Molecular Mechanism Of Cotton Resistance To Verticillium Wilt Regulated By GhCML41 Protein

Verticillium wilt is one of the most devastating diseases in cotton production,which poses a great threat to our cotton industry.At present,the most economic and effective method of prevention and control this disease is breeding disease-resistant varieties.The results of the phosphorylated proteomics analysis in the early study showed that GhCML41protein was significantly phosphorylated on the resistant and susceptible cotton after inoculated the wildtype Verticillium dahliae strain Vd080.The level of phosphorylation on the disease-resistant varieties?Zhongzhimian2?was 1.5 times higher than the susceptible varieties?Jimian11?.In order to further investigate the role of GhCML41 protein on the molecular mechanism of cotton resistant Verticillium wilt.This study used resistant cotton variety Zhongzhimian2 and susceptible cotton variety Jimian11 and strain Vd080 as researchful materials by means of biology,molecular biology and cellular biology.Our research in order to provide the theoretical basis for breeding Verticillium wilt resistant cotton varieties using genetic engineering technology.The main results are as follows:1.The cotton GhCML41 gene was cloned,with a total length of 606 bp encoded 201amino acids.The protein structure was predicted that contains 4 EF-hands structures,?-helix,extended chain and random curl.Sequence homologous alignment showed that GhCML41was closely related to AtCML41 in Arabidopsis thaliana.2.Real-time quantitative analysis of GhCML41 gene showed that the gene expression of cotton Zhongzhimian2 inoculated strain Vd080 was 26.85 times of that of non-inoculated cotton after 3-hours-inoculation.After inoculated with Vd080 for 6 h,the gene expression level in the resistant variety was 7.23 times as high as that in the susceptible variety.3.Vd080 strain was inoculated,after GhCML41 gene was silenced by VIGS technology.The disease rate of control and gene silenced plants was 59.01%and 87.5%,the disease index was 22.73 and 45.31,respectively.At the same time,it was found that the lignin synthesis ability was weakened,callose deposition ability was decreased,salicylic acid content was decreased,jasmonic acid content was increased,and the expressions of defense genes PAL,POD,PPO,C4H1,PR1 and NOA1 were down-regulated in the silenced plant.4.Compared with wild-type Arabidopsis thaliana,transgenic GhCML41 gene has a lower incidence,and its specific disease resistance mechanism needs further study.Subcellular localization results showed that GhCML41 was localized in the nucleus and cell membrane.5.The results of KEGG signaling pathway analysis showed that GhCML41,CDPK28and RBOHD enhanced the plant defense response by regulating the content of NO and H₂O₂.After silencing the three genes,Vd080 strain was inoculated,and the disease index of the silenced plants was significantly higher than that of the control.At the same time,the content of NO in GhCML41 gene-silenced plants was significantly lower than that in the control plant,and the largest descended range was 12 h after inoculation reaching 71.98%.The content of NO in CDPK28 and RBOHD silenced plants was higher than that in control plants.In addition,the content of H₂O₂ in GhCML41 gene-silenced plants was significantly increased compared with that in the control plant,while the content of H₂O₂ in CDPK28 and RBOHD gene-silenced plants was significantly decreased compared with that in the control plant.