leu Leu Asp His Ser Val Leu Thr Gly Leu Ala305 310 315 320Gln Leu Ala Leu Thr Gly Thr Ala Pro Trp Leu Ala Ala Leu Met Ile 325 330 335Val Ala Met Thr Ile Asp Arg Cys Ser Val Val Ala Leu Arg Ala Arg 340 345 350Gln Leu Arg Phe Leu Gly Phe Ser Leu His Thr Phe Ile Asn Ile Phe 355 360 365Leu Leu Leu Pro Leu Lys Ala Tyr Ala Leu Cys Thr Leu Ser Asn Ile 370 375 380Ala Trp Leu Ser Ser Leu Leu Cys Trp Gln Leu Glu Ser Thr Ser Thr385 390 395 400Ala Asp Ala Arg Thr Thr Glu Cys Ser Asp Met Arg Thr Ala Ser Lys 405 410 415Leu Ser Pro Pro Pro Ser Cys Gln Ala Asn Asp Val 420 4258450PRTRhizobium sp. 8Met Asp Leu Leu Thr Thr Thr Ser Thr Val Ala Val Ala Cys Tyr Ala1 5 10 15Leu Leu Ser Thr Val Tyr Lys Gly Met Gln Ala Val Tyr Ser Leu Pro 20 25 30Pro Thr Val Ala Pro Ala Ser Glu Asp Leu Val Gly Ser Asp Leu Trp 35 40 45Pro Ser Val Asp Val Ile Ile Pro Cys Tyr Asn Glu Gly Pro Leu Thr 50 55 60Leu Ser Ala Cys Leu Asp Ser Ile Ala Asn Gln Glu Tyr Ala Gly Lys65 70 75 80Leu Arg Val Tyr Val Val Asp Asp Gly Ser Gly Asn Arg Asp Ala Val 85 90 95Ile Pro Ile His Asp Asn Tyr Ala Gly Asp Pro Arg Phe Asp Phe Ile 100 105 110Leu Leu Pro Glu Asn Val Gly Lys Arg Lys Ala Gln Ile Ala Ala Ile 115 120 125Arg Arg Ser Ser Gly Asp Leu Val Leu Asn Val Asp Ser Asp Thr Thr 130 135 140Leu Ala Ser Asp Val Ile Arg Lys Leu Ala Arg Lys Met Gln Asp Pro145 150 155 160Ala Ile Gly Ala Ala Met Gly Gln Leu Thr Ala Ser Asn Arg Ser Asp 165 170 175Thr Trp Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn 180 185 190Glu Glu Arg Ala Ala Gln Ala Arg Phe Gly Ala Val Met Cys Cys Cys 195 200 205Gly Pro Cys Ala Met Tyr Arg Arg Ser Ser Leu Leu Ser Leu Leu Asp 210 215 220Gln Tyr Glu Thr Gln Met Phe Arg Gly Lys Pro Ser Asp Phe Gly Glu225 230 235 240Asp Arg His Leu Thr Ile Leu Met Leu Glu Ala Gly Phe Arg Thr Glu 245 250 255Tyr Val Pro Asp Ala Ile Ala Val Thr Val Val Pro Asp Arg Leu Gly 260 265 270Pro Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Asp 275 280 285Thr Leu Leu Ala Leu Arg Leu Leu Pro Gly Leu Asp Arg Tyr Leu Thr 290 295 300Leu Asp Val Val Gly Gln Asn Leu Gly Pro Leu Leu Leu Ala Leu Ser305 310 315 320Val Ile Ala Gly Ile Ala Gln Phe Ala Leu Thr Ala Thr Leu Pro Trp 325 330 335Pro Thr Ile Leu Val Ile Ala Ala Met Thr Ile Ile Arg Cys Thr Val 340 345 350Thr Ala Cys Arg Ala Arg Gln Ala Arg Phe Ile Gly Phe Ser Leu His 355 360 365Thr Phe Ile Asn Ile Phe Leu Leu Leu Pro Leu Lys Ala Tyr Ala Leu 370 375 380Cys Thr Leu Ser Asn Ser Asp Trp Leu Ser Arg Lys Thr Ala Thr Leu385 390 395 400Pro Asn Ala Asp Lys Lys Gln Ile Ile Val Ala Asn Pro Ile Ala Gly 405 410 415Val Gly Thr Gly Ser Ser Gly Ser Ala Glu Ala Ile Arg Arg Thr Asp 420 425 430Leu Pro Arg Asp Ser Ser Lys Leu Val Asn Ala Asp Ser Val Cys Ser 435 440 445Ala Glu 4509424PRTRhizobium loti 9Met Asn Leu Phe Ala Ser Ala Ser Thr Val Ala Ile Cys Ser Tyr Ala1 5 10 15Leu Leu Ser Thr Val Tyr Lys Thr Ala Gln Val Phe Tyr Thr Leu Pro 20 25 30Thr Asn Val Pro Pro Thr Ser Gly Asp Pro Pro Ser Gly Glu Pro Trp 35 40 45Pro Ser Val Asp Val Ile Ile Pro Cys Tyr Asn Glu Ala Pro Arg Thr 50 55 60Leu Ser Asp Cys Leu Ala Ser Ile Ala Ser Gln Asp Tyr Ala Gly Lys65 70 75 80Leu Gln Val Tyr Val Val Asp Asp Gly Ser Ala Asn Arg Asp Ala Leu 85 90 95Val Gly Val His Glu Glu Tyr Ala Gly Asp Pro Arg Phe Asn Phe Val 100 105 110Ala Leu Pro Lys Asn Val Gly Lys Arg Lys Ala Gln Ile Ala Ala Ile 115 120 125Arg Arg Ser Cys Gly Asp Leu Val Leu Asn Val Asp Ser Asp Thr Ile 130 135 140Leu Ala Pro Asp Val Ile Thr Arg Leu Ala Leu Lys Met Gln Asp Gln145 150 155 160Ala Val Gly Ala Ala Met Gly Gln Leu Ala Ala Ser Asn Arg Ser Glu 165 170 175Thr Trp Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys Asn 180 185 190Glu Glu Arg Ala Ala Gln Ala Arg Phe Gly Ala Val Met Cys Cys Cys 195 200 205Gly Pro Cys Ala Met Tyr Arg Arg Ser Ala Leu Val Ser Leu Leu Asp 210 215 220Gln Tyr Glu Thr Gln Arg Phe Arg Gly Lys Pro Ser Asp Phe Gly Glu225 230 235 240Asp Arg His Leu Thr Ile Leu Met Leu Lys Ala Gly Phe Arg Thr Glu 245 250 255Tyr Val Pro Glu Ala Val Ala Ala Thr Val Val Pro Asn Ser Met Gly 260 265 270Pro Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Phe Arg Asp 275 280 285Thr Leu Leu Ala Phe Gln Leu Leu Arg Gly Leu Asn Ile Tyr Leu Thr 290 295 300Leu Asp Val Ile Gly Gln Asn Ile Gly Pro Leu Leu Leu Ser Leu Ser305 310 315 320Ile Leu Ala Gly Leu Ala Gln Phe Val Thr Thr Gly Thr Val Pro Trp 325 330 335Thr Ala Cys Leu Met Ile Ala Ala Met Thr Ile Val Arg Cys Ser Val 340 345 350Ala Ala Phe Arg Ala Arg Gln Leu Arg Phe Leu Gly Phe Ser Leu His 355 360 365Thr Leu Ile Asn Ile Phe Leu Leu Leu Pro Leu Lys Ala Tyr Ala Leu 370 375 380Cys Thr Leu Ser Asn Ser Asp Trp Leu Ser Arg Ser Ser Ala Ala Asn385 390 395 400Val Gln Asp Thr Gly Asp Ala Leu Pro Lys Pro Asn Leu Val Gly Ser 405 410 415Asp Ala Ala Tyr Ser Glu Gln Gln 420103971DNAArtificialT-DNA of pTJN6misc_feature(1)..(25)Left T-DNA border (complement)terminator(56)..(316)3' nos 3'UTR from nopaline synthase gene of T-DNA pTiT37 (complement)misc_feature(336)..(887)bar coding sequence of phosphinotricin acetyl transferease of Streptomyces hygroscopicus (complement)promoter(888)..(1720)P35S3 Promoter region from CaMV35S gene (complement)promoter(1769)..(2303)P35S2 Promoter region from CaMV35S gene5'UTR(2304)..(2368)5'cab22L untranslated leader sequence of cab22L gene from Petuniamisc_feature(2369)..(2474)XylT sequence coding for 35 N-terminal amino acids of beta-1,2- xylosyltransferase from Arabidopsis thalianamisc_feature(2475)..(3664)NodC coding region from A.caulinodansterminator(3666)..(3914)3'35S 3' untranslated region from the CaMV35S genemisc_feature(3947)..(3971)RB right T-DNA border (synthetic) (complement) 10cggcaggata tattcaattg taaatggctc catggcgatc gctctagagg atctgcgatc 60tagtaacata gatgacaccg cgcgcgataa tttatcctag tttgcgcgct atattttgtt 120ttctatcgcg tattaaatgt ataattgcgg gactctaatc ataaaaaccc atctcataaa 180taacgtcatg cattacatgt taattattac atgcttaacg taattcaaca gaaattatat 240gataatcatc gcaagaccgg caacaggatt caatcttaag aaactttatt gccaaatgtt 300tgaacgatct gcttcggatc ctagaacgcg tgatctcaga tctcggtgac gggcaggacc 360ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 420ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 480atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 540gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 600cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 660gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 720cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 780aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 840gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggtccatggt tatagagaga 900gagatagatt tatagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 960ttccttatat agaggaaggg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt 1020cagtggagat gtcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt 1080ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt 1140gaatgatagc ctttccttta tcgcaatgat ggcatttgta ggagccacct tccttttcta 1200ctgtcctttc gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgaaa 1260ttatcctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgacat 1320ttttggagta gaccagagtg tcgtgctcca ccatgttgac gaagattttc ttcttgtcat 1380tgagtcgtaa aagactctgt atgaactgtt cgccagtctt cacggcgagt tctgttagat 1440cctcgatttg aatcttagac tccatgcatg gccttagatt cagtaggaac taccttttta 1500gagactccaa tctctattac ttgccttggt ttatgaagca agccttgaat cgtccatact 1560ggaatagtac ttctgatctt gagaaatatg tctttctctg tgttcttgat gcaattagtc 1620ctgaatcttt tgactgcatc tttaaccttc ttgggaaggt atttgatctc ctggagattg 1680ttactcgggt agatcgtctt gatgagacct gctgcgtagg aacgcggccg ctgtacaggg 1740cccgggcata tggcgcgcca tatgcaccat acatggagtc aaaaattcag atcgaggatc 1800taacagaact cgccgtgaag actggcgaac agttcataca gagtctttta cgactcaatg 1860acaagaagaa aatcttcgtc aacatggtgg agcacgacac tctcgtctac tccaagaata 1920tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa agggtaatat 1980cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcaaa aggacagtag 2040aaaaggaagg tggcacctac aaatgccatc attgcgataa aggaaaggct atcgttcaag 2100atgcccctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa 2160aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg 2220taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt 2280catttcattt ggagaggact cgagctcatt tctctattac ttcagccata acaaaagaac 2340tcttttctct tcttattaaa ccaaaaccat gagtaaacgg aatccgaaga ttctgaagat 2400ttttctgtat atgttacttc tcaactctct ctttctcatc atctacttcg tttttcactc 2460atcgtcgttt tcaagtgtcg tagatgtgat cggtttgctt gcgactgcag cctacgtgac 2520gttggcgagc gcatacaagg tggtccagtt cattaacgtg tcgagcgtaa cggatgtcgc 2580tggtctcgaa agtgatgctt tgccgctcac tccaagggtt gacgttatcg tgccgacatt 2640caatgagaac tccagcacat tgctcgagtg cgtcgcttct atatgcgcac aagactaccg 2700cggaccaata acgattgtcg tggtagacga tgggtcgacc aacaaaacat catttcacgc 2760agtatgcgac aagtacgcga gcgacgaaag gttcatattt gtcgaacttg atcaaaacaa 2820ggggaagcgc gccgcgcaaa tggaggccat caggagaaca gacggagacc tgatactaaa 2880cgtagactcg gacacggtta tagataagga tgttgttaca aagcttgcgt cgtccatgag 2940agccccgaat gtcggtggtg tcatggggca gctcgttgca aagaatcgag aaagatcttg 3000gcttaccaga ttaatcgata tggagtactg gcttgcgtgt aacgaggagc gcattgcgca 3060gtcgaggttt ggctccgtga tgtgttgttg tgggccgtgc gccatgtata gaagatctgc 3120aattacgcca ctattggcag aatatgagca ccagacattc ctagggcgtc cgagcaactt 3180tggtgaggat cgccatctca caatcctgat gctgaaggcg ggatttcgga ccgggtacgt 3240cccaggtgcc gtagcgagga cgttggttcc ggatgggctg gcgccgtacc tgcgccagca 3300actccgctgg gcccgcagca cttatcgcga caccgccctc gccttacgta taaagaaaaa 3360tctaagcaaa tatatcacct ttgagatatg cgcacagaat ttgggtacgg ctctcttact 3420tgtgatgacc atgatttcgc tttcgctgac tacatcaggg tcgcaaacgc ccgttatcat 3480tctgggtgtc gttgtgggga tgtctataat aagatgttgt tctgtcgccc ttatagcgaa 3540agattttcgg tttctatact tcatcgttca ctcagcgttg aatgttctaa ttttaacgcc 3600gttaaaactc tatgccctgt taaccattcg ggatagtcgg tggctatcac gcgagagttc 3660ctaagctagc aagcttggac acgctgaaat caccagtctc tctctacaaa tctatctctc 3720tctattttct ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata 3780gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa 3840tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc 3900atgcatggta cagcggccaa ttgcctgcag gtcgacggcc gagtactggc aggatatata 3960ccgttgtaat t 397111431PRTArtificialNODC protein linked to XylT Golgi signal anchor sequenceSIGNAL(1)..(35)35 N-terminal amino acids of beta-1,2- xylosyltransferase from Arabidopsis thalianaMISC_FEATURE(36)..(431)NODC from A.caulinodans 11Met Ser Lys Arg Asn Pro Lys Ile Leu Lys Ile Phe Leu Tyr Met Leu1 5 10 15Leu Leu Asn Ser Leu Phe Leu Ile Ile Tyr Phe Val Phe His Ser Ser 20 25 30Ser Phe Ser Ser Val Val Asp Val Ile Gly Leu Leu Ala Thr Ala Ala 35 40 45Tyr Val Thr Leu Ala Ser Ala Tyr Lys Val Val Gln Phe Ile Asn Val 50 55 60Ser Ser Val Thr Asp Val Ala Gly Leu Glu Ser Asp Ala Leu Pro Leu65 70 75 80Thr Pro Arg Val Asp Val Ile Val Pro Thr Phe Asn Glu Asn Ser Ser 85 90 95Thr Leu Leu Glu Cys Val Ala Ser Ile Cys Ala Gln Asp Tyr Arg Gly 100 105 110Pro Ile Thr Ile Val Val Val Asp Asp Gly Ser Thr Asn Lys Thr Ser 115 120 125Phe His Ala Val Cys Asp Lys Tyr Ala Ser Asp Glu Arg Phe Ile Phe 130 135 140Val Glu Leu Asp Gln Asn Lys Gly Lys Arg Ala Ala Gln Met Glu Ala145 150 155 160Ile Arg Arg Thr Asp Gly Asp Leu Ile Leu Asn Val Asp Ser Asp Thr 165 170 175Val Ile Asp Lys Asp Val Val

Thr Lys Leu Ala Ser Ser Met Arg Ala 180 185 190Pro Asn Val Gly Gly Val Met Gly Gln Leu Val Ala Lys Asn Arg Glu 195 200 205Arg Ser Trp Leu Thr Arg Leu Ile Asp Met Glu Tyr Trp Leu Ala Cys 210 215 220Asn Glu Glu Arg Ile Ala Gln Ser Arg Phe Gly Ser Val Met Cys Cys225 230 235 240Cys Gly Pro Cys Ala Met Tyr Arg Arg Ser Ala Ile Thr Pro Leu Leu 245 250 255Ala Glu Tyr Glu His Gln Thr Phe Leu Gly Arg Pro Ser Asn Phe Gly 260 265 270Glu Asp Arg His Leu Thr Ile Leu Met Leu Lys Ala Gly Phe Arg Thr 275 280 285Gly Tyr Val Pro Gly Ala Val Ala Arg Thr Leu Val Pro Asp Gly Leu 290 295 300Ala Pro Tyr Leu Arg Gln Gln Leu Arg Trp Ala Arg Ser Thr Tyr Arg305 310 315 320Asp Thr Ala Leu Ala Leu Arg Ile Lys Lys Asn Leu Ser Lys Tyr Ile 325 330 335Thr Phe Glu Ile Cys Ala Gln Asn Leu Gly Thr Ala Leu Leu Leu Val 340 345 350Met Thr Met Ile Ser Leu Ser Leu Thr Thr Ser Gly Ser Gln Thr Pro 355 360 365Val Ile Ile Leu Gly Val Val Val Gly Met Ser Ile Ile Arg Cys Cys 370 375 380Ser Val Ala Leu Ile Ala Lys Asp Phe Arg Phe Leu Tyr Phe Ile Val385 390 395 400His Ser Ala Leu Asn Val Leu Ile Leu Thr Pro Leu Lys Leu Tyr Ala 405 410 415Leu Leu Thr Ile Arg Asp Ser Arg Trp Leu Ser Arg Glu Ser Ser 420 425 430128491DNAArtificial SequenceTDNA comprising nucleic acids encoding the F286 promoter, a Golgi retention peptide and a NODC proteinmisc_feature(1)..(25)right border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)promoter(75)..(1484)sequence including the promoter region of the chitinase F286 gene of Gossypium hirsutum (cotton) (Haigler et al., 2004)sig_peptide(1485)..(1589)gene(1590)..(2778)3'UTR(2781)..(3013)s- equence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus (Sanfacon et al., 1991)promoter(3036)..(4451)sequence including the promoter region of the chitinase F286 gene of Gossypium hirsutum (cotton) (Haigler et al., 2004)gene(4452)..(6281)coding region of the glutaminefructose-6- phosphate amidotransferase gene of Escherichia coli (Frohberg and Essigmann, 2006)3'UTR(6282)..(6555)sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(6598)..(6631)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)promoter(6632)..(7468)P35S3 sequence including the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al., 1985)gene(7469)..(8020)bar the coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces hygroscopicus (Thompson et al., 1987).3'UTR(8021)..(8304)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(8305)..(8338)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)misc_feature(8467)..(8491)left border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988) 12aattacaacg gtatatatcc tgccagtact gggccccctc gagggcgatc gctacgtacc 60tgcagggcgg ccgccaacct ctcgagctgc catattgggt ttttcactac ccacctcttc 120attaaatgta tcttcaacct ctcaactcct ttcaccacca gacgaatctt ctttagcaaa 180atcaaaatga ccttatgaaa atttagcacg tccacctcca gattcaaagg ctgtgaatcc 240ccaacttcgg aaattgttca tctccacatt caagaataat gagttcctca atttgtttta 300actgattagc cgatattaag cgagttagac tccatggaaa taaaatcacc ctaataaata 360gcaacgcttt tgaacgtctc taggttccaa gcgtgctaag gagcgccagt aacttcaatc 420caagttgtgc gaaaacgtat gaaatggaac tgagaccagc gttcaacatc gatgaaaatt 480tgttttaaca atgagaactg caaatcctcc atagtcttct aacatttcaa cattcgaaat 540ctcgaaaaga aattggcttg atatgattta tttagggtgt taattttatg tattataata 600atgcacaaat tgatatttta tgcatcacat ttaatatttt taaagtatat aatatcaaat 660cattttatga aaataaaaat accaaataat acataaattg atagttcaag tatttcatta 720aaaattttca aaatataaat atcatattga aacattttat aaaagaatag ataccaaata 780tgacatcatc ccctgttgag agtaaccaaa cactgttttc atccagccca tgagaagtat 840ttggcccaaa agcaaaagtt tcagtacaat gaattatgaa tcccaaaaaa accccaagtg 900gtccaggtcc aagccagtct agggctgagg aaagaaatgg aaaaattgaa aagtaattcc 960agggtctgat tcaattttat taaatttagt ttgattttgg tttcggttca taaatttaaa 1020aataatttta aaatgttata taaaactgtt ttttaaaaat aaattaatca ataatctaaa 1080acgataaaaa tggcgatttg aattaagctc atattttgaa aaaaaaataa aaattatctc 1140atccagaact gattaaaacc gaaccgatga atcctagaag ccaagccaag tgtgcagagt 1200aagaatagaa catcaacatt ttgctttaag cttttcgttg cttgcactct aagaagcata 1260aaacgcaagc aaaacttgac actagtgtga gtgtgagtgc ccatcattca tcaaccctga 1320aaatcgccct tcccctaatc agttctaacc tcactttcta acactttcac tgcagcactc 1380aaaaacattc gccgaatctt tactataaac tcccagtgtt ggtttctcca ctccaaaccc 1440aaaccacgac caccacattt tgcttcgtat ctttgatatc tatcatgagt aaacggaatc 1500cgaagattct gaagattttt ctgtatatgt tacttctcaa ctctctcttt ctcatcatct 1560acttcgtttt tcactcatcg tcgttttcaa gtgtcgtaga tgtgatcggt ttgcttgcga 1620ctgcagccta cgtgacgttg gcgagcgcat acaaggtggt ccagttcatt aacgtgtcga 1680gcgtaacgga tgtcgctggt ctcgaaagtg atgctttgcc gctcactcca agggttgacg 1740ttatcgtgcc gacattcaat gagaactcca gcacattgct cgagtgcgtc gcttctatat 1800gcgcacaaga ctaccgcgga ccaataacga ttgtcgtggt agacgatggg tcgaccaaca 1860aaacatcatt tcacgcagta tgcgacaagt acgcgagcga cgaaaggttc atatttgtcg 1920aacttgatca aaacaagggg aagcgcgccg cgcaaatgga ggccatcagg agaacagacg 1980gagacctgat actaaacgta gactcggaca cggttataga taaggatgtt gttacaaagc 2040ttgcgtcgtc catgagagcc ccgaatgtcg gtggtgtcat ggggcagctc gttgcaaaga 2100atcgagaaag atcttggctt accagattaa tcgatatgga gtactggctt gcgtgtaacg 2160aggagcgcat tgcgcagtcg aggtttggct ccgtgatgtg ttgttgtggg ccgtgcgcca 2220tgtatagaag atctgcaatt acgccactat tggcagaata tgagcaccag acattcctag 2280ggcgtccgag caactttggt gaggatcgcc atctcacaat cctgatgctg aaggcgggat 2340ttcggaccgg gtacgtccca ggtgccgtag cgaggacgtt ggttccggat gggctggcgc 2400cgtacctgcg ccagcaactc cgctgggccc gcagcactta tcgcgacacc gccctcgcct 2460tacgtataaa gaaaaatcta agcaaatata tcacctttga gatatgcgca cagaatttgg 2520gtacggctct cttacttgtg atgaccatga tttcgctttc gctgactaca tcagggtcgc 2580aaacgcccgt tatcattctg ggtgtcgttg tggggatgtc tataataaga tgttgttctg 2640tcgcccttat agcgaaagat tttcggtttc tatacttcat cgttcactca gcgttgaatg 2700ttctaatttt aacgccgtta aaactctatg ccctgttaac cattcgggat agtcggtggc 2760tatcacgcga gagttcctaa gctagcaagc ttggacacgc tgaaatcacc agtctctctc 2820tacaaatcta tctctctcta ttttctccat aataatgtgt gagtagttcc cagataaggg 2880aattagggtt cctatagggt ttcgctcatg tgttgagcat ataagaaacc cttagtatgt 2940atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac caaaatccag 3000tactaaaatc cagacgcgtt taattaagcg gccgccaacc tctcgagctg ccatattggg 3060tttttcacta cccacctctt cattaaatgt atcttcaacc tctcaactcc tttcaccacc 3120agacgaatct tctttagcaa aatcaaaatg accttatgaa aatttagcac gtccacctcc 3180agattcaaag gctgtgaatc cccaacttcg gaaattgttc atctccacat tcaagaataa 3240tgagttcctc aatttgtttt aactgattag ccgatattaa gcgagttaga ctccatggaa 3300ataaaatcac cctaataaat agcaacgctt ttgaacgtct ctaggttcca agcgtgctaa 3360ggagcgccag taacttcaat ccaagttgtg cgaaaacgta tgaaatggaa ctgagaccag 3420cgttcaacat cgatgaaaat ttgttttaac aatgagaact gcaaatcctc catagtcttc 3480taacatttca acattcgaaa tctcgaaaag aaattggctt gatatgattt atttagggtg 3540ttaattttat gtattataat aatgcacaaa ttgatatttt atgcatcaca tttaatattt 3600ttaaagtata taatatcaaa tcattttatg aaaataaaaa taccaaataa tacataaatt 3660gatagttcaa gtatttcatt aaaaattttc aaaatataaa tatcatattg aaacatttta 3720taaaagaata gataccaaat atgacatcat cccctgttga gagtaaccaa acactgtttt 3780catccagccc atgagaagta tttggcccaa aagcaaaagt ttcagtacaa tgaattatga 3840atcccaaaaa aaccccaagt ggtccaggtc caagccagtc tagggctgag gaaagaaatg 3900gaaaaattga aaagtaattc cagggtctga ttcaatttta ttaaatttag tttgattttg 3960gtttcggttc ataaatttaa aaataatttt aaaatgttat ataaaactgt tttttaaaaa 4020taaattaatc aataatctaa aacgataaaa atggcgattt gaattaagct catattttga 4080aaaaaaaata aaaattatct catccagaac tgattaaaac cgaaccgatg aatcctagaa 4140gccaagccaa gtgtgcagag taagaataga acatcaacat tttgctttaa gcttttcgtt 4200gcttgcactc taagaagcat aaaacgcaag caaaacttga cactagtgtg agtgtgagtg 4260cccatcattc atcaaccctg aaaatcgccc ttcccctaat cagttctaac ctcactttct 4320aacactttca ctgcagcact caaaaacatt cgccgaatct ttactataaa ctcccagtgt 4380tggtttctcc actccaaacc caaaccacga ccaccacatt ttgcttcgta tctttgatat 4440ctaggtctcc catgtgcgga attgttggtg ctatcgccca aagagacgtt gctgagattt 4500tgttagaggg tctgcgaagg ctagagtata gaggatatga ctccgctggt ctggctgtcg 4560ttgatgctga gggtcatatg acaaggctaa gaaggttagg aaaggttcag atgcttgctc 4620aggcagctga ggaacatcca ttgcatggag gtactggtat tgcacatacc aggtgggcta 4680ctcatgggga gccatcagaa gttaatgctc atccacatgt gagtgagcat atcgttgtag 4740ttcacaatgg gataattgaa aaccacgaac cattgaggga agagttaaag gcaagaggat 4800atacttttgt gagtgagact gacactgagg ttattgcaca tttagtgaac tgggaactca 4860aacagggggg cacattgcgt gaggctgtgt taagagctat tcctcaactt agaggtgcat 4920acggtactgt tattatggat tcaagacacc cagatactct ccttgcagct agatcaggta 4980gtcccttggt cataggactt ggaatgggtg aaaattttat cgctagcgac caattggcct 5040tattgccagt tacaagacga tttattttcc ttgaagaggg cgatattgct gagattacta 5100gaaggtctgt gaacatcttt gataagactg gcgctgaggt taaacgtcag gatatcgagt 5160ctaaccttca atacgatgct ggtgataaag gaatttacag gcattatatg caaaaggaaa 5220tttatgaaca accaaatgct atcaaaaaca cacttactgg ccgtatttct catggacagg 5280tcgatttaag cgagcttggt cctaatgcag acgaactgct atcaaaagtt gagcacatac 5340agatactggc atgcggaact agttataatt caggaatggt gtctagatac tggttcgaaa 5400gcttggcagg tataccttgt gatgtagaga tcgcttctga gtttaggtat agaaagtctg 5460ctgtgcgtag aaattcatta atgattacat tatctcaatc cggagaaaca gcagatacac 5520tggctggatt gaggctttct aaggaactcg gatatctggg ttcacttgct atttgtaatg 5580taccaggttc ctcattggtt cgtgaatcag atctagcact tatgacaaat gcaggaactg 5640aaataggtgt ggcaagtacc aaggctttca caacccaact gaccgtactt ttaatgttgg 5700tagcaaaact cagtcgatta aaggggctag atgcatctat cgaacatgat attgttcacg 5760ggcttcaagc tctcccttca agaattgaac aaatgctttc acaagataag agaatagagg 5820cattggctga agatttttcc gacaaacatc acgcattgtt tcttggacgt ggcgatcaat 5880atccaattgc attggaagga gctttgaagt tgaaagaaat aagttacatt cacgcagaag 5940catatgcagc tggagaactc aagcatggtc ctttggcact catcgacgct gacatgcccg 6000tgatcgtagt ggctcctaat aacgaactgc tcgaaaagct taaatcaaat atcgaagagg 6060ttcgagctag aggaggtcag ctttacgttt tcgctgaaca agatgctgga ttcgtgtcaa 6120gcgataatat gcatataatt gaaatgcctc acgttgaaga agtgattgca cctatatttt 6180atacagtccc attgcaactt ctagcttacc atgttgcact tattaaagga actgatgttg 6240atcagcctag aaacctagca aaatctgtaa cagtcgaata aacgcgtggc gcgccgaagc 6300agatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 6360gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 6420catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 6480cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 6540tatgttacta gatcggaatt cgatatcatt accctgttat ccctaaagct tattaatata 6600acttcgtata gcatacatta tacgaagtta tgtttcctac gcagcaggtc tcatcaagac 6660gatctacccg agtaacaatc tccaggagat caaatacctt cccaagaagg ttaaagatgc 6720agtcaaaaga ttcaggacta attgcatcaa gaacacagag aaagacatat ttctcaagat 6780cagaagtact attccagtat ggacgattca aggcttgctt cataaaccaa ggcaagtaat 6840agagattgga gtctctaaaa aggtagttcc tactgaatct aaggccatgc atggagtcta 6900agattcaaat cgaggatcta acagaactcg ccgtgaagac tggcgaacag ttcatacaga 6960gtcttttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacactc 7020tggtctactc caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt 7080ttcaacaaag gataatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact 7140tcatcgaaag gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 7200gaaaggctat cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 7260cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 7320gtgacatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 7380cctctatata aggaagttca tttcatttgg agaggacacg ctgaaatcac cagtctctct 7440ctataaatct atctctctct ctataacaat ggacccagaa cgacgcccgg ccgacatccg 7500ccgtgccacc gaggcggaca tgccggcggt ctgcaccatc gtcaaccact acatcgagac 7560aagcacggtc aacttccgta ccgagccgca ggaaccgcag gagtggacgg acgacctcgt 7620ccgtctgcgg gagcgctatc cctggctcgt cgccgaggtg gacggcgagg tcgccggcat 7680cgcctacgcg ggcccctgga aggcacgcaa cgcctacgac tggacggccg agtcgaccgt 7740gtacgtctcc ccccgccacc agcggacggg actgggctcc acgctctaca cccacctgct 7800gaagtccctg gaggcacagg gcttcaagag cgtggtcgct gtcatcgggc tgcccaacga 7860cccgagcgtg cgcatgcacg aggcgctcgg atatgccccc cgcggcatgc tgcgggcggc 7920cggcttcaag cacgggaact ggcatgacgt gggtttctgg cagctggact tcagcctgcc 7980ggtaccgccc cgtccggtcc tgcccgtcac cgagatctga gatcacccgt tctaggatcc 8040gaagcagatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt 8100cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg 8160taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt 8220taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg 8280tcatctatgt tactagatcg aaacataact tcgtatagca tacattatac gaagttatca 8340aaacgtcgtg agacagtttg gttaactata acggtcctaa ggtagcgatc gaggcattac 8400ggcattacgg cactcgcgag ggtccgaatc tatgtcgggt gcggagaaag aggtaatgaa 8460atggcaattt acaattgaat atatcctgcc g 8491139567DNAArtificial SequenceTDNA comprising nucleic acid sequences encoding the gluc1A promoter, a Golgi retention peptide and a NODC proteinmisc_feature(1)..(25)right border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)promoter(26)..(2025)sequence including the promoter region of the beta-1,3-endoglucanase gene gluc1 of the A subgenome of Gossypium hirsutum (cotton)(Engelen and Arioli, 2008)sig_peptide(2026)..(2130)gene(2131)..(3321)3'UTR(3322)..(355- 4)sequence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus (Sanfacon et al., 1991)promoter(3555)..(5527)Pgluc1(A1.9) sequence including the promoter region of the beta-1,3-endoglucanase gene gluc1 of the A subgenome of Gossypium hirsutum (cotton)(Engelen and Arioli, 2008)gene(5528)..(7357)gfaEc-1Pb coding region of the glutamine fructose-6-phosphate amidotransferase gene of Escherichia coli (Frohberg and Essigmann, 2006)3'UTR(7358)..(7631)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(7674)..(7707)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)promoter(7708)..(8544)P35S3 sequence including the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al., 1985)gene(8545)..(9096)bar coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces hygroscopicus (Thompson et al., 1987).3'UTR(9097)..(9380)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(9381)..(9414)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)misc_feature(9543)..(9567)left border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988) 13aattacaacg gtatatatcc tgccagtact gggccccctc gagggcgatc gctacgtacc 60tgcagggcgg ccgcgattat ataaataggg ggcgaatcta gggagctggc atgaccccta 120aaatagaatt ttctattttg acctatcaaa atttttaaaa ttttaaatta gtaaaggtaa 180atttgtactt taacctctta aaatgataaa attttacttt aatcctttaa aatttacatt 240tttactatca taaaaattac aatttgattt tacccctaaa atttttttct agcttagccc 300tgtatataaa tatattattt ataattttta tatttaaaat ataaagtttt taattataca 360aataattaaa atctgatatt taaaactaaa gtaatttctt ttttcttttt actttttttt 420aattgcaaca taatggttta aatatctata taacgtatga agtaatttga tataaatttt 480attttaattt attattatat aaattcattt agtaaaaact tttaatagaa tcaaaatttt 540tatttgtaaa ttcgataact tttcttatca agtatatttg tgagaaccaa atatttagta 600aaattaatat tcttatttat aaatatgata aatcttataa aaaaatattt aaaatgaaaa 660aaattgtaca aatattataa aaaaatattt aaaatgaaaa acattgtaca aaggctatat 720aagaagttca aaagtttctt cgaccatgta ctcttataga gattatagat agattataaa 780actatatgta gtttctctta acttttaaat aagaggataa atgtatttta atgtactcaa 840acttatatat ttttatattg acaataatat caatatcaac ctaattaaga ttcattctaa 900cattaatgtt gaagattttt aataaaagaa aaggttaata aattaattag aacacaaaca 960aacacaaatt taagtggtat gtaaggtcct tgacccaaag gaaaaatttg ttacgtcgat 1020taaattataa attaatttaa agtaaaatta cattttaacc taaaaaaaga gaaaagtata 1080tctaatttct tcgaaaatgg aaagaaaatt ataaatttat ggcatttcta aaaaaattct 1140gaattcgcta ctaaaagatg aaattataaa atccgaagca ttaccagaag atggatcacc 1200aaatcacaaa caatcaatga aaagtaatga taattaattg aaagtgagca tttaattttg 1260atagccatat acttcctgct gaatttatag gttctcatta atgcaattaa attatattcg 1320acaccttttg aatgaaataa aatgacacaa gaggaaagac ggttcatcta ttttttcttt 1380caatcgccca tcaaaatacc aaaaatgtaa ctacatgcaa aaaatcaaat atgaaaaata 1440ttcatatttt gatattttaa tatattgtgt gttcaaaacg taaatgtatt gaaaaattat 1500gatggtgttg ttgctgtatg

tccataaaat tcaatgtact cacatttatc aaatgtatac 1560tttgagagaa gttattttga taatactcaa gtttttttta tagatgggaa aattttttaa 1620attatttttt gattttgatg aaatgtatat ataaatttta attcgataca tataaatata 1680tatgtaaatt ttaaatttaa atttaataat atacaattaa gaaaataatt tacataaata 1740tatatcctaa taaaaataaa attagaaaga ggaaatgtca aaacctcttc attatataca 1800aatatgatgg gacacgatac cctcatgcat tgatatctca tgttgtccaa aaactcggaa 1860tcctttttga aaaaaaactt ccagagagag tatataaatc cagcagtagg cacaagaaac 1920gagcaccagt tattgacttt cctttgtaaa aaaaaaaaag tgctgagatc aagaaatata 1980gtgaaatatg ggtccaagat tttctgggtt tttaatctaa gcaccatgag taaacggaat 2040ccgaagattc tgaagatttt tctgtatatg ttacttctca actctctctt tctcatcatc 2100tacttcgttt ttcactcatc gtcgttttca agtgtcgtag atgtgatcgg tttgcttgcg 2160actgcagcct acgtgacgtt ggcgagcgca tacaaggtgg tccagttcat taacgtgtcg 2220agcgtaacgg atgtcgctgg tctcgaaagt gatgctttgc cgctcactcc aagggttgac 2280gttatcgtgc cgacattcaa tgagaactcc agcacattgc tcgagtgcgt cgcttctata 2340tgcgcacaag actaccgcgg accaataacg attgtcgtgg tagacgatgg gtcgaccaac 2400aaaacatcat ttcacgcagt atgcgacaag tacgcgagcg acgaaaggtt catatttgtc 2460gaacttgatc aaaacaaggg gaagcgcgcc gcgcaaatgg aggccatcag gagaacagac 2520ggagacctga tactaaacgt agactcggac acggttatag ataaggatgt tgttacaaag 2580cttgcgtcgt ccatgagagc cccgaatgtc ggtggtgtca tggggcagct cgttgcaaag 2640aatcgagaaa gatcttggct taccagatta atcgatatgg agtactggct tgcgtgtaac 2700gaggagcgca ttgcgcagtc gaggtttggc tccgtgatgt gttgttgtgg gccgtgcgcc 2760atgtatagaa gatctgcaat tacgccacta ttggcagaat atgagcacca gacattccta 2820gggcgtccga gcaactttgg tgaggatcgc catctcacaa tcctgatgct gaaggcggga 2880tttcggaccg ggtacgtccc aggtgccgta gcgaggacgt tggttccgga tgggctggcg 2940ccgtacctgc gccagcaact ccgctgggcc cgcagcactt atcgcgacac cgccctcgcc 3000ttacgtataa agaaaaatct aagcaaatat atcacctttg agatatgcgc acagaatttg 3060ggtacggctc tcttacttgt gatgaccatg atttcgcttt cgctgactac atcagggtcg 3120caaacgcccg ttatcattct gggtgtcgtt gtggggatgt ctataataag atgttgttct 3180gtcgccctta tagcgaaaga ttttcggttt ctatacttca tcgttcactc agcgttgaat 3240gttctaattt taacgccgtt aaaactctat gccctgttaa ccattcggga tagtcggtgg 3300ctatcacgcg agagttccta agctagcaag cttggacacg ctgaaatcac cagtctctct 3360ctacaaatct atctctctct attttctcca taataatgtg tgagtagttc ccagataagg 3420gaattagggt tcctataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg 3480tatttgtatt tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca 3540gtactaaaat ccagacgcgt ttaattaagc ggccgcgatt atataaatag ggggcgaatc 3600tagggagctg gcatgacccc taaaatagaa ttttctattt tgacctatca aaatttttaa 3660aattttaaat tagtaaaggt aaatttgtac tttaacctct taaaatgata aaattttact 3720ttaatccttt aaaatttaca tttttactat cataaaaatt acaatttgat tttaccccta 3780aaattttttt ctagcttagc cctgtatata aatatattat ttataatttt tatatttaaa 3840atataaagtt tttaattata caaataatta aaatctgata tttaaaacta aagtaatttc 3900ttttttcttt ttactttttt ttaattgcaa cataatggtt taaatatcta tataacgtat 3960gaagtaattt gatataaatt ttattttaat ttattattat ataaattcat ttagtaaaaa 4020cttttaatag aatcaaaatt tttatttgta aattcgataa cttttcttat caagtatatt 4080tgtgagaacc aaatatttag taaaattaat attcttattt ataaatatga taaatcttat 4140aaaaaaatat ttaaaatgaa aaaaattgta caaatattat aaaaaaatat ttaaaatgaa 4200aaacattgta caaaggctat ataagaagtt caaaagtttc ttcgaccatg tactcttata 4260gagattatag atagattata aaactatatg tagtttctct taacttttaa ataagaggat 4320aaatgtattt taatgtactc aaacttatat atttttatat tgacaataat atcaatatca 4380acctaattaa gattcattct aacattaatg ttgaagattt ttaataaaag aaaaggttaa 4440taaattaatt agaacacaaa caaacacaaa tttaagtggt atgtaaggtc cttgacccaa 4500aggaaaaatt tgttacgtcg attaaattat aaattaattt aaagtaaaat tacattttaa 4560cctaaaaaaa gagaaaagta tatctaattt cttcgaaaat ggaaagaaaa ttataaattt 4620atggcatttc taaaaaaatt ctgaattcgc tactaaaaga tgaaattata aaatccgaag 4680cattaccaga agatggatca ccaaatcaca aacaatcaat gaaaagtaat gataattaat 4740tgaaagtgag catttaattt tgatagccat atacttcctg ctgaatttat aggttctcat 4800taatgcaatt aaattatatt cgacaccttt tgaatgaaat aaaatgacac aagaggaaag 4860acggttcatc tattttttct ttcaatcgcc catcaaaata ccaaaaatgt aactacatgc 4920aaaaaatcaa atatgaaaaa tattcatatt ttgatatttt aatatattgt gtgttcaaaa 4980cgtaaatgta ttgaaaaatt atgatggtgt tgttgctgta tgtccataaa attcaatgta 5040ctcacattta tcaaatgtat actttgagag aagttatttt gataatactc aagttttttt 5100tatagatggg aaaatttttt aaattatttt ttgattttga tgaaatgtat atataaattt 5160taattcgata catataaata tatatgtaaa ttttaaattt aaatttaata atatacaatt 5220aagaaaataa tttacataaa tatatatcct aataaaaata aaattagaaa gaggaaatgt 5280caaaacctct tcattatata caaatatgat gggacacgat accctcatgc attgatatct 5340catgttgtcc aaaaactcgg aatccttttt gaaaaaaaac ttccagagag agtatataaa 5400tccagcagta ggcacaagaa acgagcacca gttattgact ttcctttgta aaaaaaaaaa 5460agtgctgaga tcaagaaata tagtgaaata tgggtccaag attttctggg tttttaatct 5520aagcaccatg tgcggaattg ttggtgctat cgcccaaaga gacgttgctg agattttgtt 5580agagggtctg cgaaggctag agtatagagg atatgactcc gctggtctgg ctgtcgttga 5640tgctgagggt catatgacaa ggctaagaag gttaggaaag gttcagatgc ttgctcaggc 5700agctgaggaa catccattgc atggaggtac tggtattgca cataccaggt gggctactca 5760tggggagcca tcagaagtta atgctcatcc acatgtgagt gagcatatcg ttgtagttca 5820caatgggata attgaaaacc acgaaccatt gagggaagag ttaaaggcaa gaggatatac 5880ttttgtgagt gagactgaca ctgaggttat tgcacattta gtgaactggg aactcaaaca 5940ggggggcaca ttgcgtgagg ctgtgttaag agctattcct caacttagag gtgcatacgg 6000tactgttatt atggattcaa gacacccaga tactctcctt gcagctagat caggtagtcc 6060cttggtcata ggacttggaa tgggtgaaaa ttttatcgct agcgaccaat tggccttatt 6120gccagttaca agacgattta ttttccttga agagggcgat attgctgaga ttactagaag 6180gtctgtgaac atctttgata agactggcgc tgaggttaaa cgtcaggata tcgagtctaa 6240ccttcaatac gatgctggtg ataaaggaat ttacaggcat tatatgcaaa aggaaattta 6300tgaacaacca aatgctatca aaaacacact tactggccgt atttctcatg gacaggtcga 6360tttaagcgag cttggtccta atgcagacga actgctatca aaagttgagc acatacagat 6420actggcatgc ggaactagtt ataattcagg aatggtgtct agatactggt tcgaaagctt 6480ggcaggtata ccttgtgatg tagagatcgc ttctgagttt aggtatagaa agtctgctgt 6540gcgtagaaat tcattaatga ttacattatc tcaatccgga gaaacagcag atacactggc 6600tggattgagg ctttctaagg aactcggata tctgggttca cttgctattt gtaatgtacc 6660aggttcctca ttggttcgtg aatcagatct agcacttatg acaaatgcag gaactgaaat 6720aggtgtggca agtaccaagg ctttcacaac ccaactgacc gtacttttaa tgttggtagc 6780aaaactcagt cgattaaagg ggctagatgc atctatcgaa catgatattg ttcacgggct 6840tcaagctctc ccttcaagaa ttgaacaaat gctttcacaa gataagagaa tagaggcatt 6900ggctgaagat ttttccgaca aacatcacgc attgtttctt ggacgtggcg atcaatatcc 6960aattgcattg gaaggagctt tgaagttgaa agaaataagt tacattcacg cagaagcata 7020tgcagctgga gaactcaagc atggtccttt ggcactcatc gacgctgaca tgcccgtgat 7080cgtagtggct cctaataacg aactgctcga aaagcttaaa tcaaatatcg aagaggttcg 7140agctagagga ggtcagcttt acgttttcgc tgaacaagat gctggattcg tgtcaagcga 7200taatatgcat ataattgaaa tgcctcacgt tgaagaagtg attgcaccta tattttatac 7260agtcccattg caacttctag cttaccatgt tgcacttatt aaaggaactg atgttgatca 7320gcctagaaac ctagcaaaat ctgtaacagt cgaataaacg cgtggcgcgc cgaagcagat 7380cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg 7440attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg 7500acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg 7560atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg 7620ttactagatc ggaattcgat atcattaccc tgttatccct aaagcttatt aatataactt 7680cgtatagcat acattatacg aagttatgtt tcctacgcag caggtctcat caagacgatc 7740tacccgagta acaatctcca ggagatcaaa taccttccca agaaggttaa agatgcagtc 7800aaaagattca ggactaattg catcaagaac acagagaaag acatatttct caagatcaga 7860agtactattc cagtatggac gattcaaggc ttgcttcata aaccaaggca agtaatagag 7920attggagtct ctaaaaaggt agttcctact gaatctaagg ccatgcatgg agtctaagat 7980tcaaatcgag gatctaacag aactcgccgt gaagactggc gaacagttca tacagagtct 8040tttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acactctggt 8100ctactccaaa aatgtcaaag atacagtctc agaagaccaa agggctattg agacttttca 8160acaaaggata atttcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat 8220cgaaaggaca gtagaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa 8280ggctatcatt caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 8340gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 8400catctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 8460tatataagga agttcatttc atttggagag gacacgctga aatcaccagt ctctctctat 8520aaatctatct ctctctctat aacaatggac ccagaacgac gcccggccga catccgccgt 8580gccaccgagg cggacatgcc ggcggtctgc accatcgtca accactacat cgagacaagc 8640acggtcaact tccgtaccga gccgcaggaa ccgcaggagt ggacggacga cctcgtccgt 8700ctgcgggagc gctatccctg gctcgtcgcc gaggtggacg gcgaggtcgc cggcatcgcc 8760tacgcgggcc cctggaaggc acgcaacgcc tacgactgga cggccgagtc gaccgtgtac 8820gtctcccccc gccaccagcg gacgggactg ggctccacgc tctacaccca cctgctgaag 8880tccctggagg cacagggctt caagagcgtg gtcgctgtca tcgggctgcc caacgacccg 8940agcgtgcgca tgcacgaggc gctcggatat gccccccgcg gcatgctgcg ggcggccggc 9000ttcaagcacg ggaactggca tgacgtgggt ttctggcagc tggacttcag cctgccggta 9060ccgccccgtc cggtcctgcc cgtcaccgag atctgagatc acccgttcta ggatccgaag 9120cagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 9180cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 9240gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 9300acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 9360ctatgttact agatcgaaac ataacttcgt atagcataca ttatacgaag ttatcaaaac 9420gtcgtgagac agtttggtta actataacgg tcctaaggta gcgatcgagg cattacggca 9480ttacggcact cgcgagggtc cgaatctatg tcgggtgcgg agaaagaggt aatgaaatgg 9540caatttacaa ttgaatatat cctgccg 9567149621DNAArtificial SequenceTDNA comprising nucleic acid sequences encoding the gluc1D promoter, a Golgi retention peptide and a NODC proteinmisc_feature(1)..(25)right border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)promoter(26)..(2052)sequence including the promoter region of the beta-1,3-endoglucanase gene gluc1 of the D subgenome of Gossypium hirsutum (cotton)(Engelen and Arioli, 2008)sig_peptide(2053)..(2157)gene(2158)..(3348)3'UTR(3349)..(358- 1)sequence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus (Sanfacon et al., 1991)promoter(3582)..(5581)Pgluc1(D2.0) sequence including the promoter region of the beta-1,3-endoglucanase gene gluc1 of the D subgenome of Gossypium hirsutum (cotton)(Engelen and Arioli, 2008)gene(5582)..(7411)gfaEc-1Pb coding region of the glutamine fructose-6-phosphate amidotransferase gene of Escherichia coli (Frohberg and Essigmann, 2006)3'UTR(7412)..(7685)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(7728)..(7761)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)promoter(7762)..(8598)P35S3 sequence including the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al., 1985)gene(8599)..(9150)bar coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces hygroscopicus (Thompson et al., 1987).3'UTR(9151)..(9434)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(9435)..(9468)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)misc_feature(9597)..(9621)left border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988) 14aattacaacg gtatatatcc tgccagtact gggccccctc gagggcgatc gctacgtacc 60tgcagggcgg ccgcttcgaa cataatgcta ataaaaaatt tcctaatcat tattaaatca 120tttgtataaa ctataaagaa attgatatat tgtaaattaa acttttaact attcaatttt 180ttcttaatag tcaataaatt aatcataata attcataatt aatatataat taacataacc 240ataacataga attttttatt ttggcccatt aaaattttta aaattttaaa ttagtaaagg 300aaaaattaca ctttgacccc ttaaaaatga taaaatttta ttttaatcct ttaaaattga 360catttttact attgtaaaaa ttacaattta attttgcccc cctaaaaaat ttttctagct 420tcgcccttgt gtataaatat attaattaca atttttatat ttgaattata taaataatta 480aattttgata tttaaaacta aagtaatctc tttttttttt actttttttt aattgaaaca 540taatggttta aatatctata ttacgtatga agtaatttaa tataaatttt attttaattt 600attattatat aaattcattt agtaaaaact tttaatagaa tcaaaatttt tatttgtaaa 660ttcgataact tttcttatca agtaaatttg ttgaattaaa tatttagtaa aattaatatt 720tttatttata aatatgataa atcttataaa aaataaaaaa atatttaaaa tgaaaaacat 780tgtacaaagg ctatataaga agttcaaaag tttcttcgac cctgtactct aatagagatt 840atagatagat tatagaacta ttcatagttt ctcttaacct ttaaataaga attttagtgt 900actcaaactt acatattttt atattgataa taatgtcaat accagccgag ttaagattca 960ctcgacatta atgttgaaaa tttttaataa aagaaaatgt tgataagtta attagaacac 1020aagcaagcac aaatttaagt ggtaagtaag gtccttgacc ctaatggaaa aattgttatg 1080ttgattaaat tataaattaa tttaaggtaa aattatattt tgacctaaaa aaatgaaaaa 1140aatatatcta gtttcttcga aaatgaaaag aaaataataa attgatacat tataaaattt 1200atggcatttc taaaaaaatt ctgaatttga tgaaattata ataaaaaaaa agtttaaaaa 1260catatagatt tcaagaatag tgggaaaatt atatttgaac aacactgaag aaatccaaag 1320cattagcaga aaatggatca ccaaatcaca aacaatcagt gaaaagtaat gataattaat 1380tgaaagtgag catttaaatt tgatagccat atacttcctg ctgaatttat aggttctcat 1440taatgcaatt aaattatatt tgtcactttt tgaatgaaat aaatgacaca gttcatctat 1500tttttttctt tcaatcgccc atcaaaatac cgaaaatgta actacattaa aaaagatcga 1560aaaatattca tattttgata ttttaataga ttgtgtgttc aaggcgtaat gtactaaaaa 1620attatgatgg tgttgtcgct gtatgtccat aaaattcaat gtattcgcat gtatcaaatg 1680taaattttga cacaagttat tctaataata atcaagttat ttttatacat gagatacatc 1740tcaaaattat ttttatatat ccgaaaaatc ataacgtacg atcaaactag aaagaggaag 1800tgtcaaaacc tattcattat atgcaaatat gatgggacac gataccctca tgcattgata 1860tctcatattg tccaaaaact cagaatcctt tttgaaaaaa aaaaattcca gagagagtgt 1920ataaatccag cagtgtgcac aagaaacgag caccagttat tgacattcct ttgtaaaaaa 1980aaaaagaagc tgagatcaag aaatatagtg aaatatgggt ccaacatttt ctgggttttt 2040aatctcagca ccatgagtaa acggaatccg aagattctga agatttttct gtatatgtta 2100cttctcaact ctctctttct catcatctac ttcgtttttc actcatcgtc gttttcaagt 2160gtcgtagatg tgatcggttt gcttgcgact gcagcctacg tgacgttggc gagcgcatac 2220aaggtggtcc agttcattaa cgtgtcgagc gtaacggatg tcgctggtct cgaaagtgat 2280gctttgccgc tcactccaag ggttgacgtt atcgtgccga cattcaatga gaactccagc 2340acattgctcg agtgcgtcgc ttctatatgc gcacaagact accgcggacc aataacgatt 2400gtcgtggtag acgatgggtc gaccaacaaa acatcatttc acgcagtatg cgacaagtac 2460gcgagcgacg aaaggttcat atttgtcgaa cttgatcaaa acaaggggaa gcgcgccgcg 2520caaatggagg ccatcaggag aacagacgga gacctgatac taaacgtaga ctcggacacg 2580gttatagata aggatgttgt tacaaagctt gcgtcgtcca tgagagcccc gaatgtcggt 2640ggtgtcatgg ggcagctcgt tgcaaagaat cgagaaagat cttggcttac cagattaatc 2700gatatggagt actggcttgc gtgtaacgag gagcgcattg cgcagtcgag gtttggctcc 2760gtgatgtgtt gttgtgggcc gtgcgccatg tatagaagat ctgcaattac gccactattg 2820gcagaatatg agcaccagac attcctaggg cgtccgagca actttggtga ggatcgccat 2880ctcacaatcc tgatgctgaa ggcgggattt cggaccgggt acgtcccagg tgccgtagcg 2940aggacgttgg ttccggatgg gctggcgccg tacctgcgcc agcaactccg ctgggcccgc 3000agcacttatc gcgacaccgc cctcgcctta cgtataaaga aaaatctaag caaatatatc 3060acctttgaga tatgcgcaca gaatttgggt acggctctct tacttgtgat gaccatgatt 3120tcgctttcgc tgactacatc agggtcgcaa acgcccgtta tcattctggg tgtcgttgtg 3180gggatgtcta taataagatg ttgttctgtc gcccttatag cgaaagattt tcggtttcta 3240tacttcatcg ttcactcagc gttgaatgtt ctaattttaa cgccgttaaa actctatgcc 3300ctgttaacca ttcgggatag tcggtggcta tcacgcgaga gttcctaagc tagcaagctt 3360ggacacgctg aaatcaccag tctctctcta caaatctatc tctctctatt ttctccataa 3420taatgtgtga gtagttccca gataagggaa ttagggttcc tatagggttt cgctcatgtg 3480ttgagcatat aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa 3540atttctaatt cctaaaacca aaatccagta ctaaaatcca gacgcgttta attaagcggc 3600cgcttcgaac ataatgctaa taaaaaattt cctaatcatt attaaatcat ttgtataaac 3660tataaagaaa ttgatatatt gtaaattaaa cttttaacta ttcaattttt tcttaatagt 3720caataaatta atcataataa ttcataatta atatataatt aacataacca taacatagaa 3780ttttttattt tggcccatta aaatttttaa aattttaaat tagtaaagga aaaattacac 3840tttgacccct taaaaatgat aaaattttat tttaatcctt taaaattgac atttttacta 3900ttgtaaaaat tacaatttaa ttttgccccc ctaaaaaatt tttctagctt cgcccttgtg 3960tataaatata ttaattacaa tttttatatt tgaattatat aaataattaa attttgatat 4020ttaaaactaa agtaatctct ttttttttta ctttttttta attgaaacat aatggtttaa 4080atatctatat tacgtatgaa gtaatttaat ataaatttta ttttaattta ttattatata 4140aattcattta gtaaaaactt ttaatagaat caaaattttt atttgtaaat tcgataactt 4200ttcttatcaa gtaaatttgt tgaattaaat atttagtaaa attaatattt ttatttataa 4260atatgataaa tcttataaaa aataaaaaaa tatttaaaat gaaaaacatt gtacaaaggc 4320tatataagaa gttcaaaagt ttcttcgacc ctgtactcta atagagatta tagatagatt 4380atagaactat tcatagtttc tcttaacctt taaataagaa ttttagtgta ctcaaactta 4440catattttta tattgataat aatgtcaata ccagccgagt taagattcac tcgacattaa 4500tgttgaaaat ttttaataaa agaaaatgtt gataagttaa ttagaacaca agcaagcaca 4560aatttaagtg gtaagtaagg tccttgaccc taatggaaaa attgttatgt tgattaaatt 4620ataaattaat ttaaggtaaa attatatttt gacctaaaaa aatgaaaaaa atatatctag 4680tttcttcgaa aatgaaaaga aaataataaa ttgatacatt ataaaattta tggcatttct 4740aaaaaaattc tgaatttgat gaaattataa taaaaaaaaa gtttaaaaac atatagattt 4800caagaatagt gggaaaatta tatttgaaca acactgaaga aatccaaagc attagcagaa 4860aatggatcac caaatcacaa acaatcagtg aaaagtaatg ataattaatt gaaagtgagc 4920atttaaattt gatagccata tacttcctgc tgaatttata ggttctcatt aatgcaatta 4980aattatattt gtcacttttt gaatgaaata aatgacacag ttcatctatt ttttttcttt 5040caatcgccca tcaaaatacc gaaaatgtaa ctacattaaa aaagatcgaa aaatattcat 5100attttgatat tttaatagat tgtgtgttca aggcgtaatg tactaaaaaa ttatgatggt 5160gttgtcgctg tatgtccata

aaattcaatg tattcgcatg tatcaaatgt aaattttgac 5220acaagttatt ctaataataa tcaagttatt tttatacatg agatacatct caaaattatt 5280tttatatatc cgaaaaatca taacgtacga tcaaactaga aagaggaagt gtcaaaacct 5340attcattata tgcaaatatg atgggacacg ataccctcat gcattgatat ctcatattgt 5400ccaaaaactc agaatccttt ttgaaaaaaa aaaattccag agagagtgta taaatccagc 5460agtgtgcaca agaaacgagc accagttatt gacattcctt tgtaaaaaaa aaaagaagct 5520gagatcaaga aatatagtga aatatgggtc caacattttc tgggttttta atctcagcac 5580catgtgcgga attgttggtg ctatcgccca aagagacgtt gctgagattt tgttagaggg 5640tctgcgaagg ctagagtata gaggatatga ctccgctggt ctggctgtcg ttgatgctga 5700gggtcatatg acaaggctaa gaaggttagg aaaggttcag atgcttgctc aggcagctga 5760ggaacatcca ttgcatggag gtactggtat tgcacatacc aggtgggcta ctcatgggga 5820gccatcagaa gttaatgctc atccacatgt gagtgagcat atcgttgtag ttcacaatgg 5880gataattgaa aaccacgaac cattgaggga agagttaaag gcaagaggat atacttttgt 5940gagtgagact gacactgagg ttattgcaca tttagtgaac tgggaactca aacagggggg 6000cacattgcgt gaggctgtgt taagagctat tcctcaactt agaggtgcat acggtactgt 6060tattatggat tcaagacacc cagatactct ccttgcagct agatcaggta gtcccttggt 6120cataggactt ggaatgggtg aaaattttat cgctagcgac caattggcct tattgccagt 6180tacaagacga tttattttcc ttgaagaggg cgatattgct gagattacta gaaggtctgt 6240gaacatcttt gataagactg gcgctgaggt taaacgtcag gatatcgagt ctaaccttca 6300atacgatgct ggtgataaag gaatttacag gcattatatg caaaaggaaa tttatgaaca 6360accaaatgct atcaaaaaca cacttactgg ccgtatttct catggacagg tcgatttaag 6420cgagcttggt cctaatgcag acgaactgct atcaaaagtt gagcacatac agatactggc 6480atgcggaact agttataatt caggaatggt gtctagatac tggttcgaaa gcttggcagg 6540tataccttgt gatgtagaga tcgcttctga gtttaggtat agaaagtctg ctgtgcgtag 6600aaattcatta atgattacat tatctcaatc cggagaaaca gcagatacac tggctggatt 6660gaggctttct aaggaactcg gatatctggg ttcacttgct atttgtaatg taccaggttc 6720ctcattggtt cgtgaatcag atctagcact tatgacaaat gcaggaactg aaataggtgt 6780ggcaagtacc aaggctttca caacccaact gaccgtactt ttaatgttgg tagcaaaact 6840cagtcgatta aaggggctag atgcatctat cgaacatgat attgttcacg ggcttcaagc 6900tctcccttca agaattgaac aaatgctttc acaagataag agaatagagg cattggctga 6960agatttttcc gacaaacatc acgcattgtt tcttggacgt ggcgatcaat atccaattgc 7020attggaagga gctttgaagt tgaaagaaat aagttacatt cacgcagaag catatgcagc 7080tggagaactc aagcatggtc ctttggcact catcgacgct gacatgcccg tgatcgtagt 7140ggctcctaat aacgaactgc tcgaaaagct taaatcaaat atcgaagagg ttcgagctag 7200aggaggtcag ctttacgttt tcgctgaaca agatgctgga ttcgtgtcaa gcgataatat 7260gcatataatt gaaatgcctc acgttgaaga agtgattgca cctatatttt atacagtccc 7320attgcaactt ctagcttacc atgttgcact tattaaagga actgatgttg atcagcctag 7380aaacctagca aaatctgtaa cagtcgaata aacgcgtggc gcgccgaagc agatcgttca 7440aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 7500atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 7560tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 7620aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 7680gatcggaatt cgatatcatt accctgttat ccctaaagct tattaatata acttcgtata 7740gcatacatta tacgaagtta tgtttcctac gcagcaggtc tcatcaagac gatctacccg 7800agtaacaatc tccaggagat caaatacctt cccaagaagg ttaaagatgc agtcaaaaga 7860ttcaggacta attgcatcaa gaacacagag aaagacatat ttctcaagat cagaagtact 7920attccagtat ggacgattca aggcttgctt cataaaccaa ggcaagtaat agagattgga 7980gtctctaaaa aggtagttcc tactgaatct aaggccatgc atggagtcta agattcaaat 8040cgaggatcta acagaactcg ccgtgaagac tggcgaacag ttcatacaga gtcttttacg 8100actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacactc tggtctactc 8160caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 8220gataatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcgaaag 8280gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggctat 8340cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat 8400cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgacatctc 8460cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 8520aggaagttca tttcatttgg agaggacacg ctgaaatcac cagtctctct ctataaatct 8580atctctctct ctataacaat ggacccagaa cgacgcccgg ccgacatccg ccgtgccacc 8640gaggcggaca tgccggcggt ctgcaccatc gtcaaccact acatcgagac aagcacggtc 8700aacttccgta ccgagccgca ggaaccgcag gagtggacgg acgacctcgt ccgtctgcgg 8760gagcgctatc cctggctcgt cgccgaggtg gacggcgagg tcgccggcat cgcctacgcg 8820ggcccctgga aggcacgcaa cgcctacgac tggacggccg agtcgaccgt gtacgtctcc 8880ccccgccacc agcggacggg actgggctcc acgctctaca cccacctgct gaagtccctg 8940gaggcacagg gcttcaagag cgtggtcgct gtcatcgggc tgcccaacga cccgagcgtg 9000cgcatgcacg aggcgctcgg atatgccccc cgcggcatgc tgcgggcggc cggcttcaag 9060cacgggaact ggcatgacgt gggtttctgg cagctggact tcagcctgcc ggtaccgccc 9120cgtccggtcc tgcccgtcac cgagatctga gatcacccgt tctaggatcc gaagcagatc 9180gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 9240ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 9300cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 9360tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 9420tactagatcg aaacataact tcgtatagca tacattatac gaagttatca aaacgtcgtg 9480agacagtttg gttaactata acggtcctaa ggtagcgatc gaggcattac ggcattacgg 9540cactcgcgag ggtccgaatc tatgtcgggt gcggagaaag aggtaatgaa atggcaattt 9600acaattgaat atatcctgcc g 9621157343DNAArtificial SequenceTDNA comprising nucleic acid sequences encoding the expansin promoter, a Golgi retention peptide and a NODC proteinmisc_feature(1)..(25)right border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)promoter(26)..(913)sequence including the promoter region of the expansin gene of Gossypium hirsutum (cotton)sig_peptide(914)..(1018)gene(1019)..(2209)3'UTR(2210)..(- 2442)sequence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus (Sanfacon et al., 1991)promoter(2465)..(3303)Pcwp sequence including the promoter region of the expansin gene of Gossypium hirsutum (cotton)gene(3304)..(5133)gfaEc coding region of the glutaminefructose- 6-phosphate amidotransferase gene of Escherichia coli (Frohberg and Essigmann, 2006)3'UTR(5134)..(5407)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(5450)..(5449)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)promoter(5484)..(6320)P35S3 sequence including the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al., 1985)gene(6321)..(6872)bar the coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces hygroscopicus (Thompson et al., 1987).3'UTR(6873)..(7156)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(7157)..(7190)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)misc_feature(7319)..(7343)left border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988) 15aattacaacg gtatatatcc tgccagtact gggccccctc gagggcgatc gctacgtacc 60tgcagggcgg ccgcgggctg gtatcttttg attggcacaa acagtgcgaa caaagaagac 120cacacaataa caattttaac aatatactaa tttaaatgaa aaattttcaa taatttaata 180agttaaccga ggaaaactta ctaagagtta gttacccctg ttaaaataac tttcatgaag 240taatagaaac ttttagtacg tatcatctta tatagaacaa tttctatttt cagaaagtca 300agaaaattgt attctagaaa atggcgactt cttcaccttc agtccttccc tgatcggcgc 360ttgtgaaaaa cgaaaaacct gagtctgatt ggctgactga aaatgaacct actcatcacc 420attcactatt accaacttca aatgataggg gaattaactg gtaaagtgta actccaccga 480tggttgaggt ggttggctgg agttaaatga gattttttta gttttgtttc aagtggcttc 540aattgcaagc aattaggaga ctgcgctgga ataacccctc gctcaacctt ccgccattgt 600tatggtttaa ttaaacatta tgtttccatc catctatatt tatatccatt aaaacaagcc 660gttgagcaaa taatggatac tggataccat catatctatg attaaaattt tgcatgtgcc 720cttttaatgt atagcttaag ccttaattat cctccaaatt tgtactcttt caccacttaa 780ttggctacgt acggtactta gcgttgcttg tcatcttctg tactacaaac tctttctcat 840tttgtataaa tagctataca ctttttctct cctcaaatca ataaggttag gtcagccaat 900tgtttgagct accatgagta aacggaatcc gaagattctg aagatttttc tgtatatgtt 960acttctcaac tctctctttc tcatcatcta cttcgttttt cactcatcgt cgttttcaag 1020tgtcgtagat gtgatcggtt tgcttgcgac tgcagcctac gtgacgttgg cgagcgcata 1080caaggtggtc cagttcatta acgtgtcgag cgtaacggat gtcgctggtc tcgaaagtga 1140tgctttgccg ctcactccaa gggttgacgt tatcgtgccg acattcaatg agaactccag 1200cacattgctc gagtgcgtcg cttctatatg cgcacaagac taccgcggac caataacgat 1260tgtcgtggta gacgatgggt cgaccaacaa aacatcattt cacgcagtat gcgacaagta 1320cgcgagcgac gaaaggttca tatttgtcga acttgatcaa aacaagggga agcgcgccgc 1380gcaaatggag gccatcagga gaacagacgg agacctgata ctaaacgtag actcggacac 1440ggttatagat aaggatgttg ttacaaagct tgcgtcgtcc atgagagccc cgaatgtcgg 1500tggtgtcatg gggcagctcg ttgcaaagaa tcgagaaaga tcttggctta ccagattaat 1560cgatatggag tactggcttg cgtgtaacga ggagcgcatt gcgcagtcga ggtttggctc 1620cgtgatgtgt tgttgtgggc cgtgcgccat gtatagaaga tctgcaatta cgccactatt 1680ggcagaatat gagcaccaga cattcctagg gcgtccgagc aactttggtg aggatcgcca 1740tctcacaatc ctgatgctga aggcgggatt tcggaccggg tacgtcccag gtgccgtagc 1800gaggacgttg gttccggatg ggctggcgcc gtacctgcgc cagcaactcc gctgggcccg 1860cagcacttat cgcgacaccg ccctcgcctt acgtataaag aaaaatctaa gcaaatatat 1920cacctttgag atatgcgcac agaatttggg tacggctctc ttacttgtga tgaccatgat 1980ttcgctttcg ctgactacat cagggtcgca aacgcccgtt atcattctgg gtgtcgttgt 2040ggggatgtct ataataagat gttgttctgt cgcccttata gcgaaagatt ttcggtttct 2100atacttcatc gttcactcag cgttgaatgt tctaatttta acgccgttaa aactctatgc 2160cctgttaacc attcgggata gtcggtggct atcacgcgag agttcctaag ctagcaagct 2220tggacacgct gaaatcacca gtctctctct acaaatctat ctctctctat tttctccata 2280ataatgtgtg agtagttccc agataaggga attagggttc ctatagggtt tcgctcatgt 2340gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt ctatcaataa 2400aatttctaat tcctaaaacc aaaatccagt actaaaatcc agcccgggtt aattaagcgg 2460ccgcgggctg gtatcttttg attggcacaa acagtgcgaa caaagaagac cacacaataa 2520caattttaac aatatactaa tttaaatgaa aaattttcaa taatttaata agttaaccga 2580ggaaaactta ctaagagtta gttacccctg ttaaaataac tttcatgaag taatagaaac 2640ttttagtacg tatcatctta tatagaacaa tttctatttt cagaaagtca agaaaattgt 2700attctagaaa atggcgactt cttcaccttc agtccttccc tgatcggcgc ttgtgaaaaa 2760cgaaaaacct gagtctgatt ggctgactga aaatgaacct actcatcacc attcactatt 2820accaacttca aatgataggg gaattaactg gtaaagtgta actccaccga tggttgaggt 2880ggttggctgg agttaaatga gattttttta gttttgtttc aagtggcttc aattgcaagc 2940aattaggaga ctgcgctgga ataacccctc gctcaacctt ccgccattgt tatggtttaa 3000ttaaacatta tgtttccatc catctatatt tatatccatt aaaacaagcc gttgagcaaa 3060taatggatac tggataccat catatctatg attaaaattt tgcatgtgcc cttttaatgt 3120atagcttaag ccttaattat cctccaaatt tgtactcttt caccacttaa ttggctacgt 3180acggtactta gcgttgcttg tcatcttctg tactacaaac tctttctcat tttgtataaa 3240tagctataca ctttttctct cctcaaatca ataaggttag gtcagccaat tgtttgagct 3300accatgtgcg gaattgttgg tgctatcgcc caaagagacg ttgctgagat tttgttagag 3360ggtctgcgaa ggctagagta tagaggatat gactccgctg gtctggctgt cgttgatgct 3420gagggtcata tgacaaggct aagaaggtta ggaaaggttc agatgcttgc tcaggcagct 3480gaggaacatc cattgcatgg aggtactggt attgcacata ccaggtgggc tactcatggg 3540gagccatcag aagttaatgc tcatccacat gtgagtgagc atatcgttgt agttcacaat 3600gggataattg aaaaccacga accattgagg gaagagttaa aggcaagagg atatactttt 3660gtgagtgaga ctgacactga ggttattgca catttagtga actgggaact caaacagggg 3720ggcacattgc gtgaggctgt gttaagagct attcctcaac ttagaggtgc atacggtact 3780gttattatgg attcaagaca cccagatact ctccttgcag ctagatcagg tagtcccttg 3840gtcataggac ttggaatggg tgaaaatttt atcgctagcg accaattggc cttattgcca 3900gttacaagac gatttatttt ccttgaagag ggcgatattg ctgagattac tagaaggtct 3960gtgaacatct ttgataagac tggcgctgag gttaaacgtc aggatatcga gtctaacctt 4020caatacgatg ctggtgataa aggaatttac aggcattata tgcaaaagga aatttatgaa 4080caaccaaatg ctatcaaaaa cacacttact ggccgtattt ctcatggaca ggtcgattta 4140agcgagcttg gtcctaatgc agacgaactg ctatcaaaag ttgagcacat acagatactg 4200gcatgcggaa ctagttataa ttcaggaatg gtgtctagat actggttcga aagcttggca 4260ggtatacctt gtgatgtaga gatcgcttct gagtttaggt atagaaagtc tgctgtgcgt 4320agaaattcat taatgattac attatctcaa tccggagaaa cagcagatac actggctgga 4380ttgaggcttt ctaaggaact cggatatctg ggttcacttg ctatttgtaa tgtaccaggt 4440tcctcattgg ttcgtgaatc agatctagca cttatgacaa atgcaggaac tgaaataggt 4500gtggcaagta ccaaggcttt cacaacccaa ctgaccgtac ttttaatgtt ggtagcaaaa 4560ctcagtcgat taaaggggct agatgcatct atcgaacatg atattgttca cgggcttcaa 4620gctctccctt caagaattga acaaatgctt tcacaagata agagaataga ggcattggct 4680gaagattttt ccgacaaaca tcacgcattg tttcttggac gtggcgatca atatccaatt 4740gcattggaag gagctttgaa gttgaaagaa ataagttaca ttcacgcaga agcatatgca 4800gctggagaac tcaagcatgg tcctttggca ctcatcgacg ctgacatgcc cgtgatcgta 4860gtggctccta ataacgaact gctcgaaaag cttaaatcaa atatcgaaga ggttcgagct 4920agaggaggtc agctttacgt tttcgctgaa caagatgctg gattcgtgtc aagcgataat 4980atgcatataa ttgaaatgcc tcacgttgaa gaagtgattg cacctatatt ttatacagtc 5040ccattgcaac ttctagctta ccatgttgca cttattaaag gaactgatgt tgatcagcct 5100agaaacctag caaaatctgt aacagtcgaa taaacgcgtg gcgcgccgaa gcagatcgtt 5160caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 5220tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 5280tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag 5340aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 5400tagatcggaa ttcgatatca ttaccctgtt atccctaaag cttattaata taacttcgta 5460tagcatacat tatacgaagt tatgtttcct acgcagcagg tctcatcaag acgatctacc 5520cgagtaacaa tctccaggag atcaaatacc ttcccaagaa ggttaaagat gcagtcaaaa 5580gattcaggac taattgcatc aagaacacag agaaagacat atttctcaag atcagaagta 5640ctattccagt atggacgatt caaggcttgc ttcataaacc aaggcaagta atagagattg 5700gagtctctaa aaaggtagtt cctactgaat ctaaggccat gcatggagtc taagattcaa 5760atcgaggatc taacagaact cgccgtgaag actggcgaac agttcataca gagtctttta 5820cgactcaatg acaagaagaa aatcttcgtc aacatggtgg agcacgacac tctggtctac 5880tccaaaaatg tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa 5940aggataattt cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa 6000aggacagtag aaaaggaagg tggctcctac aaatgccatc attgcgataa aggaaaggct 6060atcattcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc 6120atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgacatc 6180tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata 6240taaggaagtt catttcattt ggagaggaca cgctgaaatc accagtctct ctctataaat 6300ctatctctct ctctataaca atggacccag aacgacgccc ggccgacatc cgccgtgcca 6360ccgaggcgga catgccggcg gtctgcacca tcgtcaacca ctacatcgag acaagcacgg 6420tcaacttccg taccgagccg caggaaccgc aggagtggac ggacgacctc gtccgtctgc 6480gggagcgcta tccctggctc gtcgccgagg tggacggcga ggtcgccggc atcgcctacg 6540cgggcccctg gaaggcacgc aacgcctacg actggacggc cgagtcgacc gtgtacgtct 6600ccccccgcca ccagcggacg ggactgggct ccacgctcta cacccacctg ctgaagtccc 6660tggaggcaca gggcttcaag agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg 6720tgcgcatgca cgaggcgctc ggatatgccc cccgcggcat gctgcgggcg gccggcttca 6780agcacgggaa ctggcatgac gtgggtttct ggcagctgga cttcagcctg ccggtaccgc 6840cccgtccggt cctgcccgtc accgagatct gagatcaccc gttctaggat ccgaagcaga 6900tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 6960gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 7020gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 7080gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 7140gttactagat cgaaacataa cttcgtatag catacattat acgaagttat caaaacgtcg 7200tgagacagtt tggttaacta taacggtcct aaggtagcga tcgaggcatt acggcattac 7260ggcactcgcg agggtccgaa tctatgtcgg gtgcggagaa agaggtaatg aaatggcaat 7320ttacaattga atatatcctg ccg 73431610829DNAArtificial SequenceTDNA comprising nucleic acid sequences encoding the E6 promoter, a Golgi retention peptide and a NODC proteinmisc_feature(1)..(25)right border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)promoter(26)..(2712)sequence including the promoter region of the E6 gene of Gossypium barbadense (cotton)sig_peptide(2713)..(2817)gene(2818)..(4008)3'UTR(4009)..(4244)seq- uence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus (Sanfacon et al., 1991)promoter(4262)..(6901)Pcwp sequence including the promoter region of the E6 gene of Gossypium barbadense (cotton)gene(6902)..(8731)gfaEc coding region of the glutaminefructose- 6-phosphate amidotransferase gene of Escherichia coli (Frohberg and Essigmann, 2006)3'UTR(8732)..(9005)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(9048)..(9085)lox sequence including the 34bp recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)promoter(9086)..(9918)P35S3 sequence including the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al., 1985)gene(9919)..(10470)bar the coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces hygroscopicus (Thompson et al., 1987).3'UTR(10471)..(10750)3'nos sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982)misc_feature(10751)..(10788)lox sequence including the 34bp

recognition sequence for the Cre recombinase of bacteriophage P1 (Hoess and Abremski, 1985)misc_feature(10805)..(10829)left border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988) 16aattacaacg gtatatatcc tgccagtact gggccccctc gagggcgatc gctacgtacc 60tgcagggcgg ccgcgtcaac ggatcaacat tcaattacaa taaagagtat agatagatac 120atcaatacta tccagccctc tttcatgatg atgttgcaag ttttgcactc cacccccata 180ttattatgaa gaggagaaat tcctgttaca acattaattg agctttattt tctaagaaat 240gacttctaac attaataaat ttgaatcaat ttatagctat ttctcgtact ctttcgtagt 300atagctttct ttttatgcta aaacaggaga tgattataag acgtgaagtt gtcttaagat 360taacaaggca gttagatgca tttaaattgg tatttaagga tatgtctgta tataactaga 420actaattaag attacaatac actgcaaaat gattccacct tttttttttt tttggttctc 480tcttttatat gatcaaatac aatgttgata tgaacaaggt tttgcagttg tagaaaatcg 540tggaggactt tttttttaaa aaaagaaaga taaaattcat aaaaaaatgt gaagttaagc 600atatttagtg atgggtgggg tatggggtgg tttgctaaca tggaatgcgc atggcagatt 660ggcactttaa agaagggatg gggccgaggg ggcgggagtt gttaaatcct cggcgtagaa 720aaaggtcagt aagcgtgtcc ctggcattag acaaggagag ggggtagcac atgcacagcc 780caactatatc tctttatttt atgtcccact ccactccccc tcatctctgc cgcaacatta 840aataccttat gcatccttac tattcataat ggtttttttt gttgggttga tgttacaaaa 900ttaaattttt tattatagat gtattaatat ttttaaaaat atataagatt ttttttaagt 960aggagtttaa tctttggtgg tgatgttgat tttagatata ttccccaccc gcaagtagat 1020atcatacatt ctaatataat ttaaaaaaaa gtataactaa aatatatttt atattttttt 1080atattttttt gaatttttaa attttaaaaa attaattaaa tgttcatgtg tcatctacat 1140gtatgcaacg ttagcgaagt ttaaaatata ttaatttttt cattcgtgat ttgaaaaaaa 1200aaaggtaagt ttaaaggtta aacaagcata aatctaaata aatagttaaa ataatttttt 1260ttataaagtt agagaattaa ataaattatt attttattta aaaataattt tcaataaatt 1320actaatttag tcacataatc taatataatt taaaaaataa ttattaacat tttaatttgt 1380atgaattctt gttatgtatg gattcaaacc cgttcgttcc atcaacaaat tgatctgcat 1440gagacttaag ggttagaatt ttgtgtaacc ctttttcctc ctaattttta cttttaaaaa 1500gaaattgcaa tacaattttt tttttataga attctcctat ttttatttat ttatttgctt 1560aggaagtttt actgacactg cttttatttt tccatcaatc aaatttaaga gacaattcac 1620tttttataat taacaaaaaa aaacaaaaag aaaataaaag aaattacttt tttctttttc 1680gtgttcgata caagatagat gaaatatgaa aaataaaatg aaatgaaaat atattactag 1740tgatatatga cctccattat gtaggggaaa gaaataaaaa ttatattaat ttatgatact 1800tccataatgt ggttaaaaat aattatctag tatttttttg taaaaaaaaa aaagttgata 1860tctatgctac taatgaggtt tcttagtgag tttgttacta ctaataaagt ttatttgcat 1920ggttgagacc ttatgctttt caaataccca tatttgaatt ttaaaaattg tgaattttta 1980ttatatttaa aaaacaagtt atttatataa ctagtaatgt attattttga ctttttttta 2040atcgagttaa tgttggttat ttcgttatac caattcaata aaatatttta tttatattaa 2100attatagcat acctcacgat gtgggtgaag taaaattatt taacaaatat attttgaaaa 2160attgataaaa atactaaatg aggttttggt tgaatagtaa gatataatta ttacaaatta 2220taaatatgta ggttcaaaat ctatcatgtg tatatttgta ctattattct atataaattg 2280ataaccttat aaaagtatct aatttagttt atggttgatt gatcgataat accaaattta 2340ttaaaaatta atattagtaa agatatatag tacaaaacta aacataaaat tttatatgtt 2400aaggaaatag cggaaaaaat atcatatttg tagaactgtt tagcagtgtg ggagaatggg 2460atcattacaa ggaaaaatga aatatatatc attaatacca aacataaaag aaagcgtctt 2520ttgataaagt tgttattggt gtaatgtgaa gggaccacaa tcatcaccat tcaccacttg 2580ctcctaattg agttgaaatc tttttacaac atagaaaact agaagatcgc cctttcttgc 2640ttcatatata tagattttgt atcatcgcaa tttcacatca cacacacaag taaagcatta 2700gcaaccatag ccatgagtaa acggaatccg aagattctga agatttttct gtatatgtta 2760cttctcaact ctctctttct catcatctac ttcgtttttc actcatcgtc gttttcaagt 2820gtcgtagatg tgatcggttt gcttgcgact gcagcctacg tgacgttggc gagcgcatac 2880aaggtggtcc agttcattaa cgtgtcgagc gtaacggatg tcgctggtct cgaaagtgat 2940gctttgccgc tcactccaag ggttgacgtt atcgtgccga cattcaatga gaactccagc 3000acattgctcg agtgcgtcgc ttctatatgc gcacaagact accgcggacc aataacgatt 3060gtcgtggtag acgatgggtc gaccaacaaa acatcatttc acgcagtatg cgacaagtac 3120gcgagcgacg aaaggttcat atttgtcgaa cttgatcaaa acaaggggaa gcgcgccgcg 3180caaatggagg ccatcaggag aacagacgga gacctgatac taaacgtaga ctcggacacg 3240gttatagata aggatgttgt tacaaagctt gcgtcgtcca tgagagcccc gaatgtcggt 3300ggtgtcatgg ggcagctcgt tgcaaagaat cgagaaagat cttggcttac cagattaatc 3360gatatggagt actggcttgc gtgtaacgag gagcgcattg cgcagtcgag gtttggctcc 3420gtgatgtgtt gttgtgggcc gtgcgccatg tatagaagat ctgcaattac gccactattg 3480gcagaatatg agcaccagac attcctaggg cgtccgagca actttggtga ggatcgccat 3540ctcacaatcc tgatgctgaa ggcgggattt cggaccgggt acgtcccagg tgccgtagcg 3600aggacgttgg ttccggatgg gctggcgccg tacctgcgcc agcaactccg ctgggcccgc 3660agcacttatc gcgacaccgc cctcgcctta cgtataaaga aaaatctaag caaatatatc 3720acctttgaga tatgcgcaca gaatttgggt acggctctct tacttgtgat gaccatgatt 3780tcgctttcgc tgactacatc agggtcgcaa acgcccgtta tcattctggg tgtcgttgtg 3840gggatgtcta taataagatg ttgttctgtc gcccttatag cgaaagattt tcggtttcta 3900tacttcatcg ttcactcagc gttgaatgtt ctaattttaa cgccgttaaa actctatgcc 3960ctgttaacca ttcgggatag tcggtggcta tcacgcgaga gttcctaagc tagcaagctt 4020ggacacgctg aaatcaccag tctctctcta caaatctatc tctctctatt ttctccataa 4080taatgtgtga gtagttccca gataagggaa ttagggttcc tatagggttt cgctcatgtg 4140ttgagcatat aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa 4200atttctaatt cctaaaacca aaatccagta ctaaaatcca gcccgggtta attaagcggc 4260cgcgtcaacg gatcaacatt caattacaat aaagagtata gatagataca tcaatactat 4320ccagccctct ttcatgatga tgttgcaagt tttgcactcc acccccatat tattatgaag 4380aggagaaatt cctgttacaa cattaattga gctttatttt ctaagaaatg acttctaaca 4440ttaataaatt tgaatcaatt tatagctatt tctcgtactc tttcgtagta tagctttctt 4500tttatgctaa aacaggagat gattataaga cgtgaagttg tcttaagatt aacaaggcag 4560ttagatgcat ttaaattggt atttaaggat atgtctgtat ataactagaa ctaattaaga 4620ttacaataca ctgcaaaatg attccacctt tttttttttt ttggttctct cttttatatg 4680atcaaataca atgttgatat gaacaaggtt ttgcagttgt agaaaatcgt ggaggacttt 4740ttttttaaaa aaagaaagat aaaattcata aaaaaatgtg aagttaagca tatttagtga 4800tgggtggggt atggggtggt ttgctaacat ggaatgcgca tggcagattg gcactttaaa 4860gaagggatgg ggccgagggg gcgggagttg ttaaatcctc ggcgtagaaa aaggtcagta 4920agcgtgtccc tggcattaga caaggagagg gggtagcaca tgcacagccc aactatatct 4980ctttatttta tgtcccactc cactccccct catctctgcc gcaacattaa ataccttatg 5040catccttact attcataatg gttttttttg ttgggttgat gttacaaaat taaatttttt 5100attatagatg tattaatatt tttaaaaata tataagattt tttttaagta ggagtttaat 5160ctttggtggt gatgttgatt ttagatatat tccccacccg caagtagata tcatacattc 5220taatataatt taaaaaaaag tataactaaa atatatttta tattttttta tatttttttg 5280aatttttaaa ttttaaaaaa ttaattaaat gttcatgtgt catctacatg tatgcaacgt 5340tagcgaagtt taaaatatat taattttttc attcgtgatt tgaaaaaaaa aaggtaagtt 5400taaaggttaa acaagcataa atctaaataa atagttaaaa taattttttt tataaagtta 5460gagaattaaa taaattatta ttttatttaa aaataatttt caataaatta ctaatttagt 5520cacataatct aatataattt aaaaaataat tattaacatt ttaatttgta tgaattcttg 5580ttatgtatgg attcaaaccc gttcgttcca tcaacaaatt gatctgcatg agacttaagg 5640gttagaattt tgtgtaaccc tttttcctcc taatttttac ttttaaaaag aaattgcaat 5700acaatttttt ttttatagaa ttctcctatt tttatttatt tatttgctta ggaagtttta 5760ctgacactgc ttttattttt ccatcaatca aatttaagag acaattcact ttttataatt 5820aacaaaaaaa aacaaaaaga aaataaaaga aattactttt ttctttttcg tgttcgatac 5880aagatagatg aaatatgaaa aataaaatga aatgaaaata tattactagt gatatatgac 5940ctccattatg taggggaaag aaataaaaat tatattaatt tatgatactt ccataatgtg 6000gttaaaaata attatctagt atttttttgt aaaaaaaaaa aagttgatat ctatgctact 6060aatgaggttt cttagtgagt ttgttactac taataaagtt tatttgcatg gttgagacct 6120tatgcttttc aaatacccat atttgaattt taaaaattgt gaatttttat tatatttaaa 6180aaacaagtta tttatataac tagtaatgta ttattttgac ttttttttaa tcgagttaat 6240gttggttatt tcgttatacc aattcaataa aatattttat ttatattaaa ttatagcata 6300cctcacgatg tgggtgaagt aaaattattt aacaaatata ttttgaaaaa ttgataaaaa 6360tactaaatga ggttttggtt gaatagtaag atataattat tacaaattat aaatatgtag 6420gttcaaaatc tatcatgtgt atatttgtac tattattcta tataaattga taaccttata 6480aaagtatcta atttagttta tggttgattg atcgataata ccaaatttat taaaaattaa 6540tattagtaaa gatatatagt acaaaactaa acataaaatt ttatatgtta aggaaatagc 6600ggaaaaaata tcatatttgt agaactgttt agcagtgtgg gagaatggga tcattacaag 6660gaaaaatgaa atatatatca ttaataccaa acataaaaga aagcgtcttt tgataaagtt 6720gttattggtg taatgtgaag ggaccacaat catcaccatt caccacttgc tcctaattga 6780gttgaaatct ttttacaaca tagaaaacta gaagatcgcc ctttcttgct tcatatatat 6840agattttgta tcatcgcaat ttcacatcac acacacaagt aaagcattag caaccatagc 6900catgtgcgga attgttggtg ctatcgccca aagagacgtt gctgagattt tgttagaggg 6960tctgcgaagg ctagagtata gaggatatga ctccgctggt ctggctgtcg ttgatgctga 7020gggtcatatg acaaggctaa gaaggttagg aaaggttcag atgcttgctc aggcagctga 7080ggaacatcca ttgcatggag gtactggtat tgcacatacc aggtgggcta ctcatgggga 7140gccatcagaa gttaatgctc atccacatgt gagtgagcat atcgttgtag ttcacaatgg 7200gataattgaa aaccacgaac cattgaggga agagttaaag gcaagaggat atacttttgt 7260gagtgagact gacactgagg ttattgcaca tttagtgaac tgggaactca aacagggggg 7320cacattgcgt gaggctgtgt taagagctat tcctcaactt agaggtgcat acggtactgt 7380tattatggat tcaagacacc cagatactct ccttgcagct agatcaggta gtcccttggt 7440cataggactt ggaatgggtg aaaattttat cgctagcgac caattggcct tattgccagt 7500tacaagacga tttattttcc ttgaagaggg cgatattgct gagattacta gaaggtctgt 7560gaacatcttt gataagactg gcgctgaggt taaacgtcag gatatcgagt ctaaccttca 7620atacgatgct ggtgataaag gaatttacag gcattatatg caaaaggaaa tttatgaaca 7680accaaatgct atcaaaaaca cacttactgg ccgtatttct catggacagg tcgatttaag 7740cgagcttggt cctaatgcag acgaactgct atcaaaagtt gagcacatac agatactggc 7800atgcggaact agttataatt caggaatggt gtctagatac tggttcgaaa gcttggcagg 7860tataccttgt gatgtagaga tcgcttctga gtttaggtat agaaagtctg ctgtgcgtag 7920aaattcatta atgattacat tatctcaatc cggagaaaca gcagatacac tggctggatt 7980gaggctttct aaggaactcg gatatctggg ttcacttgct atttgtaatg taccaggttc 8040ctcattggtt cgtgaatcag atctagcact tatgacaaat gcaggaactg aaataggtgt 8100ggcaagtacc aaggctttca caacccaact gaccgtactt ttaatgttgg tagcaaaact 8160cagtcgatta aaggggctag atgcatctat cgaacatgat attgttcacg ggcttcaagc 8220tctcccttca agaattgaac aaatgctttc acaagataag agaatagagg cattggctga 8280agatttttcc gacaaacatc acgcattgtt tcttggacgt ggcgatcaat atccaattgc 8340attggaagga gctttgaagt tgaaagaaat aagttacatt cacgcagaag catatgcagc 8400tggagaactc aagcatggtc ctttggcact catcgacgct gacatgcccg tgatcgtagt 8460ggctcctaat aacgaactgc tcgaaaagct taaatcaaat atcgaagagg ttcgagctag 8520aggaggtcag ctttacgttt tcgctgaaca agatgctgga ttcgtgtcaa gcgataatat 8580gcatataatt gaaatgcctc acgttgaaga agtgattgca cctatatttt atacagtccc 8640attgcaactt ctagcttacc atgttgcact tattaaagga actgatgttg atcagcctag 8700aaacctagca aaatctgtaa cagtcgaata aacgcgtggc gcgccgaagc agatcgttca 8760aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 8820atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 8880tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 8940aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 9000gatcggaatt cgatatcatt accctgttat ccctaaagct tattaatata acttcgtata 9060gcatacatta tacgaagtta tgtttcctac gcagcaggtc tcatcaagac gatctacccg 9120agtaacaatc tccaggagat caaatacctt cccaagaagg ttaaagatgc agtcaaaaga 9180ttcaggacta attgcatcaa gaacacagag aaagacatat ttctcaagat cagaagtact 9240attccagtat ggacgattca aggcttgctt cataaaccaa ggcaagtaat agagattgga 9300gtctctaaaa aggtagttcc tactgaatct aaggccatgc atggagtcta agattcaaat 9360cgaggatcta acagaactcg ccgtgaagac tggcgaacag ttcatacaga gtcttttacg 9420actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacactc tggtctactc 9480caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 9540gataatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcgaaag 9600gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggctat 9660cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat 9720cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgacatctc 9780cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 9840aggaagttca tttcatttgg agaggacacg ctgaaatcac cagtctctct ctataaatct 9900atctctctct ctataacaat ggacccagaa cgacgcccgg ccgacatccg ccgtgccacc 9960gaggcggaca tgccggcggt ctgcaccatc gtcaaccact acatcgagac aagcacggtc 10020aacttccgta ccgagccgca ggaaccgcag gagtggacgg acgacctcgt ccgtctgcgg 10080gagcgctatc cctggctcgt cgccgaggtg gacggcgagg tcgccggcat cgcctacgcg 10140ggcccctgga aggcacgcaa cgcctacgac tggacggccg agtcgaccgt gtacgtctcc 10200ccccgccacc agcggacggg actgggctcc acgctctaca cccacctgct gaagtccctg 10260gaggcacagg gcttcaagag cgtggtcgct gtcatcgggc tgcccaacga cccgagcgtg 10320cgcatgcacg aggcgctcgg atatgccccc cgcggcatgc tgcgggcggc cggcttcaag 10380cacgggaact ggcatgacgt gggtttctgg cagctggact tcagcctgcc ggtaccgccc 10440cgtccggtcc tgcccgtcac cgagatctga gatcacccgt tctaggatcc gaagcagatc 10500gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 10560ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 10620cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 10680tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 10740tactagatcg aaacataact tcgtatagca tacattatac gaagttatat tcgagcatgg 10800agccatttac aattgaatat atcctgccg 10829

Claims

1. A fiber from a cotton plant consisting essentially of plant cells comprising a chimeric gene comprising: 1. a plant-expressible promoter; 2. a DNA region coding for a Nodulation C protein fused to a signal anchor sequence for targeting to the membranes of the Golgi-apparatus; and 3. a transcription termination and polyadenylation region.

2. A yarn or a fabric made from a cotton fiber according to claim 1.

3. A method of dying cotton fibers, yarn or fabric comprising providing the fiber of claim 1 or the yarn or fabric of claim 2 and applying a dye reactive to said fibers, yarn or fabric.

4. The fiber according to claim 1, wherein said signal anchor sequence is selected from the signal anchor sequence of a rat sialyl transferase, the signal anchor sequence of a human galactosyl transferase, the signal anchor sequence of the Arabidopsis homologue of the yeast HDEL receptor (AtERD2), the signal anchor sequence of the .alpha.-2,6-sialyltransferase, the signal anchor sequence of .beta.1,2-xylosyltransferase from Arabidopsis thaliana, the signal anchor sequence of N-acetylglucosaminyl transferase I from tobacco or the amino acid sequence YYHDL or LKLEI.

5. The fiber according to claim 1 or 4, wherein said DNA region encodes a nodulation C protein obtainable from a Rhizobium species, an Azorhizobium species, a Bradyrhizobium species, a Mesorhizobium species, a Ralstonia species, a Streptomyces species, a Burkholderia species, a Cupriavidus species, a Sinorhizobium species, a Desulfobacterium species, a Dokdonia species, a Methylobacterium species, a Phyllobacterium species or a Psychroflexus species.

6. The fiber according to claim 5, wherein said nodulation C protein comprises the amino acid sequence of SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, 1615305C, 1615305D, 1615305E, AAA26226, AAA63602, AAB16897, AAB24745, AAB34509, AAB47353, AAB51164, AAB71694, AAB91695, AAB95329, AAC80567, AAD11313, AAD11315, AAD11317, AAD11319, AAD11321, AAD11323, AAD11325, AAD11327, AAD11329, AAD11331, AAD11333, AAD11335, AAD11337, AAD11339, AAD11341, AAD11343, AAD11345, AAD11347, AAD11349, AAD11351, AAD11353, AAD11355, AAD11357, AAD11359, AAD11361, AAD11363, AAD11365, AAD11367, AAD11369, AAD11371, AAD11373, AAD11375, AAD11377, AAD11379, AAD11381, AAD11383, AAD11385, AAD11387, AAD11389, AAD11391, AAD11393, AAD11395, AAD11397, AAD11399, AAD11401, AAD11403, AAD11405, AAG60998, AAK00157, AAK39956, AAK39957, AAK39958, AAK39959, AAK39960, AAK39961, AAK39962, AAK39963, AAK39964, AAK39965, AAK39966, AAK39967, AAK50872, AAK65131, AAL88670, AAN62903, AAS91748, AAU11338, AAU11339, AAU11340, AAU11341, AAU11342, AAU11343, AAU11344, AAU11345, AAU11346, AAU11347, AAU11348, AAU11349, AAU11350, AAU11351, AAU11352, AAU11353, AAU11354, AAU11355, AAU11356, AAU11357, AAU11358, AAU11359, AAU11360, AAU11361, AAU11362, AAU11363, AAU11364, AAU11365, AAX30049, AAX30050, AAY44091, AAY44092, AAY44093, AAY89044, AAZ81541, ABC40958, ABC67303, ABD39006, ABD39007, ABD39008, ABD39009, ABD39010, ABD39011, ABD39012, ABD39013, ABD39014, ABD39015, ABD39016, ABD39017, ABD39018, ABD39019, ABD39020, ABD39021, ABD39022, ABD39023, ABD39024, ABD39025, ABD39026, ABD39027, ABD39028, ABD39029, ABD39030, ABD39031, ABD39032, ABD39033, ABD39034, ABD39035, ABD39036, ABD39037, ABD39038, ABD67413, ABD67416, ABD67419, ABD67422, ABD67425, ABD67428, ABD67431, ABD67434, ABD73319, ABD73320, ABD73321, ABD73322, ABD73323, ABD73324, ABD73325, ABD73326, ABD73327, ABD73328, ABD73329, ABD73330, ABD94161, ABD94162, ABD94163, ABD94164, ABD94165, ABF93198, ABF93199, ABF93200, ABF93201, ABF93202, ABM69186, ABM69187, ABM69188, ABM69189, ABM69190, ABN09217, ABN09218, ABN09219, ABN11177, ABN11178, ABN11179, ABP93834, ABS85176, ABS85177, ABS85178, ABS85179, ABS85180, ABS85181, ABS85182, ABU69044, ABU69045, ABU69046, ABU69047, ABU69048, ABU69049, ABU69050, ABU69051, ABU69052, ABU69053, ABU69054, ABU69055, ABU69056, ABU69057, ABU69058, ABU69059, ABU69060, ABU69061, ABU89879, ABV25689, ABV25690, ABV25691, ABV25692, ABV25693, ABV25694, ABW96196, ABW96197, ABW96198, ABW96199, ABW96200, ABW96201, ABW96202, ABW96203, ABW96204, ABW96205, ABW96206, ABW96207, ABW96208, ABW96209, ABW96210, ABW96211, ABY59633, ABY59634, ABY59635, ABY59636, ABY59637, ACA80309, ACA80310, ACA80311, ACA80312, ACA80313, ACC77565, ACD39337, ACD39338, ACD39339, ACD39340, ACD39341, ACD39342, ACD39343, ACD39344, ACD39345, ACD39346, ACD39347, ACD62595, ACD63093, ACD63094, ACD63095, ACD63096, ACD63097, ACD63098, ACD63099, ACD63100, ACD63101, ACD63102, ACD63103, ACD63104, ACF19762, ACF19763, ACF19764, ACF19765, ACF19766, ACF19767, ACF19768, ACF19769, ACF19770, ACH91221, ACH91222, ACH91223, ACH91224, ACH91225, ACH91226, ACH91227, ACH91228, ACH91229, ACH91230, ACH91231, ACH91232, ACH91233, ACH91242, ACH91243, ACH91244, ACH91245, ACH91246, ACH91247, ACH91248, ACH91249, ACI47333, ACI47334, ACI47335, ACI47336, ACI47337, ACI47338, ACI47339, ACI47340, ACI47341, ACI47342, ACI47343, ACI47344, ACI47345, ACL12058, ACL12059, ACL50517, ACL50518, ACL50519, ACL50520, ACL50521, ACL50522, ACL50523, ACM69382, ACM79634, ACM79635, ACM79636, ACM79637, ACM79638, ACM79639, ACM79640, ACM79641, ACM79642, ACM79643, ACM79644, ACM79645, ACM79646, ACN17701, ACN69201, ACN69202, ACN69203, ACN69204, ACN69205, ACN69206, ACN69207, ACN69208, ACN69209, ACN69210, ACN69211, ACN69212, ACN69213, AC058664, AC058665, AC058666, AC058667, AC058668, AC058669, AC058670, AC058671, AC058672, AC058673, AC058674, AC058675, ACP40990, ACS35430, ACS35434, ACT34091, ACT34094, ACT34097, ACT34100, ACT34101, ACT34104, ACT34107, ACT34110, ACT34113, ACT34116, ACT34119, ACT34122, ACT34125, ACT34128, ACT34131, ACT34134, ACT34137, ACT34140, ACT34143, ACV52950, ACV52951, ACV52952, ACV52953, ACV52954, ACV52955, ACX47326, ACX47327, ACX47328, ACX47329, ACX47330, ACX47331, ACX47332, ACX47333, ACX47334, ACY02884, ACY78518, ACZ52692, ACZ52693, ACZ52694, ACZ52695, ACZ52696, ACZ52697, ADD20957, ADD20958, ADD20959, ADD20960, ADD20961, ADD20962, ADD20963, ADD20964, ADD20965, ADD20966, ADD20967, ADD20968, ADD20969, ADD20970, ADD20971, ADG63645, ADG63646, ADG63647, ADG63648, ADG63649, ADG63650, ADG63651, ADG63652, ADJ18191, ADJ18192, BAA06082, BAA06083, BAA06084, BAA06085, BAA06086, BAA06087, BAA06088, BAA06089, BAA06090, BAA24092, BAB52500, C26813, CAA25810, CAA25811, CAA25814, CAA26310, CAA26311, CAA51773, CAA51774, CAA608779, CAA67139, CAB56055, CAC42489, CAD29949, CAD29950, CAD29951, CAD29952, CAD29953, CAD29954, CAD29955, CAD29956, CAD29957, CAD31533, CAD43933, CAD90257, CAD90583, CAD90584, CAD90585, CAD90586, CAD90587, CAD90588, CAH04369, CAN84684, CAP64017, EAQ38847, EAS72439, NP_106714, NP_435719, NP_443883, P04340, P04341, P04677, P04678, P04679, P06234, P06235, P17862, P24151, P26024, P50357, P53417, P72334, Q07755, Q53513, YP_001796208, YP_002605865, ZP_01050448 or ZP_01252570.

7. The fiber according to claim 1, wherein said NODC fused to the Golgi signal anchor sequence comprises the amino acid sequence of SEQ ID No. 11.

8. The fiber according to claim 1, wherein said plant-expressible promoter is a fiber-specific promoter.

9. The fiber according to claim 8, wherein said plant-expressible promoter is a fiber-specific promoter selected from the fiber-specific promoter of a beta tubulin gene from cotton, a fiber-specific promoter from an actin gene from cotton, a fiber specific promoter from a lipid transfer protein gene from cotton, a promoter from the seed coat and fiber-specific protease from cotton, a promoter from fiber-specific R2R3 MYB gene from cotton, a promoter from an expansin gene from cotton or a promoter from a chitinase gene in cotton.