Construction Of VDAG₀9582.1 Gene Knockout Vector And Gene Function Analysis Of Verticillum Dahliae

Verticillium dahliae?Kleb?.is a kind of soil spread plant pathogen,causes wilt diseases in hundreds of dicotyledonous plant species,leading to enormous economic losses worldwide.At present,the pathogenic mechanism and genetic characteristics of V.dahliae are not clear.Therefore,in order to control verticillium wilt effectively,we need to explore the genes related to pathogenicity and its functions at the molecular level,so can lay a foundation for further understanding the pathogenic mechanism of verticillium wilt and provide a new approach to control verticillium wilt.At present,the researches on genes related to pathogenicity of V.dahliae had made great progress,multiple genes had been proven to relate to pathogenicity of V.dahliae.These genes had been studied in other species,had known its specific function.But there are still some unknown genes associated with certain functions in the organism genome,these genes coded protein called hypothetical protein,it was protein coded by new gene which had unknown gene functions and was not identified.Recognition of these unknown protein will help to find more pathogenic related genes of V.dahliae and analyze its features and functions can provide more theoretical basis to the pathogenic mechanism of verticillium wilt.In this experiment,we used a random insertion mutant library of V.dahliae which was constructed by others,we selected No.22 mutant to make experiment because the mutant grew more hyphae than wild type,then we got to know the insertion site of the mutant was VDAG₀9582.1 gene of V.dahliae by TAILPCR,the gene encode a hypothetical proteins through analysis sequence.So I used gene knockout technology to study its function by constructing the gene knockout vector and transformed to V.dahliae,then compared the characteristics of wildtype and mutant strains.The main results of this research were showed in following:1.By using the method of fusion PCR,we got the homologous recombinant fragments after two rounds of PCR,then the fragment connected with pPK2.Finally,after double enzyme digestion and PCR detection,we got a homologous recombination vector pPK2-UhphD successfully2.Transformed homologous recombination vector pPK2-UhphD into V.dahliae by Agrobacterium-mediated T-DNA insertion in V.dahliae V991 transformation method?Agrobacterium tumefaciens-mediated transformation,ATMT?,and got a lot of transformants,it was proved at DNA and RNA level.3.Compared the VDAG₀9582.1 gene knockout mutant strains,random insertion mutant strains,complemented mutant strains and wild type of V.dahliae in colony growth rate,spore capacity,pressure sensitivity,microsclerotial production,hyphea growth and pathogenicity.VDAG₀9582.1 is related to microscertial production and hyphea growth,but has nothing to do with pathogenicity.