Isolation Of The Cell Wall Synthesis Defective Mutants And Characterization Of Bcchs Ⅵ Gene In Botrytis Cinerea

The purpose of this study was to investigate the functions of cell wall synthesis genes in Botrytis cinerea by T-DNA insert mutants library technology and gene knockout technology.By utilizing the Agrobacterium tumefaciens containing the binary vector ATMT1 to transform Botrytis cinerea B05.10,screening hygromycin resistance mutants,sigle spore separation,and generation culturing,234 stabilized T-DNA inserting mutants were obtained.These T-DNA insertion mutants of Botrytis cinerea were further screened by using Calcofluor White,and three sensitive mutants(B-117、B-169、D-9)and one resistant mutant(B-62)were obtained.The CFW sensitive mutants were further studied by Sorbitol、SDS、NaCl tests,the results revealed that the growth of B-117、D-9 was rescued by 1.2 M sorbitol,and B-117 was also suppressed by NaCl.In the spore germination assay,the mycelium of B-62 appeared branched and its diameter and length increased evidently,the spores of D-9 appeared swollen and its hyphae were thickening,lengthily and no bend.In the tomato leaf infection experiment,D-9 mutant was found significantly reduced its virulence while B-62 mutant was enhanced its virulence.These results indicated that B-62 and D-9 mutant was probably related to cell wall defect and pathogenicity.Since chitin synthase V and VI are very important to the cell wall chitin synthesis in the filamentous fungi,Bcchs V was been investigated by our laboratory in the early experiment,the results revealed that in the knockout mutant of Bcchs V chitin content increased by 31%and pathogenicity to hosts was not affected.In order to characterize the function of Bcchs VI gene,this experiment constructed Bcchs VI knockout binary vector pCAMBIA-Bar-△chs6 by the Gateway system,through agrobacterium tumefaciens mediated transformation,obtained 9 hygromycin resistant mutants from Botrytis cinerea B05.10.The phenotypic and molecular identification revealed that the mutants except△4 mutant were the BcchsⅥ gene knockout mutants,and△4 mutant may be the ectopic insertion mutant.The studies based on △2 BcchsⅥ knockout mutants revealed that the chitin content of mutant was declined 22.5%,and the mutant was sensitive to CFW,rescued by Sorbitol,sporulation reduced significantly,pathogenicity to the tomato leaves decreased obviously.Further analysis of the BcchsⅥ gene function are still in progress.