Analysis Function Of Cotton Resistance Genes By Virus-Induced Gene Silencing(VIGS)

Cotton (Gossypium spp.) serves as a economically important crop. However, cotton production is hindered by various biotic and abiotic stresses. The genetic and molecular mechanisms mediating cotton stress responses remain poorly understood. By using RNA-seq and VIGS approach, this studies uncovered that DPC regulated cotton growth via GhCPS and GhKS which were the possible functional sites; GhOST1and GhMPK6could phosphorylate each other to regulate ABA-dependent signaling pathway positively; GhBAK1play positive role in Verticillium wilt resistance and cell death; Cotton VIGS cDNA library was constructed to dissection genes.1. GhCPS and GhKS genes were cloned by homology cloning strategy. The genetic Function of those two was claimed by VIGS. The full length of GhCPS including two conserved domains DXDD and SAYDTAW was2466bp, which had50%amino acid similarity with other species CPS by homology analysis. Phylogenetic analysis indicated GhCPS and PtCPS belonged to the same clade; the full length of GhKS including two conserved domains DDXXD and YDTAWVA was2343bp, which had50%amino acid similarity with other species KS by homology analysis. Phylogenetic analysis indicated GhKS and CsKS belonged to the same clade; DPC treatment could decrease the transcriptional level of GhCPS and GhKS. By loss-of-function analysis, our studies showed that the elongation of cotton internode was inhibited by VIGS-GhCPS or VIGS-GhKS. The inhibition could compromise by GA3treatment.2. This study analysized the transcripton change of cotton upon drought treatment with RNA-seq approach.102and169genes up-regulated by2days or4days drought stress were identified.25genes up-regulated by both2days and4days drought stress; Part of those up-regulated genes were confirmed by semi-quantitative RT-PCR.3. Two genes GhOSTl and GhWRKY30involving in drought tolerance were identified by VIGS. Cotton seedlings of VIGS-GhOSTl or VIGS-GhWRKY30were sensitive to drought. Water lost in cotton leaves of VIGS-GhOST1or VIGS-GhWRKY30were significantly increased; and relative water content of leaves were significantly decreased; cotton VIGS-GhWRKY30were sensitive to PEG but not NaCl; cotton VIGS-GhOST1were sensitive to both treatment.4. This study identified the interaction of two protein kinase GhOST1and GhMPK6by in vitro assay. Co-IP and Pull down assay indicated that ABA could induce the interaction between GhOST1and GhMPK6; In vitro kinase assay indicated that there is a phosphorylation event happened between GhOST1and GhMPK6.5. This study analysized the function of GhBAKl by homology blastn and VIGS. Two BAK1 orthologs were identified in the cotton genome; this study indicated GhBAK1is required for CA4002resistancing to Verticillium wilt by loss-of-function analysis; silencing of GhBAK1is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton.6. A detailed protocol of construction of a VIGS library from diploid cotton G raimondii was established. This library contained2×106clones; sequencing of the representative colonies revealed that the library covers a significant percentage of cotton unique genes; based on the number of colonies in the primary library, it is estimated that this VIGS library is about50×coverage of predicted cotton protein-coding genes; each clone contained diverse gene fragment.