Cloning And Functional Analysis Of Two Genes Related To Verticillium Wilt Resistance In Cotton

Cotton is one of the most important commercial crop for natural textile fiber and oil in China.Recently,Verticillium wilt is a destructive disease on cotton production and cause enormous losses in cotton yield and fiber quality.Screening disease related genes via molecular biology and modern genetic engineering supplies new chances to disease resistance breeding.In this study,two differentially expressed genes related to verticillium wilt resistance in cotton were screened out from the verticillium wilt induced transcriptome database constructed by the research group.Their functions in plant resistance to Verticillium wilt were verified by virus-induced gene silencing（VIGS）and transgenic methods,which provided assistance for cotton disease resistance breeding.GhVIP1 Gene was cloned from Sea Island Cotton Hai7124 by homologous cloning.According to the sequencing results,the full length of the ORF of the gene was 1014 bp and the number of amino acids the gene contained was 337,and it was a type of hydrophilic unstable protein.Based on the bioinformatics analysis,GbVIP1 protein contained conserved domain bZIP,which was a transcription factor of bZIP.The tissue expression specificity showed that the highest expression tissue of VIP1 gene existed in cotton roots.VIP1 was significantly up regulated in disease-resistant varieties after inoculation.The results showed that VIP1 gene was significantly up regulated by exogenous hormone ET,which suggested that the expression of GbVIP1 gene might be related to ET.VIGS was utilized to silence gene GbVIP1 in cotton.After treatment with Verticillium dahliae,symptoms such as yellowing and shedding of leaves showed up in the plants with gene silencing,while the untreated plants were much healthier.Through the detection of gene expression,the statistics of disease index and the experiments of pathogenic bacteria reisolation,trypan blue dyeing and root longitudinal section,it was further confirmed that plants with gene silencing were more susceptible to disease than the untreated ones,which indicated that gene GbVIP1 played an important role in the process of Cotton Verticillium wilt resistance.Transgenic tobaccos with gene GbVIP1 were cultivated via tissue culture.It was found that transgenic tobacco plants were more resistant to Verticillium wilt than wild plants,which was consistent with the results of VIGS.The expression of disease resistance related genes in transgenic tobacco plants was detected.According to the results,gene PR1,PR1-like and HSP70 was significantly up regulated after induced by Verticillium dahliae.Therefore,it was speculated that the up-regulated expression of gene PR1,PR1-like and HSP70 enhanced the resistance of tobacco plants to Verticillium wilt.GhNOT2b Gene was cloned from Sea Island Cotton Hai7124.According to the sequencing results,the full length of the ORF of the gene was 1839 bp and the number of amino acids the gene contained was 612,and it was a type of hydrophilic unstable protein.There are 73 number of potential phosphorylation sites.Based on the structural analysis,protein GbNOT2 b contained conserved domain NOT,which belonged to type NOT2₃₅.It was found that the gene was highly expressed in disease-resistant varieties induced by Verticillium wilt using qRT-PCR,and significant expression of thegene was also induced in cotton leaves treated with exogenous hormone SA and ET.The GbNOT2 b gene in cotton was silenced by technology VIGS,which reduced the resistance of cotton plants to Verticillium wilt,and provided a reference for the functional verification of subsequent transgenic plants.