Evaluation Of T-DNA Inserted Transformation Library Of Verticillium Dahliae (XJ2008) And Study Of Pathogenic-related Genes

Hygromycin B resistance of568transformants selected randomLy from the Verticilliumdahliae（XJ2008） mutants library were tested. There were104transformants with stableresistence to HygB. Through analyzing phenotype (growth rate, spore yield and pathogenicity)of these transformants, a transformants2-736whose pathogenicity was significantly lowerthan wild-type strain XJ2008was obtained. Its flanking sequence was amplified and thepathogenic-related gene was found. We constructed complementary plasmid and carried outthe functional complementation analysis.The results of phenotypic analysis showed that28transformants were different fromtwild-type strain XJ2008, accounting for26.9%. Among them, there were12velum type,accounting for11.5%. the growth rate of7transformants was significantly slower than thewild-type strain, accounting for6.7%, of which2-729and2-293only0.7mm d-1and0.6mm d-1, compared with the wild-type strain decreased of41.7%and50.0%; there were16transformants growing significantly faster than the wild-type strain, accounting for15.4%, ofwhich2-1103reached2.1mm d-1, compared with the wild-type strain increased by75%.Spore yield of56.7%transformants changed obviously, of which2-1045and2-1103can notproduce spores in the PDB and Czapek medium. All transformants on BMM culturing for30days can produce microsclerotia. The results of pathogenicity showed that22transformants’virulence decreased significantly, accounting for26.2%.Through evaluation of the mutants library, we got a pathogenicity decrease obviouslytransformants2-736, but its growth rate, spore yield and colony morphology was similar tothe wild-type strain. The flanking sequence of transformants2-736was cloned by hiTaiI-PCR.After sequencing, it was blasted with Vdls.17which has been sequenced already. The resultsshowed that the length of gene was2518bp containing2introns. It located in Chromosome8 and coded747amino acid. But there was no gene function annotation.Complementary vector was successfully constructed using binary shuttle vector pFL2and transformed into the protoplasts of transformants2-736by PEG-mediated transformation.Finally we screened31complementary transformants.13transformants selected from themwere detected by PCR, the results showed that65%of complementary transformants werepositive.