Upland cotton GhCM2 protein as well as coding gene and application thereof

A protein and gene technology, applied to GhCM2 protein and its encoding gene and application fields, can solve the problems of high difficulty of disease-resistant varieties and lack of disease-resistant varieties, and achieve the effect of improving genetic improvement

Active Publication Date: 2021-05-28
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

However, because its disease resistance is a quantitative trait controlled by multiple genes, coupled with the complexity of its epidemic damage, it is difficult to breed disease-resistant varieties. For a long time, my country lacks disease-resistant varieties (strains), especially the The disease-resistant varieties of upland cotton with 99.9% of the area are even more scarce, which is also an important reason for the epidemic of the disease from time to time

Method used

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  • Upland cotton GhCM2 protein as well as coding gene and application thereof
  • Upland cotton GhCM2 protein as well as coding gene and application thereof
  • Upland cotton GhCM2 protein as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cloning of Example 1 Upland Cotton GhCM2 Gene

[0035] 1. Extraction of RNA

[0036] RNAprep Pure plant polyphenol polysaccharide total RNA extraction kit was used to extract RNA from cotton KV3 leaf samples.

[0037] 2. Synthesis of cDNA

[0038] 2.1 Synthesis of intermediate fragment cDNA

[0039] The cDNA of the intermediate fragment was synthesized using the FastQuant cDNA First Strand Synthesis Kit to reverse transcribe the cDNA.

[0040] 2.2 Synthesis of cDNA at the 3' end

[0041] The synthesis system of cDNA at the 3' end is as follows:

[0042] Table 1

[0043]

[0044] 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.

[0045] 3.3. Synthesis of cDNA at the 5' end

[0046] The cDNA synthesis system of 5'RACE is as follows:

[0047] first step:

[0048] Table 2

[0049]

[0050] Mix well, centrifuge briefly and plac...

Embodiment 2

[0117] Example 2 Effect of Silencing Verticillium Wilt-resistant Upland Cotton Variety KV3 on Verticillium Wilt Disease Resistance under the Stress of Verticillium dahliae

[0118] (1) Material

[0119] The wild-type Verticillium wilt-resistant upland cotton variety Zhiqian KV3 and the Verticillium wilt-resistant upland cotton variety Zhiqian KV3 with GhCM2 gene silenced were used as experimental materials.

[0120] (2) Experimental grouping

[0121] Control group: Zhongzhi Cotton KV3 (provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences);

[0122] Experimental group: GhCM2 gene-silenced GhCM2 gene resistant to Verticillium dahliae cotton planting KV3, transforming empty vector (CLCrV-00) in planting cotton KV3 (refer to Zhang Huachong's master's thesis "VIGS technology analysis of genes related to Verticillium wilt resistance in upland cotton Preliminary identification of GHB2 function", Graduate School of Chinese Academy of Agricultural S...

Embodiment 3

[0131] Example 3 Verticillium dahliae Transgenic Arabidopsis Verticillium Wilt Resistance Detection under Verticillium dahliae Stress

[0132] (1) Material

[0133] The second-generation Arabidopsis thaliana and wild-type Arabidopsis transgenic with the GhCM2 gene were used as experimental materials (acquisition method Zhang Huachong's master's thesis "VIGS technology analyzes the gene related to Verticillium wilt resistance in upland cotton, namely the preliminary identification of GHB2 function", Graduate School of Chinese Academy of Agricultural Sciences , 2016; and Ren Yuhong's master's thesis "Cotton Verticillium wilt control technology and research on the function of two resistance-related genes", Graduate School of Chinese Academy of Agricultural Sciences, 2018).

[0134] (2) Cultivation substrate

[0135] Medium: 1 / 2MS medium; 1 / 2MS+50mg / L Kanamycin medium

[0136] Cultivation substrate: vermiculite + 1 / 4MS nutrient solution

[0137] (3) Experimental Design and Dete...

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Abstract

The invention discloses upland cotton GhCM2 protein as well as a coding gene and application thereof, and belongs to the technical field of molecular biology. The invention specifically discloses a protein comprising: 1) a protein composed of an amino acid sequence as shown in SEQ ID NO: 2, and 2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence as shown in the SEQ ID NO: 2, is related to plant verticillium wilt resistance and is derived from the SEQ ID NO: 2. The cotton GhCM2 gene is cloned, the gene is a typical DCD superprotein family gene and mediates programmed death of cells, and gene silencing and overexpression analysis prove that the verticillium wilt resistance can be improved through synergistic expression of the gene in leaves and roots of highly verticillium wilt resistant varieties, so that the verticillium wilt resistance is improved, and great significance is achieved for genetic improvement of verticillium wilt resistance of high-yield susceptible cotton or other crop varieties.

Description

technical field [0001] The invention relates to the technical field of molecular biology, relates to upland cotton GhCM2 protein and its encoding gene and application, in particular to a GhCM2 protein related to upland cotton Verticillium wilt resistance, its encoding gene and application. Background technique [0002] Cotton is an important economic crop and occupies an important position in the national economy. It is not only an important raw material for textile, chemical, pharmaceutical and national defense industries, but also an important export commodity. Among them, upland cotton (Gossypium. hirsutum) is the main planting species, accounting for more than 99% of the cotton area in my country. Verticillium wilt is one of the most important diseases in cotton production. It was introduced into my country in the 1930s, and occurred and caused damage in a few areas of my country’s cotton provinces in the early 1950s after liberation. The harm in the main cotton-producin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/82A01H5/12A01H5/06A01H6/60A01H6/20
CPCC07K14/415C12N15/8282
Inventor 简桂良卢美光杨武贺浪张华崇
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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