Screening Of Abnormal Mutants Of Verticillium Dahliae And Identification Of Its Related Genes

In order to research functional mechanism about microsclerotia development gene of Verticillium dahliae, then clone those genes to proclaim the mechanism about the formation and development of microsclerotia. T-DNA insertional mutagenesis to identify mutants was carried out. Two Verticillium dahliae strains, the sclerotium type strain V08DF1 and the intermediate type strain VBp2, were mutated through Agrobacterium tumefaciens-mediated transformation, and two Verticillium dahliae mutant libraries were constructed including the V08DF1 library contained 2000 transformants and the VBp2 library contained 1000 transformants. Compared to the wide type strain V08DF1 which produced a large quantity of microsclerotia,14 mutants（1C2、2H3、5G4、6I7、7E6、9G4、10B5、10E1、11A6、11B7、11C3、 11G3、12C8、1213） without the ability to develop microsclerotia on Czapek-Dox medium were screened out from the V08DF1 library. In phenotype test of the transformants in the VBp2 library,4 mutants producing haematochrome on Czapek-Dox medium （7C8,7H4,9B1,9C10） and 5 mutants （7F4,10B10,10C6, 10E12,10G8） which couldn’t grew on Czapek-Dox medium were selected. The Hygromycin B resistance gene was PCR amplified from all of the mutants with abnormal phenotype of microsclerotia development, and this result showed that T-DNA had inserted into their genome. The southern blot result of the mutants with abnormal microsclerotia development from V08DF1 library indicated that 7 mutants were the result of single copy T-DNA insertion,5 mutants were the result of two copies T-DNA insertion, and 2 mutants were the result of three copies T-DNA insertion.7 single copy insertion mutants were 617、10B5、10E1、12C8、12I3、1C2 and 2H3. Only 3 mutants （7F4,9B1,10E12） with abnormal microsclerotia development from VBp2 library were gotten the southern blot result, and were all two copies T-DNA insertion. These results play an important role on study the molecular mechanisms of microsclerotial development related genes in V. dahliae.We should pay more attention to research those single copy T-DNA insertion mutants, because they are easier to find the T-DNA insertion site. The single copy T-DNA insertion mutants 2H3 and 1C2 were randomly selected. Firstly, we used the method of TAIL-PCR to find the insertion sites of T-DNA, then use the BROAD datebase which published online. The datebase obtained the whole genome sequence of Verticillium dahliae. We can BLAST on the NCBI website to find and analyze the T-DNA insertion flanking sequences and genes which related to microsclerotia. Based on T-DNA sequence analysis, the gene destroyed by T-DNA insertion in mutant 2H3 was a conidial yellow pigment biosynthesis polyketide synthase gene, its gene numbers is VDAG₀0190.1. In order to cinfirm the gene function, the experiments of gene knockout and genetic complement were carried out. For mutant 2H3, we obtained 7 complement transformants and 6 deletion transformants. The destoied gene in mutant 1C2 which related about microsclerotium development was a hypothetical protein gene, its gene number is VDAG₀1680.1. We obtained 1 complement transformant of mutant 1C2. In addition, we studied the mutants 5G4 and 9G4. Because the mutants 5G4 and 9G4 were two copies T-DNA insertion, only one of the T-DNA insertion sites was found via the methods TAIL-PCR or plasmid rescue. The result explained that the damaged gene of mutant 5G4 is the downstream G931P828RE17. The gene expressed sequence tags.9G4 mutant gene is also assumes that the protein may be broken, gene number was VDAG₀9865.1.