| Citrus is the most productive fruit in the world,and citrus tristeza virus is one of the most serious diseases affecting the quality and yield of citrus.The previous research have found that the different citrus varieties and different infection time have different molecular mechanisms of interaction between citrus and citrus tristeza virus.Shiranuhi is a very good quality citrus variety,but its quality and yield are greatly reduced due to the infection of citrus tristeza virus during production.Identifying the valuable disease resistance genes will provide a theoretical basis for Shiranuhi resistance and citrus tristeza virus resistance breeding.In this study,we firstly used gene chips to study the early differential genes after Shiranuhi infection with citrus tristeza virus by the GO enrichment and visual pathways enrichment analysis,and identified some early responsive genes.Then,some citrus tristeza virus late responsive genes were isolated and identified through using the classic suppression subtractive hybridization technique.The CDS sequence of a calmodulin-binding protein gene,which expressed in both the early and late stages of citrus tristeza virus infecting,was cloned and analyzed by bioinformatics.The overexpressive and RNAi transgenic plants were tested for the disease resistance in citrus and tobacco.For the overexpressive transgenic citrus plants,the content of the defensive hormone SA was measured,and the transcriptome sequencing was used to analyze the transcriptome changes after inoculation with phytophthora parasitica.The main results are as follows:1.The early responsive genes of citrus with the infection of citrus tristeza virusThrough gene chip detection,the early differentially expressed genes after the infection of Shiranuhi with the citrus tristeza virus were detected.The GO analyis showed that the genes related with the process of cell wall remodeling,polysaccharide catabolism,flavonoid biosynthesis and chitin metabolism were significantly enriched in the early responsive process.Pathways enrichment analysis results showed that differentially expressed genes were related to cell wall remodeling,secondary metabolite formation,hormone signal transduction,proteolysis,redox reaction,signal transduction and other pathways.The differential genes of pathways analysis were consistent with GO enrichment analysis.2.The late responsive genes of citrus with the infection of citrus tristeza virusUsing the classic suppression subtractive hybridization technique,the late responsive genes of Shiranuhi after three years of infection with citrus tristeza virus was studied.A total of 77 non-repetitive sequences were obtained after clustering and splicing 192 valid sequences.Homology comparison analysis and BLAST2 GO functional classification showed that the functions of these genes were mainly related to stress and defense,metabolic pathways,reverse transcription,transport,and protein fate.The real-time fluorescent quantitative PCR were conducted to verify four genes including oxymethyltransferase gene,class I chitinase gene,calmodulin-binding protein gene,and Jasmonate ZIM-domain protein 1(JAZ1 protein gene).The results showed that under the long-term induction of citrus tristeza virus,the expression levels of these four genes in the leaves of Shiranuhi were all up-regulated.By comparing the early and late responsive genes of citrus tristeza virus,it is found that the differential genes are mainly concentrated in stress and defense pathways,metabolic pathways,cell wall remodeling,protein modification and hydrolysis pathways,redox pathways,jasmonic acid synthesis and Genes related to signal transduction may be suppressed during the early and late responsive genes of citrus tristeza virus3.Cloning and bioinformatics analysis of CsCaMBP geneThe promoter sequence and full-length CDS sequence of a calmodulin-binding protein gene in citrus were cloned.Bioinformatics analysis revealed that the promoter sequence region have MYB and MYC transcription factor action sites,have cis-acting elements that respond to salicylic acid and methyl jasmonate,have light response cis-acting element and ABRE cis-acting element related to abscisic acid response.The molecular phylogenetic tree showed CsCaMBP is closely related to the calmodulin-binding protein genes in Populus trichocarpa and Theobroma cacao.Real-time PCR analysis found that CsCaMBP could be induced by drought stress,citrus tristeza virus and phytophthora parasitica.CsCaMBP was mainly located in the cell membrane and nucleus.4.The disease resistance of up and down-regulated expression of CsCaMBP in tobacco and citrusWhen leaves of the transgenic tobacco and Jincheng orange plants were inoculated with Alternaria alternate and phytophthora parasitica respectively,compared with the control,the leaves of overexpressing tobacco showed symptoms of chlorosis and necrosis.The leaves of transgenic Jincheng orange plants showed necrotic lesions that were significantly larger than the control lesions.The leaves of the RNA interference transgenic Jincheng orange plant after inoculation with phytophthora parasitica was not significantly different from that of the control plant with the empty vector.These results showed that overexpression of CsCaMBP gene made the plants more sensitive to pathogens.5.The changes of hormone content and transcriptome sequencing analysis of CsCaMBP overexpressing citrus plant inoculating with phytophthora parasiticaThe salicylic acid content of CsCaMBP overexpressing leaves of citrus is lower than that of control plants,and the genes related with the salicylic acid hormone signal transduction pathway were significantly down-regulated in the leaves of CsCaMBP overexpressing citrus plant.The results showed that the decrease of salicylic acid content of over-expression CsCaMBP citrus plant might be the main reason why citrus plants were more sensitive to phytophthora parasitica. |
