野油菜黄单胞菌野油菜致病变种一个新的致病相关基因的克隆和鉴定  

Cloning and identification of a novel virulence gene of Xanthomonas campestris pv campestris

作　　者：陆光涛[1] 唐纪良[2] 何勇强[2] 陈保善[2] 危广宁[2] 

机构地区：[1]浙江大学植物保护系,浙江杭州310029 [2]广西大学分子遗传研究所,广西南宁530005

出　　处：《浙江农业大学学报》2003年第6期591-598,共8页

基　　金：国家自然科学基金重点资助项目(30130010)。

摘　　要：野油菜黄单胞菌野油菜致病变种(Xcc)是十字花科植物重要的病原菌,能引起十字花科作物发生黑腐病。本课题组采用转座子Tn5gusA5对Xcc野生型菌株8004诱变,获得17280株Tn5gusA5突变体,并从中筛选到一批致病缺陷突变体,采用TAIL-PCR对致病缺陷突变体的Tn5gusA5插入位点进行定位,发现5株突变体的Tn5gusA5插在8004菌株的基因组ORF1857内(未发表资料),该ORF功能尚未见报道。序列分析表明,ORF1857演绎的编码产物与铜绿假单胞菌(Pseudomonaseaeruginosa)的wbpL基因演绎的编码产物具有33%的一致性和45%的相似性,并具有第4家族的糖基转移酶功能域,因此本文暂将ORF1857命名为wbxL基因。对wbxL基因进行缺失,获得的缺失突变体用剪叶法接种到寄主萝卜叶片时,缺失突变体完全丧失致病性,一段PCR合成的包含wbxL基因的DNA片段能互补缺失突变体,恢复缺失突变体的致病性。这证实wbxL基因是Xcc的一个新的致病相关基因。Xanthomonas campestris pv. campestris (Xcc) is the pathogenic agent of black rot disease in cruciferous plants. The wild-type strain 8004 of Xcc was mutagenized with transposon Tn5gusA5, and 17280 strain of Tn5gusA5 mutants were obtained. From these Tn5gusA5 mutants, a number of virulence-deficient mutants were isolated. The insertion sites of these virulence-deficient mutants were analyzed using TAIL-PCR, and the corresponding genes were identified by homology blast to the completely sequenced genome of Xcc 8004 strain (data not published). A novel gene, wbxL, identified from 5 strains of virulence-deficient mutants, was found to be disrupted by the insertion of Tn5gusA5 in ORF1857 of the genome. This gene showed 33% identity and 45% similarity to the wbpL gene of Pseudomonase aeruginosa at amino acid level, with characteristics of glycostransferase 4th family domain. wbxL gene was deleted and the virulence of the deletion mutant disappeared completely following innoculation into radish by clipping. The deletion mutant could be complemented in trans with a PCR fragment containing an intact wbxL gene so that the virulence of the mutant was restored. The data confirmed the involvement of wbxL gene in the virulence of Xcc.

关 键 词：野油菜黄单胞菌野油菜致病变种 wbxL基因 致病相关基因 缺失突变体 致病性 克隆 鉴定 

分 类 号：Q786[生物学—分子生物学]