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Application of cotton ghvln4 gene in resistance to Verticillium wilt

A technology for resistance to verticillium wilt and verticillium wilt, which is applied in the field of genetic engineering and can solve the problems of lack of research and application of microfilament binding protein genes.

Active Publication Date: 2021-01-01
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few studies and applications on the relationship between the actin skeleton and plant disease resistance and the application of actin-binding protein genes in plant disease resistance.

Method used

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  • Application of cotton ghvln4 gene in resistance to Verticillium wilt
  • Application of cotton ghvln4 gene in resistance to Verticillium wilt
  • Application of cotton ghvln4 gene in resistance to Verticillium wilt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The upland cotton fluff protein GhVLN4 gene is the basis of the present invention, so the cloning and obtaining process of the gene are briefly introduced as follows.

[0044] 1. Cotton GhVLN4 gene cloning

[0045] In this study, Li-1 fiber non-elongating mutants and wild-type WT were used as materials to compare the differences of -3, 0, 4, and 8DPA proteomes and the results of mass spectrometry analysis. In this paper, the matching EST sequence (DW488595) was identified according to mass spectrometry, and the full-length sequence of the gene was obtained by 3' RACE-PCR.

[0046] First, use the CTAB method to extract cotton total RNA. The specific steps are: add 4 mL of CTAB buffer solution to a 10 mL centrifuge tube, take an appropriate amount of ovule sample, grind it with liquid nitrogen and add it to the above tube, shake and mix, ice bath for 10 minutes, and 65 °C water bath for 15 minutes , and mix by inversion 3 times. Add 4mL of chloroform and mix well, and l...

Embodiment 2

[0078] Based on the understanding of the basic characteristics of the gene GhVLN4 in the previous examples, the inventors further analyzed the expression pattern of this gene in cotton tissue and the tissue expression after stress. The relevant experiments are briefly introduced as follows.

[0079] 1. Expression analysis of cotton GhVLN4 gene in different tissues (root, stem, leaf, ovule, fiber)

[0080] The expression of GhVLN4 in different tissues (root, stem, leaf, ODPA ovule, 3 and 6DPA fiber) of wild-type TM-1 and linter mutant Li-1 were analyzed by real-time quantitative PCR. Collect roots, stems and leaves from three-leaf age cotton seedlings, and collect 0d ovules, 3d and 6d fibers at full flowering stage. Cotton total RNA was extracted using the plant polysaccharide and polyphenol RNATIQU kit of Beijing Biotech Biotechnology Co., Ltd., and the reverse transcription kit PrimeScript of TaKaRa Company was used. TM 1st Strand cDNASynthesisKit synthesizes cDNA by reverse...

Embodiment 3

[0091] The inventors used VIGS technology to silence the GhVLN4 gene in cotton plants, and inoculated against Verticillium wilt, so as to study the relationship between GhVLN4 gene and Verticillium wilt. The relevant experimental process is introduced as follows. 1. Construction of VIGS vector containing GhVLN4 gene

[0092] First, design the primer sequence and perform PCR amplification to obtain the GhVLN4 gene sequence. The specific primer sequence is:

[0093] pTRV2-GhVLN4-F: 5'-GCTCTAGAGCTAGAAACCTTTCTACTCCGCCAC-3'

[0094] pTRV2-GhVLN4-R: 5'-CGGGATCCGAACAGTTGAAGAGCCATTTTTAGC-3'

[0095] Secondly, the TRV2 empty vector and the GhVLN4 gene sequence fragment obtained by PCR amplification were double-digested with BamHI and Xba I respectively, and the digested products were recovered and purified, and the above-mentioned digested products were ligated with T4 ligase to construct a recombinant plasmid .

[0096] Transform the above-mentioned ligation product into DH5α compe...

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Abstract

The invention belongs to the technical field of genetic engineering, and concretely relates to application of a cotton GhVLN4 gene in resistance to verticillium wilt. The GhVLN4 gene is related to verticillium wilt resistance of plants. An inventor finds that when the plants are infected by verticillium lecanii, the gene expression level is remarkably increased, and when part type of hormone treatment is adopted, the gene expression level is remarkably increased again. Furthermore, after the gene is silenced, the verticillium wilt resistance of the plants is reduced, and after the gene is excessively expressed, the verticillium wilt resistance of the plants is increased. Based on the characteristics, GhVLN4 overexpression plants can be cultivated or screened, and a better reference can beprovided for obtaining a new plant species with better verticillium wilt resistance, so that a better application value is realized on new plant species cultivation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to the application of upland cotton gene GhVLN4 in resisting verticillium wilt. Background technique [0002] Cotton is one of the most important economic crops in the world and occupies an important position in the national economy. However, cotton Verticillium wilt has become a disaster in various cotton areas in my country, and it has become the first major disease in cotton production since Fusarium wilt was effectively controlled in my country, and it is one of the main obstacles to sustainable cotton production. At present, the conventional control methods can be partially controlled but cannot be effectively controlled, and the traditional disease-resistant breeding strategy is used to breed disease-resistant varieties with slow results. Breeding disease-resistant varieties is the most effective way to solve cotton Verticillium wilt. [0003] After long-term evolut...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/60
CPCC07K14/415C12N15/8282
Inventor 刘康张培培葛冬冬张中起韩明亚张晓彤吕芬妮李丽凤
Owner NANJING AGRICULTURAL UNIVERSITY
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