WO2021051883A1 - Upland cotton fiber strength gene ghubx and use thereof - Google Patents

Abstract

Provided are the fiber strength gene GhUBX from tetraploid upland cotton (Gossypium hirsutum), wherein the nucleotide sequence of this gene in tetraploid upland cotton Prema is as shown in SEQ ID NO. 1 and the nucleotide sequence of this gene in tetraploid upland cotton 86-1 is as shown in SEQ ID NO. 2. Also provided is the fiber strength gene GhUBX as shown in SEQ ID NO. 1 and the use of a super-expression vector and antisense expression vector of the gene in improving the strength of cotton fibers. The super-expression vector can be used for increasing the spiral degree of the cotton fibers and improving the strength of the cotton fibers. The antisense expression vector can be used for thickening the secondary walls of the cotton fibers and improving the strength of the cotton fibers.

Description

Upland cotton fiber strength gene GhUBX and its application Technical field

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

Background technique

Cotton (Gossypium) is an important economic crop, which is widely grown worldwide, and cotton fiber, as a natural fiber, is an important raw material for the textile industry. Although the different uses of cotton fiber require different varieties, the fiber quality is the only constant important selection condition. The measurement standards of cotton fiber mainly include: fiber length, breaking strength, elongation, micronaire value, etc. The breaking strength of cotton fiber is an important index to evaluate the quality of cotton fiber, and it also determines whether the cotton fiber can be processed. The main conditions. Therefore, isolating and identifying genes related to fiber strength and systematically elucidating the molecular mechanisms of cell development and regulation that determine fiber strength have important theoretical and practical significance for making full use of cotton's own genetic resources to improve fiber quality (Zhang Tianzhen, 2000; Guo Wangzhen et al. , 2003; Zhang Hui et al., 2007; Shangguan Xiaoxia et al., 2008; Lai Tongfei et al., 2008).

The definition of fiber strength is the breaking load of a single fiber divided by the cross-sectional area of a single fiber, that is, the strongest force that a unit fiber cross-sectional area can withstand, and it is a measure of the relative gravitational force of cotton fibers. The cloth woven from fiber materials with good fiber strength is strong and durable. Therefore, with the innovation of textile technology, the requirements for the quality of cotton fibers are getting higher and higher, especially for the strength of cotton fibers. Therefore, how to improve the strength of cotton fiber has become the main goal of current breeding work. Previous studies have shown that there are different ways of expressing fiber strength, and the factors that affect different ones are also different. The zero-gauge ratio strength is mainly affected by the morphology of cotton fiber, and mainly depends on the cellulose content, fiber polymerization degree, and fiber supramolecular structure, which are jointly affected by the three factors. The 3.2mm gauge ratio strength depends on the zero gauge ratio strength and the number of reverse spirals of cotton fiber. The 3.2mm gauge ratio strength and fiber fineness jointly determine the strength of the single fiber (Yao Mu et al., 1998; Liu Jihua, 1989). It can be seen that the fiber breaking strength is mainly determined by the supramolecular structure and the deposition of cellulose.

As a key link of intracellular ubiquitination, UBX protein is ubiquitous in eukaryotic organisms. The ubiquitination modification in eukaryotic cells involves a series of reactions of ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. First, in the case of ATP supply, the enzyme E1 adheres to the Cys residue at the tail of the ubiquitin molecule to activate ubiquitin, then E1 transfers the activated ubiquitin molecule to the E2 enzyme, and then the E2 enzyme and some different types of E3 Enzymes recognize the target protein together and modify it for ubiquitination. According to the relative ratio of E3 to the target protein, the target protein can be modified by monoubiquitination and polyubiquitination.

The ubiquitination of substrates by E1, E2, and E3 can form several different ubiquitination substrates. Some substrate proteins can only be monoubiquitinated, such as H2B; some substrate proteins have multiple lysine residues, which will be monoubiquitinated at multiple sites under suitable conditions; and some proteins are monoubiquitinated in a single lysine. The amino acid site will form a polyubiquitin chain, which can be divided into single, mixed and dendritic structures according to the different lysine sites connected to the ubiquitin chain.

Summary of the invention

The purpose of the present invention is to provide a cotton UBX-Domain Containing10 gene (GhUBX) sequence and its genome sequence in upland cotton 86-1, Prema and Raymond cotton; design a pair of specific primers for GhUBX Detect its temporal and spatial expression in upland cotton Prema, 86-1; and use this gene as a target gene to verify the transgene function through genetic engineering methods, so that it can be used to cultivate new germplasm lines and apply in production.

The purpose of the present invention can be achieved through the following technical solutions:

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

The cotton UBX gene (GhUBX) of the present invention has a 6-bp InDel difference between Prema (high-strength fiber) and 86-1 (low-strength fiber). The 6-bp difference is an SSR (CCTCCG), and the InDel is missing in the fiber high-strength parent Prema. The number of SSR motifs in the GhUBX gene sequence in different upland cotton varieties is different. The formula for the repeat type of SSR motifs in upland cotton is (CTCGGC) ₁ CTCTG(CCTCCG) _n=2/5/6 . Only SSR (CCTCCG) has a number difference. Among them, SSR in the UBX gene sequence of subgroup A in upland cotton Primitive repetition n=2, there are two cases of n=5 or n=6 in the D subgroup; n=5 in Prema and n=6 in 86-1. The correlation analysis between different genotypes of materials and fiber strength in upland cotton 281 species populations found that GhUBX gene is significantly related to fiber strength, but to a certain extent, it is easily affected by the environment. As shown in Table 3, Group A The genotypes of 33 varieties are the same as prema, and the genotypes of 235 varieties in group B are the same as 86-1. In the comparison of the average fiber breakage strength, A is greater than B, and significant differences in fiber strength have been detected over many years. .

The overexpression vector of the 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 into the eGFP4 expression vector Sma I and BamH I by means of gene recombination.

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

Application of the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 in improving the cotton fiber strength.

The said application preferably uses genetic engineering means to overexpress the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 to increase the degree of fiber helix and fiber strength; or to inhibit SEQ ID NO. The expression of the tetraploid upland cotton fiber strength gene GhUBX shown in 1, thickens the fiber secondary wall and increases the fiber strength.

The overexpression vector of the present invention is used in increasing the degree of cotton fiber helix and improving the strength of cotton fiber.

The antisense expression vector of the present invention is used for thickening the secondary wall of cotton fiber and improving the strength of cotton fiber.

Beneficial effects The advantages of the present invention are shown in:

(1) The gene cloned in the present invention is directly related to the quality of cotton fiber. GhUBX protein is a key protein in the ubiquitination pathway and is closely related to the degradation of key proteins during cotton fiber development. Observing the mature fibers of GhUBX transgenic plants by scanning electron microscopy (Figure 4) and transmission electron microscopy (Figure 5), we found that: compared with the transgenic receptor W0, the secondary wall of the overexpression plants became thinner, and the fibers of the antisense expressing plants became thinner. The green wall became thicker, compared with the control, the fiber breaking strength of the transgenic plants increased by 6.4-11.4%, and most of the transgenic plants showed significant differences (Table 2).

(2) The structural variation of the GhUBX gene (6-bp deletion, as shown in Figure 7) found in this study is present at the N-terminus of the gene sequence. It is speculated that the fiber high-strength material Prema lacks the 6-bp at the N-terminus. The sequence cannot specifically bind to AAA-ATPase, which in turn enhances the activity of AAA-ATPase and accelerates the metabolic process in the cell. 86-1 can produce functional GhUBX to regulate the activity of AAA-ATPase, and the cell maintains a normal metabolic process.

(3) The full-length sequence of the genome of the present invention is directly amplified by PCR technology, which has the advantages of small amount of starting template and simple test steps.

(4) The gene cloned in the present invention is more similar to plant UBX10 in terms of structure, and has not been reported in cotton. Through the sequence alignment obtained from the parent, the difference is mainly in the N-terminal short repeat sequence (SSR) (Figure 7), and there are also differences in individual amino acids. The gene structure has been comprehensively displayed and analyzed for the first time.

(5) Quantitative PCR results show that the expression of this gene is different at different stages of fiber development, as shown in Figure 1: In the critical period of fiber secondary wall thickening (20-25 days after anthesis, DPA) Prema and 86-1 There is a big difference in expression between them. In the early stage of secondary wall thickening, the expression level of GhUBX in 86-1 was significantly higher than that in Prema. It is speculated that UBX protein forms a polymer to degrade the protein involved in the synthesis of the secondary wall in plant cells, making the fibrous secondary wall of 86-1 thinner.

(6) The constructed plant antisense expression vector and overexpression vector were used for transgenic research on upland cotton W0, and it was found that the increase of the UBX protein content would lead to the enhancement of the fiber helix, and the decrease would lead to the weakening of the fiber helix.

Description of the drawings

Figure 1 Tissue expression analysis, quantitative PCR to detect the spatiotemporal distribution of cotton fiber strength gene (GhUBX) expression in different cotton tissues (roots, stems, leaves, 15 days, 20 days, and 25 days after flowering).

Figure 2 Quantitative PCR detection of GhUBX gene transcription level expression in cotton plants overexpressing GhUBX gene and antisense GhUBX gene, of which 120, 141, 145, and 153 are overexpression plants, W0 is the control group, and 159, 163, 177, and 181 are antisense Righteous plant. The samples are fibers at 15 days, 20 days, and 25 days after flowering.

Figure 3 A and B are the Western Blot detection of overexpression transgenic cotton plants with UBX gene and antisense UBX vector transgenic cotton. Among them, 120, 141, 145, and 153 are different lines of overexpression plants, W0 is the control group, 159, 163, 177 and 181 are different lines of antisense plants. C and D are the gray-scale scanning statistical results of Western Blot hybridization bands. The samples are fibers at 15 days, 20 days, and 25 days after flowering. β-actin is an internal reference protein. After Student’s t-test test, *p<0.05; **p<0.01.

Figure 4 Electron microscopic examination of transgenic fibers, showing the spiral situation of over-expression and antisense transgenic cotton fibers.

(A): (a)-(d) are mature fibers of overexpression plants, (e)-(h) are mature fibers of antisense plants, (i) and (j) are mature fibers of control. The scanning electron microscope model is GEMINI 300, the magnification is 200 times, and the scale is 50μm.

(B): The mature fiber helix distance statistics of the overexpression line and the antisense line, the overexpression line and the wild type show a significant or extremely significant difference. After Student’s t-test test, *p<0.05; **p<0.01.

Figure 5 Transmission electron microscopy of the transgenic fiber resin section, showing the thickening of the secondary wall of the mature fiber of overexpression and antisense transgenic. Among them (A): 120, 141, 145, 153 are different strains of overexpression plants, W0 is the control group, (B): 159, 163, 177, 181 are different strains of antisense plants, and histogram of cell wall thickness statistics As shown in (C), both overexpression and antisense show significant differences compared with wild-type W0. The sample is a naturally mature dry fiber. After Student’s t-test test, *p<0.05; **p<0.01.

The electron microscope model is Hitachi H-9500 scale bar is 0.5μm.

Figure 6 Evolutionary tree of homologous genes in UBX species.

Extracted tea tree, citrus, orange, coffee, cantaloupe, winter squash, pumpkin, zucchini, durian, Asian cotton, Raymond cotton, rubber, walnut, Sichuan mulberry, European poplar, plum, plum, green peach, European cork The UBX10 amino acid sequence of oak, rose, cocoa, grape, jujube, castor, soybean, and Colombian mallow was analyzed by phylogenetic tree. ubx represents the post-translational amino acid sequence of SEQ ID NO.1.

Figure 7 Overview of GhUBX domains in prema and 86-1

As shown in the figure, the GhUBX amino acid sequences in prema and 86-1 are listed. Compared with the amino acid sequence of 86-1, the amino acid sequence of prema has two amino acids missing because the N-terminal short repeat sequence (SSR) has six bases missing. Base sequence, these six bases encode two amino acids alanine (Ala) and serine (Ser), so the length of the amino acid sequence prema is 470 amino acids, but 86-1 is 472 amino acids.

detailed description

Example 1

(1) Obtaining the full-length sequence of cotton fiber strength gene.

1. Take fiber samples of 15 DPA from both parents (86-1 and Prema) according to the expression amount, extract RNA, reverse transcription to obtain cDNA, design specific primers and recombinant primers (N-terminal Sma I restriction site, C-terminal BamH I enzyme Cut site), a fragment of about 1,400bp was amplified from the cDNA template by PCR and connected to the eGFP vector, transformed into DH5α, 12 hours later, a single colony was picked for testing and sequencing, and the returned sequence was repeatedly aligned. The difference in the sequence of the UBX gene between the parents was analyzed.

Table 1 PCR recombination primers

(2) The structure of GhUBX gene and preliminary analysis of bioinformatics

The gene consists of 4 exons and 3 introns, with a total length of 1,413 bp. Preliminary analysis of bioinformatics: BLAST (http://www.ncbi.nlm.nih.gov/blast) compares the amino acid sequence and finds that it has the highest homology of 88% with the amino acid sequence of Durian UBX10, which is similar to that of Cocoa UBX10. The amino acid sequence reaches 87%. The gene consists of UBA-like, UAS, and UBX domains (Figure 7).

The result of homology relationship tree analysis (ftp://ftp-igbme.u-strasbg.fr/pub/ClustalX/) is shown in Figure 6.

(3) Quantitative PCR analysis of cotton GhUBX gene

Design specific primers:

F:5'-GGTGATGAACCTGAGAAAGG-3'(SEQ ID NO.5), R:5'-TTAGTGCAGTACTGTGAAACC-3'(SEQ ID NO.6) was tested by quantitative PCR, and the results showed that the gene is in each tissue (root, stem, Leaf) and fiber development at different stages (15, 20, 25 DPA after flowering) constitutively expressed (Figure 1), but the expression level is different. In addition to the lower expression level in roots, stems and leaves, there are also differences at different stages of fiber development. The expression level of is higher (15DPA), while the expression level is lower during the period of rapid thickening of the fiber secondary wall (20, 25DPA). This result reflects that the gene of the present invention is closely related to the thickening of the secondary wall and plays a vital role in controlling the development of cotton fibers.

Example 2

(1) Validation of transgenic function of cotton GhUBX gene

eGFP4 is a traditional plant binary expression vector, and its promoter is 35S promoter. Sma I and BamH I digested the vector and ligated it with the PCR product of the GhUBX recombination primer at 37°C to obtain the eGFP4 vector containing the complete expression fragment of the GhUBX gene.

To construct a plant overexpression vector with the full length of the SEQ NO ID.1 sequence of the gene of the present invention, the specific process is as follows:

Design the recombinant primer F: 5'-GAACGATAGGGTACCCCCCCGGGATGGTTGATGTAACCGATAAATTGG-3' (SEQ ID NO.3), R: 5'-GCCCTTGCTCACCATGGATCCGTTTAGCTCCACAAAGAGGCTGG-3' (SEQ ID NO.4), and perform PCR with the T vector plasmid containing the 1410bp target fragment as a template ; The eGFP4 expression vector was digested with Sma I and BamH I, and then cut through gel running, and then inactivated at 85°C for 15 minutes and placed on ice for later use. Perform recombination according to the requirements of the kit system, transfer the recombined plasmid into E. coli competent DH5α, and select bacteria for detection after 12°C.

PBI121 is a traditional plant binary expression vector, and its promoter is 35S promoter. The antisense plant expression vector is constructed with a specific fragment of 745bp-1,171bp in the sequence of SEQ NO ID.1 of the present invention with a length of 426 bp. The specific process is as follows:

PCR was performed with the T vector plasmid containing the 1410bp target fragment as a template, using primers F:5'-GGGGATATCAGGTTCCCGTTTTGTGCAGT-3'(SEQ ID NO.7), F:5'-GGGGAGCTCTAGGTCCTTTCTCAGGTTCA-3'(SEQ ID NO.8), The amplified fragment is a 426bp fragment at 745bp-1,171bp. The amplified product was digested with EcoR V and Sac I, and small fragments were recovered; the pBI 121 expression vector was digested with Sma I and Sac I, the Gus gene was excised, and the large fragment (approximately 13 kb) was recovered. Since EcoR V and Sma I are both blunt ends, the 426bp target fragment and the large fragment of the pBI 121 expression vector recovered by restriction digestion are ligated to construct the antisense fragment into the pBI 121 vector.

Agrobacterium-mediated transformation of cotton was carried out for functional verification, and 35S promoter overexpression transgenic plants and 35S promoter antisense transgenic plants were obtained respectively. Four overexpression and four antisense transgenic plants have been identified by transgenic molecules (Figure 2). The average fiber strength of the overexpression and antisense transgenic plants was stronger than that of the control, so that the function of the gene and cotton fiber strength was directly verified.

(2) Western blot detection of cotton GhUBX

Take transgenic plants and 15, 20, 25 DPA fibers of w0, extract total protein, run SDS-PAGE electrophoresis and transfer to membrane, use the prepared ubx antibody to detect GhUBX protein, and use β-actin as the internal reference. The results are shown in Figure 3.

(3) Scanning electron microscope observation of mature fiber

Take mature fibers overexpressing 120, 141, 145, 153, control group W0 and antisense transgenes 159, 163, 177, and 181 respectively, and fix the fibers on an aluminum table with a special double-sided tape. After spraying gold, perform Scanning electron microscopy observes the distortion of the fiber, and calculates the total number of spirals and the total length of the fiber, and divides them to obtain the distance between the spirals. The results are shown in Figure 4.

(4) Observation of mature fiber by transmission electron microscope

Take mature fibers overexpressing 120, 141, 145, 153, control group W0 and antisense transgenes 159, 163, 177, and 181 respectively, and put in 2.5% glutaraldehyde fixative solution, rinse with PBS three times, 15 minutes each time, use 1 % Osmic acid was fixed for 2 hours, and then washed with PBS buffer three times for 15 minutes each time. Treat the sample with 50%, 70%, 90%, and 100% ethanol for 15 minutes to dehydrate the sample, then embed it with a resin embedding agent. After solidification, use an ultrathin microtome to cut into thin slices with a thickness of less than 0.1 μm. Use a copper mesh It is fixedly placed under a transmission electron microscope for microscopic examination. The results are shown in Figure 4.

Table 2 Detection of genetically modified cotton fiber

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

Table 3 Correlation between multi-point fiber strength and SSR sites for many years

Group A contains 33 varieties, SSR repeat motif n=5, which is the same as Prema; Group B contains 235 varieties, SSR repeat motif n=6, which is the same as 86-1

Claims (8)

1. The fiber strength gene GhUBX from tetraploid upland cotton, characterized in that the nucleotide sequence of this gene in tetraploid upland cotton (G. hirsutum) Prema is shown in SEQ ID NO.1, in tetraploid upland cotton (G. hirsutum) The nucleotide sequence of the gene in .hirsutum)86-1 is shown in SEQ ID NO.2.
2. The overexpression vector of the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1.
3. The overexpression vector according to claim 2, characterized in that the overexpression vector is the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 cloned into the eGFP4 expression vector Sma by means of gene recombination. Obtained between I and BamH I restriction sites.
4. The antisense expression vector of the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1.
5. Application of the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 in improving the cotton fiber strength.
6. The application according to claim 5, characterized in that genetic engineering means overexpression of the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 to increase the degree of fiber helix and fiber strength; or through genetic engineering The method inhibits the expression of the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1, which thickens the fiber secondary wall and increases the fiber strength.
7. The use of the overexpression vector of claim 2 or 3 in increasing the degree of cotton fiber helix and improving the strength of cotton fiber.
8. The use of the antisense expression vector of claim 4 in thickening the secondary wall of cotton fiber and improving the strength of cotton fiber.

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