Phenotype Characteristics And Flanking Sequence Analysis Of T-DNA Insertional Mutants On Verticillium Dahliae

Verticillium dahliae is the causal agent of the soil-borne vascular wilt disease, which results in severe yield and quality losses in cotton worldwide every year. Over the years, many studies were done by the domestic and foreign scholars on about the pathogenic mechanism of Verticillium dahliae, but little achievements were obtained. In this study, using the defoliated Verticillium wilt strain VD991as a wild type strain, a large number of mutants were constructed by the method of Agrobacterium tumefaciens mediaed transformation (ATMT). Subsequently, the optimizing of transformation protocol, the identification of mutants phenotype and their pathogenicity, and the flanking sequences of T-DNA insertional sites were studied. Accordingly, the technical platform on studying the T-DNA insertional mutation and the cloning of the pathogenicity related genes were built in our laboratory. This was a foundation for the study of pathogenic molecular mechanisms in Verticillium dahliae.Based on the transformation method of Fusarium oxysporum described by Mullins, the parameters of T-DNA insertional mutation for Verticillium dahliae were optimized.2628T-DNA insertional mutants were obtained and the stability of resistance heredity was testified by series culture and molecular verification. Some of the mutants were selected for the phenotype identification, the results indicated that:(1) VD991did not produce microsclerotia when cultivating at normal temperature on PDA. Some mutants, accounted for7.3%, changed from the original mycelial type into a sclerotium type, and producing large amount of microsclerotia at5-7d post cultivation.(2) The growth rate of mutants was slightly slower than that of wild-type strains on either MM or PDA medium. In shake-flask cultivation, the sporulation peak of most mutants was at5-6d post-inoculation. The sporulation capacity of mycelial type mutants was close to or slightly lower than that of wild-type, however, the sporulation capacity of sclerotium type mutants were higher than that of VD991, even up to4.9times.(3) The determination results of extracellular enzyme showed that VD991could produce extracellular protease, amylase, cellulase and pectinase, and it could grow on Czapek medium appending chitin as only carbon sources. Eight auxotrophic mutants that could not grow well on MM medium, three mutants that could not use skimmed milk powder, five mutants that could not make use of soluble starch and seven mutants with pectinase secretion reduced were selected.(4) Using the inoculation methods of root-dipping and stem puncturing with spore suspensions on cotton seedlings, the virulence of some mutants were identified. Six mutants that virulence reduced comparing with VD991were screened.The right flanking sequences of T-DNA insertional sites of30mutants were obtained by using the method of hiTAIL-PCR, and the results BLAST to the database of Verticillium dahliae VDLs.17and Verticillium albo-atrum VaMs.102genomes indicated that:(1) The flanking sequences showed95-100%identity over its full length to the corresponding site of VDLs.17, and87-94%identity to that of VaMs.102. Therefore, the genome sequence of VDLs.17could be used as a conference sequence for the functional genome research.(2) Between the sequences of VD991and VDLs.17, with the exception of the existence of a large number of single base differences and gaps, there are a small number of gene sequences showed fragments insertion and rearrangement;(3) There were7mutants with T-DNA inserted in coding region, thirteen mutants with insertional sites located in the range of500bp upstream of start codon may have direct impact on the expression of its downstream genes.(4) The downstream genes of T-DNA insertional sites of5mutants could serve as candidate genes for functional genetic research.(5) The matching sequence to H20flanking sequence could not be found in Verticillium dahliae VDLs.17, but it existed in Verticillium albo-atrum VaMs.102and shared homologous of88%. Further study should be done on how it happened on evolution.(6) Comparing the T-DNA insertional sites of30mutants showed that they were different from each other, and widely distributed in all8chromosomes. This demonstrated that the T-DNA insertional events happened randomly.