| Cotton is one of the most important crops in the world,and the stability of cotton production is of vital importance to country’s economic development.Cotton Verticillium wilt is the most important disease to cotton production.The main pathogen of Verticillium wilt is Verticillium dahliae.At present,the mechanism of cotton resistance to Verticillium wilt is still unclear,and Verticillium dahliae can survive in the soil for a long time,and the existing control agents are not effective.Therefore,it is necessary to dig out the genes related to the interaction between cotton and Verticillium dahliae,study the functions of related genes,and provide candidate genes for the cultivation of cotton resistance to Verticillium dahliae.In this thesis,based on the previous transcriptome sequencing analysis,three down-regulated genes Gh BZR1,Gh WRKY74,and Gh TGA7 were selected to study the mechanism of Verticillium wilt resistance,laying a theoretical foundation for the cultivation of cotton varieties resistant to Verticillium wilt.1)Real-time PCR technology was used to study the expression patterns of Gh BZR1,Gh WRKY74,and Gh TGA7 in Gossypium hirsutum L.The results showed that the anti-sense Verticillium dahliae infection of different materials,in the three genes susceptible comparative CK expression upregulated presentation,the three genes in the resistant varieties no significant difference in comparison CK expression or presentation downregulation trend.In resistant varieties,the expression of the three genes was no significant difference or trend downregulation.After treatment with exogenous plant hormones salicylic acid(SA)and jasmonic acid(JA),the expression of Gh TGA7 and Gh BZR1 can be induced by exogenous SA and JA,and the expression of Gh WRKY74 can be inhibited by exogenous SA and JA.2)Using virus-induced gene silencing technology,it was found that Gh BZR1,Gh WRKY74,Gh TGA7 silenced plants had enhanced disease resistance to Verticillium dahlia.In addition,the leaves of Gh BZR1,Gh WRKY74,Gh TGA7 silenced plants were stained with trypan blue,and it was found that Gh TGA7,Gh WRKY74,Gh BZR1 silenced plants had the effect of slowing down cell necrosis.3)The full length of Gh BZR1 and Gh WRKY74 genes were obtained by RACE technology.The Gh BZR1 gene had a full length of 1515 bp,3’UTR was 350 bp,5’UTR was 223 bp,ORF was 942 bp,and had a BES1＿N superfamily domain.There were two ABRE cis-acting elements in the promoter sequence that were involved in the reaction of abscisic acid and one MYB binding site was an MBSI element involved in gene regulation of flavonoid biosynthesis.The full length of Gh WRKY74 gene was1786 bp,of which 3’UTR was 514 bp,5’UTR was 291 bp,and ORF was 995 bp.Gh WRKY74 belongs to group IId WRKY member.This protein had a WRKY domain and C2H2 pattern(CX(4-5)CX(22-23)HXH)Zinc finger structure.The full length of Gh TGA7 gene was obtained by electronic cloning technology,the full length of Gh TGA7 was 1786 bp,3’UTR was 69 bp,5’UTR was348 bp,and ORF was 1074 bp.The protein had a typical b ZIP protein DNA binding domain and DOG1 protein domain.In addition,Gh BZR1,Gh WRKY74,and Gh TGA7 expression vectors were constructed by subcellular localization technology and transiently expressed in tobacco.The results showed that the three genes were all located in the nucleus,which was consistent with the characteristics of transcription factors.4)The resistance of transgenic gene Gh BZR1 Arabidopsis to Verticillium wilt was studied by overexpression technology,and the T2 generation positive plants were screened for resistance to Verticillium dahliae.The results showed that transgenic Arabidopsis overexpressing Gh BZR1 was more susceptible to Verticillium dahliae.Through the analysis of the expression of genes related to the defense pathway,it was found that the expression of At PR-1,At PR-5,At PAL1 related to the Gh BZR1 gene in Arabidopsis SA pathway was significantly up-regulated,and the expression of the related genes At PDF1.2 and At LOX2 of the JA pathway was inhibited.Therefore,the negative regulation of Gh BZR1 is involved in cotton Verticillium wilt resistance and JA-related pathways. |
