Study On Cloning And Mechanism Of Cotton GhWRKY7 Regulating Plant Defense Against Verticillium Wilt

Cotton is an important cash crop,which provides not only natural fiber,but also edible oil and protein feed.However,its production is seriously threatened by cotton Verticillium wilt,which is commonly known as"cotton cancer".Verticillium dahliae is the main pathogen of Cotton Verticillium wilt.It is a soil borne vascular semi parasitic fungus.Due to the characteristics of complex physiological races,long dormancy and survival time in soil and parasitic in plant vascular bundles,the control methods of V.dahliae are limited,and the control effect of conventional technology is not ideal.Plants have formed a series of complex defense mechanisms in the long-term competition and cooperative progress with pathogen of verticillium wilt.As one of the largest transcription factor families in plants,WRKY transcription factors play an important role in the regulation of plant defense responses.At present,breeding disease-resistant varieties is considered to be the most suitable method to control verticillium wilt,cloning and identification of disease-resistant genes have become the main means of disease-resistant breeding.Based on the eight disease resistance-related WRKY genes of cotton selected in the early stage of the laboratory,and the cotton transcription factor WRKY7 was used as the research object,the GhWRKY7 gene was cloned from the upland cotton"Zhongmian Institute No.35",and the gene function and disease resistance were preliminarily studied.Using bioinformatics,genetics and biochemistry,the induced response and resistance mechanism of GhWRKY7 to V.dahliae are revealed,which provides theoretical support for cotton disease resistance breeding,and provides candidate genes.The main results achieved are as follows:1.Using MEGA,DNAMAN and other related software and GFP reporting analysis system,GhWRKY7 was used to conduct evolutionary bioinformatics analysis and subcellular localization.The results showed that GhWRKY7 and Arabidopsis thaliana WKRY7 were highly homologous and belonged to Group II,the number of different WRKY domains and the structure type of zinc finger were suggestively involved in regulating plant disease resistance responses,the GhWRKY7 protein belonged to a hydrophilic protein with multiple phosphorylation sites and was localized in the plant nucleus,and the induced expression analysis of GhWRKY7 was performed by timed quantitative PCR（q PCR）method.The results showed that GhWRKY7 was expressed in roots,stems and leaf organs,and GhWRKY7 gene was induced by V.dahliae,Salicylic Acid（SA）and Jasmonic Acid（JA）,suggesting that its disease resistance may involve SA and JA anti-disease hormone signaling pathways.2.GhWRKY7 silencing plants were cultured by virus-induced gene silencing（VIGS）,and plants with ideal silencing effect were selected to inoculate V.dahliae.The results showed that the resistance of cotton plants with the silent GhWRKY7 gene to V.dahliae was reduced,indicating that GhWRKY7 was a transcription factor gene that positively regulated cotton resistance to Verticillium wilt.The expression levels of SA and JA hormone-related signaling pathway genes GhPR3,GhPR4 and GhPDF1.2 in the silent GhWRKY7 gene plants were significantly downregulated compared with the control plants.It shows that the GhWRKY7 gene can regulate the expression of SA and JA disease resistance signaling pathway genes,thereby improving the disease resistance of plants.3,qPCR detected the cotton homologous gene GhCYP71B36 of the antitoxin deficiency gene PAD3 that drives the synthesis of phytoprotein Camalexin in Arabidopsis thaliana,combined with the results of previous studies,we speculate that GhWRKY7 may directly regulate the transcriptional expression of GhCYP71B36 to improve the disease resistance of plants.Using GUS reporting technology and Tobacco Ben’s for the transient expression system,the results showed that GhWRKY7 can directly activate the transcriptional expression of GhCYP71B36 and improve the disease resistance of plants.In addition,the GhWRKY7 protein interaction factor was analyzed using yeast double hybridization system（Y₂H）detection and luciferase complementary experiment（LUC）.The results showed that the GhWRKY7 and GhMPK6 proteins interacted in vivo and in vivo.In summary,GhWRKY7,as a positive regulatory transcription factor,participates in cotton resistance to verticillium wilt through SA and JA signaling pathways.At the same time,GhWRKY7 and GhMPK6 proteins interact in vivo and in plants,and may be accompanied by phosphorylation modification,transcription activates the expression of the downstream disease-resistant gene GhCYP71B36,promotes the synthesis of phytapatin Camalexin,and thus regulates cotton resistance to V.dahliae.