CN110551734B

CN110551734B - Upland cotton fiber strength gene GhUBX and application thereof

Info

Publication number: CN110551734B
Application number: CN201910871799.4A
Authority: CN
Inventors: 张天真; 臧毅浩; 胡艳; 王洋坤; 宁志怨; 方磊; 朱协飞
Original assignee: Zhejiang University ZJU; Nanjing Agricultural University
Current assignee: Zhejiang University ZJU; Nanjing Agricultural University
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2022-07-19
Anticipated expiration: 2039-09-16
Also published as: AU2020348612A1; WO2021051883A1; CN110551734A

Abstract

The invention discloses a cotton fiber strength gene GhUBX of upland field and application thereof. The fiber strength gene GhUBX from tetraploid upland cotton is shown in SEQ ID NO.1 in the nucleotide sequence of the gene in tetraploid upland cotton Prema, and is shown in SEQ ID NO.2 in tetraploid upland cotton 86-1. The cloned gene of the invention has direct connection with the quality of cotton fiber. Transgenic research is carried out on the upland cotton W0 by the constructed plant antisense expression vector and the constructed overexpression vector, and the result shows that the increase of the UBX protein content can lead to the enhancement of fiber helix, and the decrease of the UBX protein content can lead to the weakening of fiber helix. The overexpression vector can be applied to increasing the cotton fiber spiral degree and improving the cotton fiber strength. The antisense expression vector can be applied to thickening the secondary wall of the cotton fiber and improving the strength of the cotton fiber.

Description

Upland cotton fiber strength gene GhUBX and application thereof

Technical Field

The invention relates to a cotton fiber strength gene (GhUBX) of upland, which is a gene sequence obtained from cotton Prema of upland, and belongs to the field of biotechnology application.

Background

Cotton (Gossypium) is an important commercial crop, widely grown worldwide, while cotton fiber, as a natural fiber, is an important raw material for the textile industry. Although the variety of the cotton fiber to be cultivated is different according to different purposes, the fiber quality is the only invariable important selection condition. The cotton fiber measurement criteria mainly include: fiber length, breaking strength, elongation, micronaire value, etc., wherein the breaking strength of cotton fibers is an important index for evaluating the quality of cotton fibers and is also a main condition for determining whether the cotton fibers can be processed. Therefore, genes related to fiber strength are separated and identified, and systematic elucidation determines the molecular mechanism of cell development regulation of fiber strength, so that the method has important theoretical value and practical significance for fully utilizing the gene resources of cotton to improve the fiber quality (Zhang Tianzhen, 2000; Guo Wang Zhen et al, 2003; Zhang Hui et al, 2007; Shang guan Xiaoxia et al, 2008; Litong Fei et al, 2008).

Fiber strength, defined as the breaking load of a single fiber divided by the cross-sectional area of the single fiber, i.e., the strongest force that can be borne per unit fiber cross-sectional area, is a measure of the relative attraction of cotton fibers. The fabric woven by the fiber material with good fiber strength is firm and durable, so with the innovation of textile technology, the requirement on the quality of cotton fiber is higher and higher, and especially the requirement on the strength of the cotton fiber is severer. Therefore, how to improve the strength of cotton fiber is a major goal of current breeding efforts. Previous studies have shown that fiber strength is expressed differently, and the factors affecting the fiber strength are different. The zero-gauge specific strength is mainly influenced by the form of cotton fibers and mainly depends on the cellulose content, the fiber polymerization degree and the fiber supermolecular structure, and the three factors are jointly influenced. While the 3.2mm gauge strength is dependent on the zero gauge strength and the number of reverse spirals of the cotton fiber. The strength of the 3.2mm gauge length and the fiber fineness jointly determine the single fiber strength (Yaomau et al, 1998; Liu Jiu, 1989). It follows that the specific breaking strength of fibers is mainly determined by the supramolecular structure and the deposition of cellulose.

UBX proteins are ubiquitous in eukaryotes as a key link for intracellular ubiquitination. Ubiquitination modification in eukaryotic cells involves a series of reactions of ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, and ubiquitin ligase E3. Firstly, in the case of ATP-powered, the enzyme E1 adheres to Cys residue in the tail of ubiquitin molecule to activate ubiquitin, then E1 transfers the activated ubiquitin molecule to E2 enzyme, and then E2 enzyme and some different E3 enzymes recognize target protein together to carry out ubiquitination modification. The target protein can be mono-ubiquitinated and poly-ubiquitinated according to the relative proportion of E3 to the target protein.

Ubiquitination of substrates by E1, E2, E3 can form several different ubiquitinated substrates. Some substrate proteins can only be monoubiquinated, such as H2B; some substrate proteins have a plurality of lysine residues and can be singly ubiquinated at multiple sites under proper conditions; still other proteins form polyubiquitin chains at a single lysine site, which can be divided into single, mixed and dendritic structures depending on the lysine site to which the ubiquitin chain is attached.

Disclosure of Invention

The invention aims to provide a UBX-Domain containment 10 gene (GhUBX) sequence of cotton and a genome sequence thereof in upland cotton 86-1, Prema and Raymond cotton; designing a pair of GhUBX specific primers to detect the space-time expression condition of the GhUBX specific primers in cotton Prema and 86-1; and the gene is used as a target gene, and the transgenic function verification is carried out by a genetic engineering method so as to be used for cultivating a new germ plasm system and applying the new germ plasm system in production.

The purpose of the invention can be realized by the following technical scheme:

the strength gene GhUBX is derived from tetraploid upland cotton fiber, and is characterized in that the nucleotide sequence of the gene in tetraploid upland cotton (G.hirsutum) Prema is shown as SEQ ID NO.1, and the nucleotide sequence of the gene in tetraploid upland cotton (G.hirsutum)86-1 is shown as SEQ ID NO. 2.

The cotton UBX gene (GhUBX) has 6-bp InDel difference in Prema (high strength fiber) and 86-1 (low fiber strength). The 6-bp difference is SSR (CCTCCG), and the InDel is deleted in the high-strength fiber parent Prema. The repetition times of the elements of SSR in the GhUBX gene sequences in different cotton variety materials of upland cotton are different. SSR primitive repetition type formula in upland cotton is (CTCGGC)₁CTCTG(CCTCCG)_n＝2/5/6Only SSRs (cctccg) differ in number, where n-2 is the repeat of the motif of SSR in the UBX gene sequence of subgroup a in upland cotton variety, and n-5 or n-6 is present in subgroup D; n is 5 in Prema and 6 in 86-1. Correlation analysis is carried out on materials with different genotypes in 281 varieties groups of upland cotton and fiber strength to find that: the GhUBX gene is significantly related to fiber strength, but is susceptible to environmental influences to some extent, as shown in table 3, 33 varieties of genotypes in group a are all consistent with prema, 235 varieties of genotypes in group B are all consistent with 86-1, a is greater than B in comparison of average fiber breakage ratio strength, and significant differences in fiber strength are detected at multiple points over the years.

An overexpression vector of tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO. 1.

The overexpression vector is preferably obtained by cloning the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 between enzyme cutting sites of eGFP4 expression vector Sma I and BamH I in a gene recombination mode.

The antisense expression vector of tetraploid uploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO. 1.

The tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 is used for improving the strength of cotton fibers.

The application preferably utilizes a genetic engineering means to increase the fiber helix degree and the fiber strength by over-expressing a tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO. 1; or inhibiting the expression of tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 by means of genetic engineering to thicken the secondary wall of the fiber and increase the fiber strength.

The overexpression vector disclosed by the invention is applied to increasing the cotton fiber spiral degree and improving the cotton fiber strength.

The antisense expression vector is applied to thickening the secondary wall of the cotton fiber and improving the strength of the cotton fiber.

The invention has the advantages that:

(1) the cloned gene of the invention has direct connection with the quality of cotton fiber. The GhUBX protein is a key protein in the ubiquitination pathway and is closely related to the degradation of the key protein in the cotton fiber development period. By observing the mature fibers of GhUBX transgenic plants by scanning electron microscopy (fig. 4) and transmission electron microscopy (fig. 5), we found that: compared with the transgenic receptor W0, the secondary wall of the over-expression plant is thinned, the secondary wall of the fiber of the antisense expression plant is thickened, compared with the control, the breaking ratio strength of the fiber of the transgenic plant is improved by 6.4-11.4%, and most transgenic plants show significant difference (Table 2).

(2) The structural variation (6-bp base deletion, as shown in figure 7) of the GhUBX gene found in the research exists at the N end of the gene sequence, and it is presumed that the fiber high-strength material Prema cannot be specifically combined with AAA-ATPase due to the deletion of the 6-bp sequence at the N end, so that the activity of the AAA-ATPase is enhanced, and the intracellular metabolic process is accelerated. 86-1 can generate functional GhUBX to regulate the activity of AAA-ATPase, and the cells are maintained in normal metabolic process.

(3) The genome full-length sequence is directly obtained by PCR technology amplification, and the technology has the advantages of small initial template amount and simple and easy test steps.

(4) The cloned gene of the invention is more similar to UBX10 of plants in structure, and is not reported in cotton. Through sequence alignment obtained from parents, the differences are mainly on a short repeat sequence (SSR) at the N terminal (figure 7), and the differences exist in individual amino acids. The gene structure is comprehensively displayed and analyzed for the first time.

(5) The quantitative PCR result shows that the expression quantity of the gene is different at different stages of fiber development, as shown in figure 1: there is a large difference in expression between Prema and 86-1 during the critical period of fibrous secondary wall thickening (20-25day after anti-hesis, DPA). In the early stage of secondary wall thickening, the expression level of GhUBX in 86-1 is obviously higher than that of Prema. It is postulated that the formation of polymers of UBX proteins degrades proteins involved in secondary wall synthesis within plant cells, resulting in thinning of the 86-1 fibrous secondary wall.

(6) Transgenic research is carried out on the upland cotton W0 by the constructed plant antisense expression vector and the constructed overexpression vector, and the result shows that the increase of the UBX protein content can lead to the enhancement of fiber helix, and the decrease of the UBX protein content can lead to the weakening of fiber helix.

Drawings

FIG. 1 is a histological expression analysis showing that the quantitative PCR detects the time-space distribution of the cotton fiber strength gene (GhUBX) in the expression of different tissues (root, stem, leaf, fiber 15 days, 20 days and 25 days after flowering) of cotton.

FIG. 2 shows quantitative PCR detection of GhUBX gene transcription level expression in cotton plants overexpressing GhUBX gene and antisense GhUBX gene, wherein 120, 141, 145 and 153 are overexpressing plants, W0 is control group, and 159, 163, 177 and 181 are antisense plants. Samples were fiber 15, 20, 25 days after flowering.

FIG. 3A, B shows Western Blot analysis of cotton plants overexpressing UBX gene and transgenic cotton plants expressing antisense UBX vector, where 120, 141, 145 and 153 are different lines of overexpressing plants, W0 is a control group, and 159, 163, 177 and 181 are different lines of antisense plants. C. D is the statistics of the gray scale scanning of the Western Blot hybridization bands, and the samples are fibers 15 days, 20 days and 25 days after flowering. Beta-actin is an internal reference protein. P <0.05 by Student's t-test; p <0.01.

FIG. 4 shows the fiber helix of the transgenic cotton with overexpression and antisense orientation detected by electron microscope.

(A) The method comprises the following steps (a) - (d) mature fiber of over-expression plant, (e) - (h) mature fiber of antisense plant, and (i), (j) mature fiber of control group. A scanning electron microscope model GEMINI 300 was used, the magnification was 200 times, and the scale bar was 50 μm.

(B) The method comprises the following steps And (4) counting the mature fiber helical distance of the overexpression strain and the antisense strain, wherein the overexpression strain and the wild type show significant difference or extremely significant difference. P <0.05 by Student's t-test; p <0.01.

FIG. 5 transmission electron microscope examination of transgenic fiber resin section shows the secondary wall thickening condition of mature fiber of overexpression and antisense transgene. Wherein (A): 120. 141, 145, 153 are different lines of over-expression plants, W0 is a control group, (B): 159. 163, 177 and 181 are different lines of antisense plants, the statistical histogram of cell wall thickness is shown in (C), and the overexpression and the antisense are obviously different compared with wild type W0. The samples were naturally matured dry fibers. P <0.05 by Student's t-test; p <0.01.

The model of the electron microscope is Hitachi H-9500 and the scale bar is 0.5 mu m.

FIG. 6UBX isogenic evolutionary tree of various species.

The UBX10 amino acid sequence of tea tree, mandarin orange, coffee, muskmelon, winter squash, pumpkin, durian, Asian cotton, Raymond cotton, rubber, walnut, Sichuan mulberry, European populus tremuloides, plum, flowering peach, European cork oak, Chinese rose, cocoa, grape, jujube, castor oil, soybean, and mallow of Columbia was extracted for evolutionary tree analysis. ubx represents the sequence of the post-translational amino acids of SEQ ID NO. 1.

FIG. 7 overview of the GhUBX domain in prema and 86-1

As shown in the figure, the GhUBX amino acid sequences in prema and 86-1 are listed, the amino acid sequence of prema is two amino acids less compared with the amino acid sequence of 86-1, because the short repeat sequence (SSR) at the N-terminus is 6 bases less, and the six bases encode two amino acids alanine (Ala) and serine (Ser), so the length of the amino acid sequence is 470 amino acids for prema, but 472 amino acids for 86-1.

Detailed Description

Example 1

Firstly, obtaining the full-length sequence of the cotton fiber strength gene.

1. Fiber samples of parents (86-1 and Prema)15DPA are measured according to expression, RNA is extracted, cDNA is obtained through reverse transcription, a specific primer and a recombinant primer (an N-terminal Sma I enzyme cutting site and a C-terminal BamH I enzyme cutting site) are designed, a fragment of about 1,400bp is amplified from a cDNA template through a PCR method and is connected to an eGFP carrier, DH5 alpha is converted, a single colony is selected after 12 hours, bacteria shaking is detected, samples are sent for sequencing, and the returned sequence is repeatedly compared to obtain the difference on the UBX gene sequence of the parents.

TABLE 1 PCR recombination primers

Preliminary analysis of structure and bioinformatics of GhUBX gene

The gene consists of 4 exons and 3 introns, and the total length is 1,413 bp. Preliminary analysis of bioinformatics: the amino acid sequence was compared with BLAST (http:// www.ncbi.nlm.nih.gov/BLAST) and found to have the highest homology of 88% with the amino acid sequence of durian UBX10 and 87% with the amino acid sequence of cocoa UBX 10. The gene consists of UBA-like, UAS, UBX domains (FIG. 7).

The results of the homology tree analysis (ftp:// ftp-igbme.u-strasbg.fr/pub/clustalX /) are shown in FIG. 6.

(III) quantitative PCR analysis of Cotton GhUBX Gene

Designing a specific primer:

the quantitative PCR detection of F: 5'-GGTGATGAACCTGAGAAAGG-3' (SEQ ID NO.5) and R: 5'-TTAGTGCAGTACTGTGAAACC-3' (SEQ ID NO.6) shows that the gene is constitutively expressed in different tissues (roots, stems and leaves) and different stages of fiber development (15, 20 and 25DPA after flowering) (figure 1), but the expression levels are different, except that the expression levels in roots, stems and leaves are lower, the expression level in the early stage is higher (15DPA) and the expression level in the stage of rapid thickening of the secondary fiber wall is lower (20 and 25 DPA). The result reflects that the gene is closely related to secondary wall thickening and plays an important role in controlling the development process of cotton fibers.

Example 2

Transgenic functional verification of cotton GhUBX gene

eGFP4 is a traditional plant binary expression vector, and its promoter is 35S promoter. Sma I and BamH I enzyme cut the carrier, and connect with GhUBX recombinant primer PCR product at 37 ℃ to obtain eGFP4 carrier containing GhUBX gene complete expression fragment.

The gene SEQ NO ID.1 sequence of the invention constructs a plant over-expression vector in full length, and the specific process is as follows:

designing a recombinant primer F: 5'-GAACGATAGGGTACCCCCGGGATGGTTGATGTAACCGATAAATTGG-3' (SEQ ID NO.3), R: 5'-GCCCTTGCTCACCATGGATCCGTTTAGCTCCACAAAGAGGCTGG-3' (SEQ ID NO.4), and carrying out PCR by using a T vector plasmid containing a 1410bp target fragment as a template; the eGFP4 expression vector is cut by Sma I and BamH I enzyme, and inactivated at 85 ℃ for 15min after running glue and identifying and cutting for later use. Recombining according to the requirements of a kit system, transferring the recombined plasmid into escherichia coli competent DH5 alpha, and carrying out bacterium selection detection at 12 ℃.

PBI121 is a traditional plant binary expression vector, and the promoter of the PBI121 is a 35S promoter. The gene SEQ ID.1 sequence 745bp-1,171bp specific fragment 426bp long construction antisense plant expression vector, the concrete process is as follows:

PCR was performed using T-vector plasmid containing 1410bp target fragment as template, and fragments of 426bp in size at 745bp to 1,171bp were amplified using primers F: 5'-GGGGATATCAGGTTCCCGTTTTGTGCAGT-3' (SEQ ID NO.7) and F: 5'-GGGGAGCTCTAGGTCCTTTCTCAGGTTCA-3' (SEQ ID NO. 8). The amplified product is cut by enzyme EcoR V and Sac I, and small fragments are recovered; the pBI121 expression vector was digested with Sma I and Sac I to excise the Gus gene, and a large fragment (about 13kb) was recovered. As the EcoR V and the Sma I are both blunt ends, the antisense fragment is constructed on the pBI121 vector by connecting the 426bp target fragment with the large fragment of the pBI121 expression vector recovered by enzyme digestion.

And transforming cotton by an agrobacterium-mediated method for functional verification to respectively obtain a 35S promoter overexpression transgenic plant and a 35S promoter antisense transgenic plant. Both 4 overexpressing and 4 antisense transgenic plants have been identified by the transgenic molecule (FIG. 2). The average fiber strength of both the over-expression and antisense transgenic plant is higher than that of the control, so that the function of the gene related to the strength of cotton fiber is verified directly.

(II) western blot detection of cotton GhUBX

Taking transgenic plants and fibers of w0 of 15, 20 and 25DPA, extracting total protein, running SDS-PAGE electrophoresis and membrane conversion, and detecting GhUBX protein by using prepared ubx antibody, wherein beta-actin is used as internal reference. The results are shown in FIG. 3.

(III) scanning electron microscope observation of mature fiber

Mature fibers of

overexpression

120, 141, 145 and 153, a control group W0 and

antisense transgenes

159, 163, 177 and 181 are respectively taken, the fibers are fixed on an aluminum platform by using special double-faced adhesive tape, after gold spraying treatment, the twisting condition of the fibers is observed by a scanning electron microscope, and the distances between the spirals are obtained by counting the total number of the spirals and the total length of the fibers and dividing the total number of the spirals and the total length of the fibers. The results are shown in FIG. 4.

(IV) mature fiber transmission electron microscope observation of

overexpression

120, 141, 145, 153, control group W0 and

antisense transgenes

159, 163, 177, 181 mature fiber, respectively, were added with 2.5% glutaraldehyde fixation solution, rinsed three times with PBS, 15min each, fixed with 1% osmic acid for 2h, and then washed three times with PBS buffer, 15min each. Treating with 50%, 70%, 90% and 100% ethanol respectively for 15min to dehydrate the sample, embedding with resin embedding medium, solidifying, cutting into slices with thickness less than 0.1 μm with ultrathin section instrument, fixing with copper net, and microscopic examination under transmission electron microscope. The results are shown in FIG. 4.

TABLE 2 transgenic Cotton fiber detection

120. 141, 149, 153 are overexpression materials, 159, 163, 177, 181 are antisense materials, and w0 is control.

TABLE 3 perennial multipoint fiber intensity correlation with SSR sites

Group a contains 33 varieties, and an SSR repeat element n is 5, which is the same as Prema; group B contains 235 varieties, and SSR repeat element n ═ 6, similar to 86-1.

Sequence listing

<110> Nanjing university of agriculture

Zhejiang university

<120> upland cotton fiber strength gene GhUBX and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1410

<212> DNA

<213> tetraploid upland cotton Prema (G. hirsutum Prema)

<400> 1

atggttgatg taaccgataa attggcatat tttcaagcca ccacgggact cgaagatccc 60

gatttgtgta ccgaaatcct tcaggcccat ggttgggacc ttgaactcgc catctcttcc 120

ttcacctctt ccactcaacc ctccgcttct tccactgtct ctgattccga ccctcccgat 180

tcacgggccc ggactgattc cgcctcggcc tctgcctccg cctccgcctc cgcctccgcc 240

tccggtccta ttaccgctcc cgcaccgggc ttagcctgga gactcgttac attaccgatt 300

tccgtgatat ccggaagttt agggttgatc tctggcgctg ttgggctggg tctatgggcc 360

gcgggtggag ttctttcgta ttcccttggg atgatcgggc taggctctgg gcagggcggg 420

gaatcatcgg ccggattggt atcggtctca gctgcggctt ctgaagcgat ggatttcgtg 480

gcggagtttg aaagggagta cggaacgaga gggctgaatt ttgtcggcga gggttttatg 540

gacgcgttgc agaggtcgag gaacgccttc aagctgttgt ttgtgtactt gcactcgccg 600

gaccacccgg attcgcccgt gttctgcgaa aggacgttgt gttcccaggc tctggcggcc 660

tttgtgaatg agaatttcgt agcgtggggc ggaagcatta gggctagtga aggttttaag 720

atgagtaata gcttgaaagc ctccaggttc ccgttttgtg cagtcgttat gcctgctacc 780

aatcagagga tcgcgcttct tcaacaggtt gagggaccta aatctcctga agaaatgctc 840

acaatactac agagagtgct tgaagaaagt gctcccgttc ttgttgctgc aagactagat 900

gcggaagaga gaagaaacaa catgcgttta agggaggagc aagatgctgc ttatagagct 960

gcgcttgaag ctgatcaggc tagggaacgc cagaggagag aggagcaaga acgtctggaa 1020

agggaagcag cagaagctga gcggaaacgt aaggaagaag aagaggctcg tgaaagagca 1080

gcacgtgaag ctgctgagaa ggaagctgcc cgagctagaa tgcggcaaga gaaagccttg 1140

tcacttggtg atgaacctga gaaaggacct aatgttacac aagttttggt acgttttcct 1200

actggagaac gcaaagaaag gaggtttcac agtactgcac taatccaagc tgtttatgat 1260

tatgttgatt cattgggttg cttagaagtt gaggattaca accttgtctc caactttcct 1320

cgagtaacat atggtacaga gaagcgctca ctgaccttga aagaggccgg attacatcct 1380

caggccagcc tctttgtgga gctaaactag 1410

<210> 2

<211> 1416

<212> DNA

<213> tetraploid upland cotton 86-1(G. hirsutum 86-1)

<400> 2

atggttgatg taaccgataa attggcatat tttcaagcca ccacgggact cgaagatccc 60

gatttgtgta ccgaaatcct tcaggcccat ggttgggacc ttgaactcgc catctcttcc 120

ttcacctctt ccactcaacc ctccgcttct tccactgtct ctgattccga ccctcccgat 180

tcacgggccc ggactgattc cgcctcggcc tctgcctccg cctccgcctc cgcctccgcc 240

tccgcctccg gtcctattac cgctcccgca ccgggcttag cctggagact cgttacatta 300

ccgatttccg tgatatccgg aagtttaggg ttgatctctg gcgctgttgg gctgggtcta 360

tgggccgcgg gtggagttct ttcgtattcc cttgggatga tcgggctagg ctctgggcag 420

ggcggggaat catcggccgg attggtatcg gtctcagctg cggcttctga agcgatggat 480

ttcgtggcgg agtttgaaag ggagtacgga acgagagggc tgaattttgt cggcgagggt 540

tttatggacg cgttgcagag gtcgaggaac gccttcaagc tgttgtttgt gtacttgcac 600

tcgccggacc acccggattc gcccgtgttc tgcgaaagga cgttgtgttc ccaggctctg 660

gcggcctttg tgaatgagaa tttcgtagcg tggggcggaa gcattagggc tagtgaaggt 720

tttaagatga gtaatagctt gaaagcctcc aggttcccgt tttgtgcagt cgttatgcct 780

gctaccaatc agaggatcgc gcttcttcaa caggttgagg gacctaaatc tcctgaagaa 840

atgctcacaa tactacagag agtgcttgaa gaaagtgctc ccgttcttgt tgctgcaaga 900

ctagatgcgg aagagagaag aaacaacatg cgtttaaggg aggagcaaga tgctgcttat 960

agagctgcgc ttgaagctga tcaggctagg gaacgccaga ggagagagga gcaagaacgt 1020

ctggaaaggg aagcagcaga agctgagcgg aaacgtaagg aagaagaaga ggctcgtgaa 1080

agagcagcac gtgaagctgc tgagaaggaa gctgcccgag ctagaatgcg gcaagagaaa 1140

gccttgtcac ttggtgatga acctgagaaa ggacctaatg ttacacaagt tttggtacgt 1200

tttcctactg gagaacgcaa agaaaggagg tttcacagta ctgcactaat ccaagctgtt 1260

tatgattatg ttgattcatt gggttgctta gaagttgagg attacaacct tgtctccaac 1320

tttcctcgag taacatatgg tacagagaag cgctcactga ccttgaaaga ggccggatta 1380

catcctcagg ccagcctctt tgtggagcta aactag 1416

<210> 3

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaacgatagg gtacccccgg gatggttgat gtaaccgata aattgg 46

<210> 4

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcccttgctc accatggatc cgtttagctc cacaaagagg ctgg 44

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggtgatgaac ctgagaaagg 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttagtgcagt actgtgaaac c 21

<210> 7

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggggatatca ggttcccgtt ttgtgcagt 29

<210> 8

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggggagctct aggtcctttc tcaggttca 29

Claims

1. Overexpression of tetraploid upland cotton fiber strength gene shown in SEQ ID NO.1GhUBXUse in increasing the degree of spiralling in cotton fibres.

2. Tetraploid upland cotton fiber strength gene shown in SEQ ID NO.1GhUBXThe use of the overexpression vector of (a) in increasing the degree of cotton fiber helicity.