CN-121975863-A - Cloning and application of island cotton GbGELP D gene

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly provides cloning and application of a sea island cotton GbGELP D gene. The invention clones and obtains GbGELP D gene of full length 1092 bp from sea-island cotton variety Xinhai No. 7 for the first time, and the gene codes a secretion type lipase protein with signal peptide and is positioned in extracellular space. Through bioinformatics analysis, phylogenetic classification, protein structure modeling and signal peptide function verification, the gene is determined to belong to the plant GELP family. Further, by utilizing virus-induced gene silencing technology, the silencing GbGELP D can obviously enhance the resistance of cotton to verticillium wilt, and the mechanism is closely related to activating ethylene synthesis and signal passage and inducing the expression of disease-resistant related genes. The resistance gene provided by the invention enriches the gene resources of cotton verticillium wilt-resistant breeding, and has important theoretical significance and application value.

Inventors

• DUAN LISHENG
• ZHAO HAORAN
• LIU HAIYANG
• YAO JU
• ZHANG RENFU
• WANG WEI
• DING RUIFENG
• LI LIN
• YAN YIBIN
• YANG JIAXIN

Assignees

• 新疆维吾尔自治区农业科学院

Dates

Publication Date

20260505

Application Date

20260227

Claims (7)

1. 1. Use of silencing GbGELP D gene, wherein said use is any of the following: A1 Application in improving verticillium wilt resistance of island cotton; a2 The application of the composition in the preparation of products for improving the verticillium wilt resistance of island cotton; a3 Application in cultivating verticillium-resistant island cotton; a4 The application of the method in preparing the products for cultivating the verticillium-resistant island cotton; a5 Application in verticillium wilt-resistant breeding of island cotton or verticillium wilt-resistant germplasm resource improvement of island cotton; The CDS sequence of GbGELP D gene is shown in SEQ ID NO. 1.
2. 2. Use of a biological material related to silencing GbGELP D gene, wherein said use is any one of the following: A1 Application in improving verticillium wilt resistance of island cotton; a2 The application of the composition in the preparation of products for improving the verticillium wilt resistance of island cotton; a3 Application in cultivating verticillium-resistant island cotton; a4 The application of the method in preparing the products for cultivating the verticillium-resistant island cotton; a5 Application in verticillium wilt-resistant breeding of island cotton or verticillium wilt-resistant germplasm resource improvement of island cotton; the biomaterial is any one of the following B1) to B4): B1 A nucleic acid molecule that inhibits or reduces expression of the GbGELP D gene; b2 An expression cassette comprising the nucleic acid molecule of B1); B3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2); B4 A recombinant microorganism comprising B1) said nucleic acid molecule, or a recombinant microorganism comprising B2) said expression cassette, or a recombinant microorganism comprising B3) said recombinant vector, said microorganism being Agrobacterium; The CDS sequence of GbGELP D gene is shown in SEQ ID NO. 1.
3. 3. The use according to claim 2, wherein the GbGELP D gene encodes a protein having the amino acid sequence shown in SEQ ID No. 2.
4. 4. A method for cultivating verticillium wilt-resistant island cotton is characterized in that GbGELP D genes are silenced in the island cotton to obtain the island cotton with high verticillium wilt resistance, and CDS sequences of GbGELP D genes are shown as SEQ ID NO. 1.
5. 5. The method of claim 4, wherein silencing GbGELP D gene in gossypium barbadense is to reduce the expression level of GbGELP D gene using gene silencing techniques.
6. 6. The method of claim 5, wherein the reducing the expression level of GbGELP D gene using gene silencing technology is performed using a virus-induced gene silencing vector constructed by forward integration of the nucleic acid molecule shown in SEQ ID No. 3 into a tobacco embrittlement virus-based plasmid vector.
7. 7. The method of claim 5, wherein the primer pair for cloning the nucleic acid molecule set forth in SEQ ID NO. 3 is set forth in SEQ ID NO. 12 and SEQ ID NO. 13.

Description

Cloning and application of island cotton GbGELP D gene Technical Field The invention belongs to the technical field of plant genetic engineering, and relates to cloning and application of a sea island cotton GbGELP D gene. Background Xinjiang is the most important cotton planting base in China, cotton yield is over nine times of the country, the cotton industry becomes an important support for promoting the economic development of Xinjiang, strong power is injected for village plain and peasant income increasing, and the safety significance of the production of the Xinjiang cotton is ensured. The verticillium wilt is the most serious disease causing the yield reduction of the Xinjiang cotton, is called as cotton cancer, and has the advantages of high mutation speed, wide spread range and large prevention and control difficulty. The most effective prevention and control measures at present are to select disease-resistant varieties, however, the production lacks of high verticillium wilt resistance germplasm, and the traditional breeding period is long and the cost is high, so that the disease-resistant breeding work process is slow. Biological breeding has become a great strategic choice and effective means for enhancing the core competitiveness of the agricultural industry by virtue of the advantage of improving crop disease resistance rapidly, accurately and efficiently, and is also a trend of cotton verticillium wilt-resistant variety cultivation. Therefore, the method utilizes the molecular design breeding technology to precisely and directionally improve the plant characteristics by excavating the disease-resistant key genes of the cotton and analyzing the resistance regulation mechanism, thereby being the best means for cultivating the germplasm of the cotton with high verticillium resistance at present. The nature of cotton resistance to verticillium is the expression of resistance genes, which involve pathways such as lipid metabolism, secondary metabolism and hormone signal transduction, and encompass relevant resistance genes such as defensive enzymes, secondary metabolites and plant hormones. Lipid metabolism is the core of the metabolic regulation network in plants responding to stress defense systems. Lipids are not only the major components that make up the biofilm, but also signal molecules or substrates for signal molecule production. Lipid-based substances are involved in very complex regulatory processes for plant disease resistance, including several aspects, (1) formation of the stratum corneum as a first physical barrier, (2) formation of extracellular vesicles as signal molecules to establish a link in intercellular communication or acquired resistance of the system, and (3) formation of oxygenins as antibacterial or toxic compounds against invading pathogens. Lipases are metabolic bands of lipids and oxidized lipids, and lipid-related defensive reactions are mainly promoted by activating lipases, so that plant lipases play a key role in defensive reactions. Plant lipase gene GELPs is a key gene in the disease-resistant, stress-resistant, defensive metabolism regulation network. Biosynthesis of plant cutins, waxes is part of the lipid metabolic pathway. GDSL lipase is involved in plant immune responses including hormone-mediated immune responses and regulation of cell wall component synthesis. Based on the above, the study uses Xinhai No. 7 of Xinjiang island cotton variety as a test material, clones and obtains a GDSL-type esterase/lipase gene with the total length of 1092 bp, and the GDSL-type esterase/lipase gene is named GbGELP D. The method comprises the steps of utilizing a bioinformatics method to comprehensively analyze the gene structure, physicochemical properties of encoded proteins, conserved structural domains and phylogenetic relations of the genes, determining the functional execution position of the encoded proteins of the genes through protein subcellular localization and signal peptide secretion experiments, and analyzing the biological functions of the encoded proteins in the cotton verticillium wilt resisting process by means of a VIGS technology. Disclosure of Invention The invention aims to clone and identify a brand new cotton verticillium resistance negative regulation gene GbGELP D, and create a new method and a new tool for improving cotton disease resistance by inhibiting the expression of the gene based on the functional discovery of the gene, and finally serve for breeding disease resistant varieties. In order to achieve the above purpose, the present invention provides the following technical solutions: the first aspect of the invention provides GbGELP D gene cloning, which comprises the steps of taking Xinjiang sea island cotton variety Xinhai No. 7 as a test material, extracting cotton leaf total RNA according to a polysaccharide polyphenol plant total RNA extraction kit instruction book, obtaining a cDNA first strand through reverse transcription, design