The Screening Of T-DNA Insertional Mutants And Construction Of The Pathogenicity-related Gene Knockout Vector For Verticillium Dahliae On Cotton

The cotton verticillium wilt caused by verticillium dahliae is a kind of soil-borne disease,which is one of the most important diseases of cotton production on almost all main cotton regions worldwide and caused serious economic losses.Planting resistant varieties is an effective measure to control of cotton verticillium wilt but there is no cotton varieties with high resistance to verticillium wilt currently.As V.dahaliae in the field is a population with highly differentiation and variation in virulence,and easy to form a strong virulent type,along with the unstable resistance of cotton varieties,the cotton verticillium wilt occurs frequently in our country.V.dahliae is a typical soil-borne fungi and has complicated mechanism of pathogenesis.Although with some progresses recent years at home and abroad,the researches on mechanism of pathogenesis still relatively lag behind,compared with other phytopathogenic fungi.Therefore,the study on molecular mechanism of the micro sclerotium formation and identification of major pathogenic genes will help us to reveal the mechanism of pathogenesis and provide the candidate targets of new fungicide for the design and screening.And further study of V.dahliae pathogenesis and molecular mechanism of the micro sclerotium forming,identified the key to control pathogenic genes,and helps to reveal its pathogenic mechanism,the design of the new fungicide for control of cotton verticillium wilt bacteria and screening the candidate targets.1.Previously,a T-DNA insertion mutant library of V.dahliae had been constructed in our laboratory.In this study,from some transformants,randomly picked up and measured in terms of biology characteristics,mutants were obtained with defects on speed of colony growth,ability to generate micro sclerotium,quantity of spore production,and pathogenicity,etc.The transformants were divided to three types,which are selerotium type,hypha type,and middle type,based on the characters of colony culture.The result showd that most of the transformants on thermostatic cultivation under 25? of colony morphology in PDA medium had no obvious difference,in which 71.88% of them belonged to selerotium type,47 of the transformants decreased in mico sclerotium ability significantly,24.48% of the total found their way to the middle type,and 7 of them with no micro sclerotium belonged to the type of hypha including transformants V874-2-1,V923-1-2,V923-1-1,V534-1,V966-1,V909-1,and V945-1,respectively.Colony growth rate determination showed that the diameter of most transformants kept the same level with the wild type strains of FGH2.Among them,16 of transformants grew slowly.Test in spore production showed that 34 transformants reduced and 14 of them increased significantly with a rate of 17.7% and 7.29%,respectively.Measurement on pathogenicity showed that 95 of transformants had similar pathogenicity with wild type strain FGH2 in disease index above 80,but 8 of them with attenuated virulence in an index below 50,which included V923-1-1,V898-1,V782-1,V700-1,V923-2,V641-1,V835-1,and V910-1,respectively.2.Preliminary functional identification of two mutants V1009 and V546 in the lab.Both of the mutants exhibited decreased pathogenicity significantly,while the ability to form the micro sclerotium was reduced in mutant V1009,but lost in mutant V546.Early research had shown that T-DNA inserted in VDAG₀1673.1 and VDAG₀7282.1 coding region of V.dahliae genome,which encoded a peroxidase membrane protein and unknown function product,respectively.In this study,the Split-marker method using hygromycin gene for selection markers was used to generate two fragments through fusion PCR for each candidate gene.At the same time,two knockout constructs were generated with p KOV21 plasmid as the backbone,which contained both hygromycin and neomycin genes,through cut-ligation method in multiple cloning sites.The upstream and downstream of the target gene sequence were connected with the upstream and downstream of hygromycin gene sequences,respectively.The resulting knockout constructs were designated as PEX16 p1009KOV21 and VDAG₀7282.1 p546KOV21,respectively.These works above were the basic to further knock the pathogenic related genes out.3.Drislease and lysing enzymes were applied to produce protoplast from fresh mycelia of verticillium wilt fungus.It's a plenty of protoplast generated for subsequent genetic transformation through PEG-CaCl₂ approach.