Abstract:

【Objective】 The objective of the experiment is to identify the phenotype characteristics of T-DNA insertional mutants and constructing a platform of genetic transformation and functional genomics research in Verticillium dahliae. 【Method】 There were 2628 T-DNA insertional mutants of Verticillium dahliae strain V991 and flanking sequences of 15 mutants were obtained, and the insertional sites of some mutants were analyzed by using Agrobacterium tumefaciens mediated transformation and high-efficiency TAIL-PCR, respectively.【Result】 The mutant colony could be divided into three forms, which were hyphal type, sclerotium type and intermediate type. The sclerotium type colonies accounted for 7.3%. Culturing in flask, the sporulation peak of mutants was in the first 5-6 days after inoculation, and the sporulation ability of sclerotium mutants were generally higher than showing that of hyphal type mutants; VD991 could produce extracellular protease, amylase, cellulase and pectinase. Three mutants showing deleted protease secretion ability deleted, one mutant showing reduced amylase secretion ability and 6 mutants showing declined pectinase production were obtained; The virulence of the sclerotium type mutants was generally higher than that of the wild-type. Six mutants with reduced virulence were found from the virulence test of 26 mutants; The BLAST results showed that the flanking sequences of VD991 insertional mutants were 95%-100% identity to homologous sequences of Verticillium dahliae VDLs.17, and 87%-94% identity to Verticillium albo-atrum VaMs.102. 【Conclusion】 Agrobacterium tumefaciens mediated T-DNA random insertion could be used to Verticillium dahliae mutagenesis, and the production of microsclerotia is related to virulence and sporulation. The American strain VDLs.17 genome could be used as a reference sequence on genetic functional research of VD991.

Key words: Verticillium dahliae, T-DNA insertional mutagenesis, phenotype identification, flanking sequence analysis