Cloning Of GbRvd From Gossypium Barbadense And Its Functional Analysis For Verticillium Wilt

Verticillium wilt caused by Verticillium dahliae is a soilborne vascular disease, whichis difficult to control and harmful to cotton production severely in China. So to studyresistance mechanism of Verticillium wilt is important for resistance breeding in cotton.Based on a full-length cDNA library of Gossypium barbadense Pima90-53underVerticillium dahliae stress, a novel CC-NBS-LRR gene was amplified by in silico cloningand RACE technology, it was named GbRvd. The bioinformatics analysis, differentialexpression analysis and resistance functional analysis after GbRvd silencing wereresearched. The results were as follows:1. The full-length cDNA of GbRvd was3486bp including88bp5′UTR,470bp3′UTR and an open reading frame (ORF) of2928bp, encoded a protein of975amino acids.The theoretical molecular weight and isoelectric point of GbRvd protein was112.6kD and6.51，respectively; The prediction of protein structure showed that GbRvd included57.95%α-helixes,2.36%β-sheets,30.56%coil and9.13%turn. Additionally, it containeddomains of NB-ARC, Smc, PLNOO113and PLN3210, which were assumed to beinvolved in the recognition for fungal effectors. Transmembrane domain analysis indicatedthat GbRvd contained three transmembrane domains, no signal peptide, and was located onthe membrane of chloroplast.2. The expression level of GbRvd was higher in Pima90-53than in other Gossypiumhirsutum cultivars after inoculation with V. dahliae, but no significant difference in G.hirsutum. The tissue specific expression analysis in Pima90-53after inoculation with V.dahlia indicated the expression level of GbRvd was higher in leaves than in other tissues.3. Silencing of GbRvd by VIGS enhanced cotton plants susceptibility to V. dahliae,and plants exhibited higher disease index and lower content of chlorophyll than the vectorcontrol plants at15dpi and20dpi.4. To analyze the transcriptional changes in GbRvd VIGS plants infected with V.dahliae, we monitored three genes that function in the pathways for salicylic acid (SA) andjasmonic acid (JA). After inoculation, the expression quantity of PR5was higher in GbRvdsilencing plants compared to wild type. But PR4and EDS1were no change. These results demonstrated that GbRvd was involved in salicylic acid-signal transduction pathways, butnot depend on EDS1.5. GbRvd was cloned into plant overexpression vector, and then transformed intoArabidopsis thaliana. It laid the foundation for subsequent researching the function ofGbRvd.