棉花黄萎病菌插入突变体库的构建及致病相关基因DVK1的克隆与鉴定

Identification of the Pathogenicity-related Gene DVK1 in Verticillium dahliae

利用ATMT(Agrobacterium tumefaciens-mediated transformation)技术自建Verticillium dahliae强致病力落叶型菌株T-DNA插入突变体库,共获得5000个转化子;随机挑选1000个转化子进行致病力鉴定,筛选获得5株致病力衰退的突变体。挑选致病力下降最为明显的突变体d1,通过TAIL-PCR技术最终获得一段长3237 bp的DVK1全长cDNA序列,编码1079个氨基酸的蛋白。基于DNA序列比对发现,DVK1基因含有2个内含子,分别为52 bp和36 bp。进一步比对发现T-DNA插入到DVK1基因启始密码上游740 bp的启动子中。通过遗传互补实验d1恢复了致病性,进一步证明DVK1基因与黄萎菌致病性相关。

In this study,we carried out T-DNA insertional mutagenesis to identify mutants,which are effective in pathogenicity.The highly virulent,defoliating strain was mutated through Agrobacterium tumefaciens-mediated transformation,and 5000 transformants were obtained.Pathogenicity test of randomly selected 1000 transformants found that five mutants lost their virulence on a susceptible cotton cultivar.A new mutant,d1,was unable to cause full disease on cotton.Analysis of the mutation using Thermal Asymmetric Interlaced PCR（TAIL-PCR） confirmed an insertion into a gene DVK1.The full length of DVK1 genomic DNA and cDNA sequences were obtained using TAIL-PCR and RT-PCR methods.DVK1 has an open reading frame of 3325 bp interrupted by two introns with 52 bp and 36 bp,respectively,and putatively encodes a 1079 aa protein.The reduced pathogenic phenotype of d1 was fully complemented by reintroduction of the gene,indicating DVK1 is essential for pathogenicity in V.dahliae.