The Cloning Of Pathogenicity Related Genes Of Verticillium Dahliae On Cotton And Functional Analysis Of CYC8

In the current study,800T-DNA inserted mutants of high virulent cotton Verticilliumdahliae strain Vd080producing microsclerotia were conducted pathogenicity tests using amethod named vermiculate and sand in bottomless paper pot dipping in quantitative spores.Go through twice screen, we obtained25single T-DNA inserted and low virulent mutants,then the pathogenicity related genes were cloned. We focused on the special mutant T286,which showed lower yield of conidia and weaker pathogenicity without microsclerotiaproducing. And, we made comprehensive functional analysis of the pathogenicity related geneCYC8by gene knock out and complementation. The main conclusions showed as fllowing:1.800T-DNA inserted mutants of high pathogenic virulent Verticillium dahliae strainVd080producing microsclerotia were conducted pathogenicity tests. Among them, thevirulence of31mutants exhibited highly significant reduction, and analyzed there biologicalcharacteristics. The results of molecular identification showed that all the low pathogenicvirulent mutants were positive and25mutants had single T-DNA inserted. Compared with thewild type strainVd080, the25single T-DNA inserted mutant colonies showed diversevariation. Among them,8mutants were similar with wild type producing microsclerotia, therewere also3yellow mycelia,2white mycelia and12intermediate strains. Furthermore, thegrowth rate, spore yield and crude toxin of most strains changed significantly, but thecorrelation among these physiological indicators was not significant. With the aid ofTAIL-PCR technique and VdLs.17genome database, the flanking sequences of the25singleT-DNA inserted and low virulent mutants were obtained, and the pathogenicity related geneswere successfully cloned.2. Throuth the comparition with lettuce V. dahliae VdLs.17genome database, we foundthat, in T286, T-DNA was inserted into CYC8(glucose repression mediator protein). The genewas located in the fifth chromosome with3201bp in full length and contained7exons and6introns. The length of its cDNA was2679bp coding892aa. The function of CYC8was still notreported in V. dahliae yet. The result of southern blot showed that there was only one copy for CYC8on Vd080. This evidence provides a premise for researching gene function.3. CYC8gene knockout vector, which had lethal gene, HSVtk, was constructed with thecombination of Fusion PCR, Nested PCR and Gateway technology. We knocked out theCYC8gene by using Agrobacterium tumifaciens-mediated transformation，ATMT. The resultsof reverse lethal mutation, resistance screening and molecular identification showed that, wesuccessfully obtained17positive knockout mutants.4. Based on the pSULFgfp vector, which had chlorimuron-ethyl resistance and greenfluorescent protein label, we connected the CYC8gene containing its promoter and terminatorto the frame in the territory. Then we structured CYC8complementation vector through thedigestion method. And, we used ATMT to complement it into the T286genome of V. dahliae.The results of resistance screening, molecular identification and fluorescence observationshowed that, we successfully obtained16positive complementation mutants.5. Pathogenicity tests, biological characteristics and extracellular enzyme activity wereanalyzed in a part of knockout mutants and complementation mutants, to analysis the functionof CYC8in V. dahliae. The results of biological characteristics and pathogenicity tests showedthat, compared with the wild type strainVd080, two knockout mutants, CYC-55and CYC-56,present intermediate colony, irulence significantly reduced, but didn’t significant difference toT286; the spore yeild of three complementation mutants were higher than T286at eight day,and the disease index was all significantly improved than T286at24day. The results ofextracellular enzyme activity showed that, Protease and Cellulase’s activity reduced inknockout mutants, and recovered in complementation mutants. In conclusion, CYC8was notonly a pathogenicity related gene, but also controlled the production of microsclerotia andmelanin, yield of conidia and the activity of Protease and Cellulase.6. To monitor CYC8’s expression characteristic in mycelium of V. dahlia, we takesamples at4,8,12,16,20day. The results showed that, the curve of expression quantity ofCYC8in mycelial showed a bell shap, raised at first, then decreased, and the peak appeared at12th day.7. CYC8code glucose repression mediator protein. Then we made different glucoseconcentration and took samples at different time to study the relation of glucose and theexpression quantity of V. dahliae CYC8. The results of qRT-PCR showed that, lower glucoseconcentration inhibites the expression of CYC8at the earlier stage of culture; higher glucoseconcentration promotes the expression of CYC8at the later stage of culture.