The Gene Cloning And Functional Analysis Involved In Melanin Synthesis Pathway Of Verticillium Dahliae From Cotton

Cotton wilt disease, caused by the phytopathogenic fungus Verticillium dahliae Kleb., isone of the most widespread, damaging diseases in most cotton-growing countries. Thefunctional genomics research of V. dahliae has lagged behind those of other fungi due to itslong-term culturing under laboratory conditions and the high complexities of its genome, withthe result that its pathogenic mechanism has not been elucidated. Microsclerotia with melaninare the main primary inoculum of V. dahliae during disease cycle. The melanin formed ingrowth process plays an important role in the pathogenicity. Degenerate primers weredesigned based on sequences involving in the melanin synthesis pathway accessed inGenBank. PKS gene was determined from a virulent defoliating isolate V592of V. dahlae byPCR, cloning and sequencing. It comprised6742nucleotides (KC422576) including4exonsand3introns, and ORF encode2189amino acid. It showed typical PKSI geneticcharacteristics having1ketoacyl synthase (KS) domain,2acyl carrier protein transacylase(ACP) domains,1Acyl transferase (AT) domain,2Phosphopantetheine attachment site (PP)domains and1Thioesterase domain. Phylogenetic tree was performed based on alignments ofamino acid sequences of KS domain among V592and other fungi. It showed that these fungiproducing secondary metabolite including Polyketide had same phylogenetic relationships.V592clustered together with other fungi producing melanin, while other fungi producingother same Polyketide metabolite clustered together, respectively. It indicated that PKSI wasclosely related to melanin biosynthesis of V. dahlae from cotton.Ninety-one melanin-defective mutants were obtained from1144T-DNA insertionalmutants by Agrobacterium tumefaciens mediated transformation (ATMT). Southern blotanalysis indicated that T-DNA were inserted randomly into the V. dahliae genome and about20%of the20melanin-defective mutants have a single copy of the T-DNA integrated into thegenome using Digoxin-labeled EGFP-specific DNA probe. Genomic DNA flankingsequences inserted by T-DNA were cloned from10insertion mutants by thermal asymmetricinterlaced PCR (TAIL-PCR) protocol. It showed that the annotated genes associated withthose tagged mutant genes were involved in cell cycle regulation by bioinformatics analysis.Gene knockout is the most direct way to identify the gene function. Gene knockout vectorswere constructed which will be used to disrupt five targeted genes by homologousrecombination.