Screening And Cloning Resistance Genes To Cotton Verticillium Wilt Based On The Transcriptome Sequencing

Cotton verticillium wilt seriously affects the yield and quality of cotton, hindering the development of cotton production in China. With the rapid development of molecular biology and genetic engineering, useing the biotechnology to research the resistance mechanism of verticillium wilt and cloning resistance genes related to verticillium wilt has become a research hotspot. This research used the transcriptome database of cotton induced by Verticillium dahliae, combined with the R genes identified in cotton D genome. Candidate resistance genes associated with cotton verticillium wilt were selected. Through the fluorescence quantitative PCR analysis the express trend of some candidate resistance genes after induced by Verticillium dahliae. Two genes were selected from the candidate resistance genes, GbDRP66319 and GbHIR42734 to be cloned and to bioinformatics analysis. finally function was verified preliminary by VIGS technique. The results were as follows:1. 64 R genes containing NBS-LRR domains were found at 800 Kb ranged both ends of molecular markers related to verticillium wilt. Through R genes as query sequnce blast with the transcriptome database, highly homologous genes were obtained. 24 Unigenes significantly different expression were choosed for fluorescent quantitative PCR analysis. Express trends of them after induced by Verticillium dahliae were reseached. The result shows that these genes presenting up-regulated expression after induced by Verticillium dahliae, but different gene showing different expression patterns. Combined the result of the expression, two cases were existed. One kind was up-regulated expression after induced 4h by Verticillium dahliae, namely transient basal resistance. One kind was up-regulated expression after induced 12 h by Verticillium dahlia, namely lagging specific resistance.2. In this paper, through homology cloning, the CDS of GbDRP66319 and GbHIR42734 were cloned, sequencing result showed that the length of GbDRP66319 CDS was 879 bp and coding 292 aa. Analysis of the trans-membrane domain by TMHMM software showed that this protein didn’t have trans-membrane domain. GbDRP66319 blast with A and D genome database, 29 homologous genes were obtained. According to analysis the conservative domain structure by SMART software, some motifs and conservative structure domains, such as P-loop, Kinase2, Kinase3, GLPL, MHDL, LRR_BAC, LRR_CC, LRR_TYP were found.3. The length of GbHIR42734 CDS was 855 bp and coding 284 aa. Analysis of the trans-membrane domain by TMHMM software showed that this protein didn’t have trans-membrane domain. GbHIR42734 blast with A and D genome database, 6 homologous genes were obtained. These kind proteins contained the conservative structure of the protein family which were induced by hypersensitive reaction, such as SPFH(Stomatins, Prohibitin, Flo-tillins, Hflk/C) and PHB(Prohibitin homologues).4. This study analysized the tissue specificity of GbDRP66319 and GbHIR42734. The result showed that the express level of GbDRP66319 in the hypocotyl and cotyledon of disease-resistant variety induced 12 h by Verticillium dahliae significantly higher than in susceptible variety, and the express level in the hypocotyl was the highest in the disease-resistant variety. So that high expression of the GbDRP66319 in hypocotyl probably might play an important role in the disease resistance of cotton; The express level of GbHIR42734 in the root of disease-resistant variety induced 12 h by Verticillium dahliae significantly higher than in susceptible variety, and the express level in the root was the highest in the disease-resistant variety. So that high expression of the GbHIR42734 in the root might play an important role in the disease resistance of cotton.5. In this study, GbDRP66319 and GbHIR42734 VIGS silence vectors were constructed and transformed into cotton plants mediated by Agrobacterium GV3101. Then the cotton plants were inoculated by Vd080, and the results showed that the incidence of diseases of VIGS plants more severely than the control plant. This result preliminary verified GbDRP66319 and GbHIR42734 play a role in resistance to verticillium wilt of cotton.