Construction Of T-DNA Inserted Transformation Library Of Verticillium Dahliae On Cotton And Analysis Of Mutative Traits

A mutant library, which initiated from a high virulent Verticillium dahliae strain Vd080, wasconstructed by agrobacterium-mediated transformation. And its quality was evaluated. Phenotype ofsome mutants which were selected randomly were analyzed. The mutant VdT286that spore yield,growth rate and pathogenicity were all lower than wild strain Vd080. And it’s flanking sequence wasamplified. The main conclusions was showed as flowing:1. The Agrobacterium tumefaciens mediated transformation after optimizing was used to construct highvirulent Verticillium dahliae strain Vd080’s mutant library that contained2000mutants. The freshagrobacterium, Verticillium dahliae spore suspension,OD600reaching about0.4,200μMAcetosyringone and48hours of co-cultivation period at25℃were optimal for the transformationsystem. Ratio could reach150~540transformants per106spores. The results of PCR and Southern blotindicated that T-DNA inserted into the genome of Vd080successfully, and T-DNA in mutants selectedrandomly were all single copy. All of the tested transformants maintained their resistance to hygromycinB. The method had some advantages,for example, simple operation, short circulation, hightransformation rate, high rate of single copy and stable transformants.2. Colony morphology of309mutants that were selected randomly from the library were observed. Theresult showed that143mutants were different from wild strain Vd080, accounting for46.3%. Amongthem, there were40mycelia mutants (containing white mycelia and yellow mycelia), accounting for28.2%. The growth rate, spore yield, crude toxin and pathogenicity of85mutants were studied. Thegrowth rate of11mutants decreased significantly, occupying12.9%; Crude toxin of43mutants variedobviously, occupying50.1%; Spore yield of31mutants changed obviously, occupying36.5%.Theresults of pathogenicity showed that27mutants’ pathogenicity changed obviously, accounting for31.8%. The correlation analysis of the mutants indicated that no relationship existed among theseindexes.3. The mutant VdT286that all of yield spore, growth rate and pathogenicity decreased significantly wasobtained during screening and was further studied in this chapter. Spore yield of VdT286was muchlower than wild strain Vd080and the difference reached4order of magnitude. The growth rate ofVdT286was much lower than Vd080. Concentration of VdT286spore suspension was used to inoculatecotton, pathogenicity of mutant VdT286decreased significantly compared with wild strain Vd080. Theresults of temperature tolerance and pH sensitivity demonstrated that the growth rate of VdT286indifferent temperature and pH reduced obviously comparing with Vd080, but their growth trendcy weresame. Activity of extracellular enzyme in the two strains were analyzed, the results showed they couldgrow in the medium containing only skim milk powder, starch or cellulose as carbon sources. Vd080could produce cellulase, amylase and protease.VdT286could produce amylase, while the ability ofcellulose and protease secretion decreased.4. Special walking primers were designed on the basis of right border and left border of T-DNA insertregion. The system of TALI-PCR and reaction program were optimized to clone flanking sequence of mutant VdT286. After sequencing, it was blasted with Verticillium dahliae Vdls.17which has beensequenced already. The results showed that the length of gene was3201bp containing7exons and6introns which code glucose repression mediator protein VdGR1. It indicated that it was the key gene inthe control of Verticillium dahliae spore yield and related pathogenic gene. The function of VdGR1wasstill not reported now. So the function of this gene VdGR1will be next research emphasis on the basis ofthis study.