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Gossypium hirsutum GhWRKY74 protein, and coding gene and application thereof

A technology of upland cotton and genes, applied in the field of molecular biology

Active Publication Date: 2021-02-26
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

How to screen more differentially expressed WRKY transcription factors is of great significance in order to provide candidate genes for cultivating cotton varieties resistant to Verticillium wilt, and to improve the poor situation of existing cotton resistance to Verticillium wilt

Method used

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  • Gossypium hirsutum GhWRKY74 protein, and coding gene and application thereof
  • Gossypium hirsutum GhWRKY74 protein, and coding gene and application thereof
  • Gossypium hirsutum GhWRKY74 protein, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Cloning of Verticillium wilt-related gene GhWRKY74 in upland cotton

[0029] Cloning the Verticillium wilt-related gene GhWRKY74 of upland cotton comprises the following steps:

[0030] 1. Extraction of RNA

[0031] RNA from cotton variety 86-1 leaf samples were extracted using RNAprep Pure Plant Polyphenol Polysaccharide Total RNA Extraction Kit.

[0032] 2. Synthesis of cDNA

[0033] 2.1 Synthesis of intermediate fragment cDNA

[0034] The cDNA of the intermediate fragment was synthesized using the FastQuant cDNA First Strand Synthesis Kit to reverse transcribe the cDNA.

[0035] 2.2 Synthesis of cDNA at the 3' end

[0036] The synthesis system of cDNA at the 3' end is shown in Table 1 below:

[0037] Table 1

[0038]

[0039] Mix according to the above system, after centrifugation, place on PCR instrument at 42°C for 60 minutes, and at 70°C for 15 minutes, cool on ice after the reaction, and store at -20°C.

[0040] 2.3 Synthesis of cDNA at the 5'...

Embodiment 2

[0111] The biological information analysis of embodiment 2GhWRKY74 protein

[0112] According to the prediction of the online tool ProtParam, the molecular weight of the protein is 36.82626kDa, the isoelectric point is 9.80, and the molecular formula is C 1606 h 2564 N 478O472 S 22 The instability index is 56.41, which belongs to unstable protein; the total average hydrophilicity-0.653, belongs to hydrophilic protein; 20 kinds of amino acids constituting protein, serine (Ser) accounts for the highest ratio (13.8%) (such as figure 2 Shown in A), containing 23 negatively charged amino acid residues and 47 positively charged amino acid residues.

[0113] SignalP 4.0 was used to predict that the protein had no signal peptide; TMHMM Server v.2.0 predicted that the protein had no transmembrane helical region; CDD predicted that the protein had zinc finger domain and WRKY domain.

[0114] Through multiple sequence alignment and application of MEGA software, the phylogenetic tree...

Embodiment 3

[0116] Example 3 Analysis of the expression level of GhWRKY74 in different tissues of cotton susceptible variety 86-1

[0117] 3.1 Experimental method:

[0118] When Upland Cotton 86-1 was cultivated until two true leaves unfolded, the roots, stems and leaves of the cotton were harvested, thoroughly cleaned and surface sterilized, frozen in liquid nitrogen and stored in a -80°C ultra-low temperature refrigerator.

[0119] RNAprep Pure plant polyphenol polysaccharide total RNA extraction kit was used to extract RNA from different cotton tissue samples, and FastQuant cDNA first-strand synthesis kit was used to reverse transcribe cDNA, and the expression of GhWRKY74 gene in different tissues was determined after reverse transcription. (All kits were purchased from Beijing Tiangen)

[0120] 3.2 Experimental results:

[0121] By extracting RNA from different tissues of roots, stems, and leaves of cotton susceptible varieties, it was confirmed by RT-PCR that it was expressed in ro...

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Abstract

The invention discloses a gossypium hirsutum GhWRKY74 protein, and a coding gene and application thereof. The cDNA full-length nucleotide sequence of the gene is disclosed as SEQ ID No: 1; and the amino acid sequence is disclosed as SEQ ID No: 2. The gene GhWRKY74 related to susceptibility verticillium wilt is obtained by cloning from the leaves of the susceptibility verticillium wilt cotton variety 86-1; the RT-PCR proves that the gene is synergistically expressed in roots, stems and leaves under the stress of verticillium wilt bacteria; and the VIGS silences the expression of the gene, whichproves that the gene can enhance the verticillium dahliae resistance of the susceptibility verticillium wilt gossypium hirsutum variety, thereby enhancing the verticillium wilt resistance of the gossypium hirsutum and providing theoretical basis for genetic improvement of the susceptibility verticillium wilt gossypium hirsutum variety.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an upland cotton GhWRKY74 protein, its coding gene and its application. Background technique [0002] Cotton is an important fiber crop and oil crop in the world. Cotton is grown in 24 of China's 31 provinces, with nearly 300 million people involved in its production, and 30% of the total sown area is used for cotton planting. Among them, Verticillium wilt (Verticillium wilt) seriously harms cotton yield and fiber quality in production. Because Verticillium wilt damages the vascular bundles of cotton, it is difficult to control it with chemical agents. Now the most effective way to control it is to cultivate and plant resistant disease species. In the past studies, the research on genes related to Verticillium wilt resistance in upland cotton has progressed slowly, and the main gene for Verticillium wilt resistance has not been reported yet. Due to the variability of Verticilli...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/60
CPCC07K14/415C12N15/8218C12N15/8282
Inventor 简桂良贺浪司宁
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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