Promoter Cloning And Functional Analysis Of Fdsarium Wilt Resistance Genes GbCHI And GbDFR In Gossypium Barbadense L

Fusarium wilt seriously damages the yield and fiber quality of Gossypium barbadense L.To study the regulation mechanism of disease resistance-related genes will provide an important theoretical basis for the subsequent analysis of the molecular mechanism of island cotton disease resistance and the use of genetic engineering methods to improve the resistance of island cotton to wilt disease.In the early stage of this research group,two genes related to Fusarium wilt resistance,GbCHI and GbDFR,were screened based on the sequencing data of island cotton transcriptome.Through qRT-PCR analysis,these genes were differentially expressed between disease resistant and susceptible materials of island cotton.In order to elucidate the resistance expression mechanism of these two genes in different resistant varieties of island cotton from the aspect of transcriptional regulation.In this study,firstly,the expression analysis of GbCHI gene and GbDFR gene under the treatment of methyl jasmonate(MeJA)and salicylic acid(SA)on the disease resistant variety "06-146" of island cotton and the susceptible variety "Xinhai 14";Then cloned the promoter sequences of the GbCHI gene and GbDFR gene of these varieties,and predicted and compared the differences in their promoters;Finally,using the Agrobacterium tumefaciens-mediated tobacco transient expression system,the induction activities of the GbCHI gene promoters from these varieties under the treatment of methyl jasmonate,salicylic acid and Fusarium wilt were analyzed.The overall results achieved are as follows:1.Under the treatment of methyl jasmonate,the expression levels of GbCHI gene and GbDFR gene in disease-resistant varieties were obviously induced at 8 h.Except for 4 h,the expression of GbCHI gene in disease-resistant varieties was higher than that of susceptible varieties in all treatment periods,and the expression of GbDFR gene in disease-resistant varieties was higher than that of susceptible varieties in all treatment periods.While in each period of salicylic acid treatment,the expression levels of GbCHI and GbDFR genes in the disease resistant and susceptible materials of island cotton were down-regulated.2.Clone the GbCHI gene promoter sequence of the the disease resistant and susceptible varieties of island cotton,and named them pRGbCHI and pSGbCHI respectively.Sequence comparison analysis found that the homology between the two sequences were up to 99.56%,and there are 8 single nucleotide sites and 1 base deletion and insertion;Transcription start site prediction found that the TSS sites of both sequences are located-49 bp upstream of the start codon ATG,and the bases are both C;Cis-acting element prediction analysis that both sequences contained pathogenic bacteria and hormone response elements related to the signal pathways that mediated plant disease resistance.The positions were basically the same except for the difference of 1 bp between individual elements.In terms of the number of elements,the pRGbCHI promoter had two fewer methyl jasmonate response elements TGACG-motif and CGTCA-motif than the pSGbCHI promoter,one less light-responsive element Box4,two fewer core elements TATA-box,one less the unknown element as-1,one more promoter enhancer element CAAT-box.The predicted transcription factors that can be combined are MYB,BBR-BPC and DOF,However,the Dof transcription factor family gene predicted to bind to the pRGbCHI promoter is one less than the pSGbCHI promoter,and the binding position differs by 1 bp.3.Clone the GbDFR gene promoter sequence of the disease resistant and susceptible varieties of island cotton,named pRGbDFR and pSGbDFR respectively.Sequence comparison analysis found that the homology between the two sequences were up to 99.67%,and there were 7 single nucleotide difference;Transcription start site prediction found that both TSS sites are located-119 bp upstream of the start codon ATG,and bases are both C;Cis-acting element prediction analysis found that both sequences contained defense and stress and hormone response elements that mediated plant disease resistance signaling pathways.The main set of TATA-box on the pRGbDFR promoter at the element position was longer than the pSGbDFR promoter.In the number of elements,the pRGbDFR promoter sequence was one light response element chs-CMA1a less than the pSGbDFR promoter sequence,one more light response element TCT-motif,and three promoter enhancer elements CAAT-box.There were five types of transcription factors that were predicted to be bound:SHN,ERF,DOF,ABR,and ABI,and the number and position of binding sites were the same.4.Using methyl jasmonate,salicylic acid,and fusarium wilt,respectively,to treat tobacco leaves transiently transformed with the pRGbCHI promoter and pSGbCHI promoter for 24 h,then performed GUS histochemical staining and Real-time PCR expression detection to determine the two promoter activity.The results showed that the pRGbCHI and pSGbCHI promoters were both active;the pRGbCHI promoter was significantly increased in the expression level of the GUS gene after being induced by methyl jasmonate,salicylic acid and fusarium wilt;The pSGbCHI promoter was induced by methyl jasmonate,salicylic acid and Fusarium wilt to drive the expression level of GUS gene to decrease;After treatment with methyl jasmonate,the expression level of GUS gene driven by pRGbCHI promoter was 3.93 times that of pSGbCHI promoter;After salicylic acid treatment,the expression level of GUS gene driven by pRGbCHI promoter was 2.09 times that of pSGbCHI promoter;After treatment with Fusarium wilt,the expression level of GUS gene driven by pRGbCHI promoter was 2.41 times that of pSGbCHI promoter.