阅读说明：本技术 棉花抗黄萎病相关基因GhCML41的应用 (Application of cotton verticillium wilt resistance-related gene GhCML41 ) 是由 冯鸿杰 赵沛 朱荷琴 赵丽红 冯自力 魏锋 师勇强 于 2019-11-05 设计创作，主要内容包括：本发明属于农业生物技术领域,具体涉及棉花抗黄萎病相关基因GhCML41的应用。棉花抗黄萎病相关基因GhCML41,其核苷酸序列如SEQ ID No.1所示,其编码的氨基酸序列如SEQ ID No.2所示。本发明的基因GhCML41的表达与棉花抗黄萎病呈正相关,可通过基因工程用于选育棉花抗病品种。(The invention belongs to the technical field of agricultural biology, and particularly relates to application of a cotton verticillium wilt resistance related gene GhCML 41. The nucleotide sequence of the cotton verticillium wilt resistance related gene GhCML41 is shown in SEQ ID No.1, and the coded amino acid sequence is shown in SEQ ID No. 2. The expression of the gene GhCML41 of the invention is positively correlated with the verticillium wilt resistance of cotton, and can be used for breeding cotton disease-resistant varieties through genetic engineering.)

1. Application of a cotton verticillium wilt resistance-related gene GhCML41, wherein the nucleotide sequence of the gene GhCML41 is shown as SEQ ID NO. 1.

2. The application of a cotton verticillium wilt resistance related gene GhCML41 in preventing and treating cotton verticillium wilt is disclosed, wherein the nucleotide sequence of the gene GhCML41 is shown as SEQ ID NO. 1.

3. The application of a cotton verticillium wilt resistance related gene GhCML41 in breeding cotton verticillium wilt resistance varieties, wherein the nucleotide sequence of the gene GhCML41 is shown as SEQ ID No. 1.

4. A method for improving the disease resistance of cotton verticillium wilt, which is characterized by comprising the step of over-expressing a cotton verticillium wilt resistance-related gene GhCML41 in cotton, wherein the nucleotide sequence of the gene GhCML41 is shown as SEQ ID NO. 1.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a cotton verticillium wilt resistance related gene GhCML 41.

Background

Cotton is an important economic crop and textile raw material in China, and has important significance for the development of the civilian and the country, however, the cotton verticillium wilt seriously harms the healthy development of the cotton industry in China. The verticillium wilt of cotton is used as a soil-borne disease, no ideal prevention and treatment measures exist, effective disease-resistant genes are screened, and the improvement of cotton varieties by means of genetic engineering is an effective means for preventing and treating the verticillium wilt of cotton.

Pathogenic bacteria and plant infection and infection resistance mechanisms are always research hotspots, and PTI and ETI disease resistance mechanisms between plants and pathogenic substances become key points for researching disease resistance breeding. Under the stress of pathogens, a series of signal transduction is activated in plants to stimulate the resistance response of the plants to resist the infection of pathogenic bacteria. Calcium is an indispensable second messenger in a plant signal network, and plant calcium sensors are diverse and are mutually connected among various signal transduction pathways to form a strict signal regulation network. Calcium signals are important signal transmission molecules and play a vital role in physiological and biochemical processes of plants. After pathogenic bacteria invade plant cells, as one of early signals of plant disease resistance response, calcium ions can flow into the plant, and when the plant faces adverse circumstances, the calcium signals can transmit extracellular signals to the inside of the plant through the change of the calcium ion concentration, so that the plant can make corresponding physiological response to the stress.

The plant calmodulin protein CaM is evolutionarily conserved eukaryotic Ca²⁺A sensor which binds calcium ions to its four EF-hand motifs. Calmodulin is an acidic protein, its Ca²⁺The binding structure resembles a "dumbbell" shape in which two spherical domains, each containing a pair of EF-hand, are connected by a flexible central helical region. CaM binds Ca with positive cooperativity²⁺Inducing a conformational change, exposing a hydrophobic region that can interact with a downstream protein of interest. The CMLs gene participates in a plurality of signal paths related to plant development and stress induction,in particular, in the face of excessive changes in temperature, moisture and soil quality in CMLs, the environmental factors perceived by plant cells are passed through Ca²⁺Ion, cyclic nucleotide monophosphate, active oxygen and other small molecule transmission, promote physiological or morphological change, and reach the optimal state of plant growth and productivity under adverse conditions. Interaction of CML18 with AtNHX1 relies on pH to lower its Na⁺/H⁺Exchange activity. Therefore, vacuolar acidification under salt stress can block the interaction between CML18 and AtNHX1 and promote Na under salt stress⁺And the damage to cells is reduced. CML9 seedlings are highly sensitive to ABA, the induction level of salt to CML9 is obviously reduced in ABA insensitive mutants and ABA synthesis deletion mutants, and CML9 is presumed to be an ABA dependent salinity tolerance negative regulator. Under drought conditions, the transgenic plant over expressing the gene of the rice OsCML4 gene has obviously improved growth performance and higher survival rate compared with a wild type, and the activity of Reactive Oxygen Species (ROS) and Catalase (CAT) and the concentration of proline are obviously increased in the transgenic plant.

Disclosure of Invention

The invention aims to provide application of a cotton verticillium wilt resistance related gene GhCML 41.

It is still another object of the present invention to provide a method for improving the disease resistance of cotton verticillium wilt.

The nucleotide sequence of the gene GhCML41 according to the embodiment of the invention is shown in SEQ ID NO. 1:

ATGGCAACTGCTAAAGTTTCCAAGGCTTCCAAATGGTTCTCTAACAAGAGCCTTAGGTTAAGCCTTCAACGTCGTGGATCAAAGTCTAGATCAACATCCTTAAGCTCTTCTCCTGGACCATCATCAACACCAATATCTCCTCGCACAATTCCTAATATGAGAACCAAAAGGCCTGAAGAAGACGAGATGAAGCAAGTCTTTGGCTATTTCGACGGGGATGGTGATGGCAAAATCTCTGCTCTTGAACTTAGGGCGTACTTTGGGTCCATCGGGGAGTACATGTCACATGAAGATGCTCAAGGGGTGATCAACGATCTTGACTCGGACGGGGATAGCATGCTGGACTATCAGGATTTCTTGAAGCTAATGAAAAGAGAACCCAGCAAGGATGATGAGGGAGATGATTATGATGATCTGAAGAAAGCGTTTGAGATGTTTGAACTGGAGAAAGGTTCGGGGTGCATAACACCCAAAGGGCTGCAAAGGATGTTGAATCGACTTGGGGATGCAAAATCATATGATGAATGCGTTGCCATGATTCGGGTATATGATATCGATGGTAATGGGGTGCTTGATTTTCATGAGTTCCATCAAATGATGGCTTAA

the protein GhPML 41 belongs to calmodulin-like protein, and the amino acid sequence is shown as SEQ ID No. 2:

the application of the gene GhCML41 in preventing and treating cotton verticillium wilt can be used for improving the disease resistance of resistant and susceptible cotton varieties to verticillium wilt.

The invention also provides a method for improving the disease resistance of cotton verticillium wilt, which comprises the step of over-expressing the gene GhCML41 in cotton varieties.

The invention has the beneficial effects that:

the invention constructs a silencing vector of the gene GhML 41, and inhibits the expression of the GhML 41 in cotton by utilizing a virus-mediated gene silencing technology. After the gene silencing plant is inoculated with the cotton verticillium wilt pathogenic bacterium Vd080 spore suspension, the silencing plant shows more susceptible diseases, and the mechanism research shows that the synthesis of xylem and callose in the silencing plant is reduced, the NO content is reduced, the SA content is reduced, the JA content is increased, and the down regulation of partial defense genes is realized. Transgenic Arabidopsis with the GhPML 41 gene shows stronger verticillium wilt resistance. Therefore, the gene GhCML41 of the invention is positively correlated with cotton verticillium wilt resistance, and the gene GhCML41 can be applied to cotton breeding for disease resistance.

Drawings

FIG. 1 shows the expression of the gene GhCML41 under pathogenic stress in resistant/susceptible varieties;

FIG. 2 shows the albinism and morbidity of cotton leaves after gene silencing;

FIG. 3 shows the accumulation of xylem in plants following gene silencing;

FIG. 4 shows the accumulation of callose in plant leaves after gene silencing;

FIG. 5 shows leaf salicylic acid accumulation in plants after gene silencing;

FIG. 6 shows the accumulation of jasmonic acid in the leaves of plants after gene silencing;

FIG. 7 shows leaf NO accumulation in plants following gene silencing;

FIG. 8 shows the relative expression of the defense genes in plants after pathogen treatment;

FIG. 9 shows a screen of GhCML41 transgenic Arabidopsis;

FIG. 10 shows the identification of disease resistance of transgenic Arabidopsis with GhCML41 gene.

Detailed Description