Function Analysis Of GhWrky53 In Gossypium Hirsutum Resistance To Verticillium Wilt

【Object】Cotton(Gossypium hirsutum L.)is an important fiber crop and textile material in the world.However,during the growth process,cotton is susceptible to infection by Verticillium wilt,which leads to a decline in quality and yield.Verticillium wilt is a soil-borne disease,which has the characteristics of wide distribution,heavy harm,and strong infection,it is extremely difficult to cure.At present,the most effective measure is to cultivate cotton varieties with high resistance to Verticillium wilt.Therefore,studying the molecular mechanism of cotton resistance to Verticillium wilt and mining cotton disease resistance genes are significance for enhancing cotton disease resistance and improving fiber quality.【Methods】In this study,the gene GhWRKY53,which was up-regulated by Verticillium dahliae,was selected by the research group in the early stage as the research object.The Bioinformatics methods were used to identify and analyze the WRKY53 genes in four cotton species.The expression pattern of GhWRKY53 gene was analyzed by qRT-PCR,Verticillium dahliae infection treatment,hormone spray treatment and other methods.Using virus-induced gene silencing technology(VIGS),qRT-PCR and other physiological and biochemical methods to clarify the function of GhWRKY53 in cotton resistance to Verticillium wilt.【Results and conclusions】1.There are 3,4,2,and 2 WRKY53 genes were identified in Gossypium hirsutum,Gossypium arboretum,Gossypium barbadense and Gossypium raimondii respectively.The WRKY53 gene in the four cotton species is highly conserved.They all contain the WRKY domain and a C2 HC zinc finger domain,which belongs to type III WRKY transcription factor.Analysis of gene structure and motifs showed that the WRKY53 gene of four cotton species consisted of 3 exons and 2 introns,containing 9-10 motifs.Evolutionary analysis showed that the evolution of WRKY53 gene tends to be stable.The prediction results of cis-response elements show that the promoter region of WRKY53 gene in cotton contains cis-response elements for hormones such as salicylic acid(SA)and methyl jasmonate(Me JA).2.The results of qRT-PCR showed that in two cotton varieties with different resistances,the expression level of GhWRKY53 gene in roots and stems was higher,and the expression level in leaves was lower.After inoculation with Verticillium dahliae,in the susceptible variety Xinluzao 7,the expression level of GhWRKY53 was significantly up-regulated at 24 h;in the disease-resistant variety Zhongzhimian 2,the expression level of GhWRKY53 was significantly up-regulated at 12 h.After SA treatment,the expression level of GhWRKY53 in Xinluzao 7 was significantly up-regulated at 24 h,and the expression level of GhWRKY53 in Zhongzhimian 2 was significantly up-regulated at 48 h;after Me JA treatment,in two varieties with different resistance.Among them,the expression level of GhWRKY53 was significantly up-regulated at 48 h.It showed that the expression level of GhWRKY53 gene responds to the induction of Verticillium dahliae,SA and Me JA.3.The expression of GhWRKY53 gene in the silenced plant was significantly lower than that of the control plant,indicating that the tobacco rattle virus(TRV)system successfully suppressed the expression of cotton GhWRKY53.After inoculation with Verticillium dahliae,The GhWRKY53 gene-silenced plants were more yellowish and wilted and more fallen leaves than control plants.The disease index of silenced plants was significantly higher than that of control plants at 14 and 21 days;the degree of browning of the stems of the silenced plants was more serious than that of the control plants;the fungal biomass of the silenced plants was significantly higher than that of the control plants.Our results indicated that inhibiting the expression of GhWRKY53 reduced cotton resistance to Verticillium dahliae.4.Compared with the control plants,in Xinluzao 7,when the GhWRKY53 gene-silenced plants were inoculated with Verticillium dahliae for 0 h,the SA content and the expression levels of pathway genes were significantly reduced;48 hours after inoculation with Verticillium dahliae,JA content and its pathway gene expression levels increased significantly.In Zhongzhimian 2,when GhWRKY53 gene-silenced plants were inoculated with Verticillium dahliae for 48 h,the SA content and its pathway gene expression levels were significantly reduced,and the JA content and its pathway gene expression levels were significantly increased.The results showed that the silencing of the GhWRKY53 gene inhibited the SA pathway and activated the JA pathway,thereby reducing the plant’s resistance to Verticillium wilt.