广藿香青枯病菌致病相关基因RsgidA的克隆及生物信息学分析  

Cloning and bioinformatics analysis of pathogenicity-related gene RsgidA in Ralstonia solanacearum isolated from Pogostemon cablin

作　　者：李巧 王亚琴 贺红[1] 张泳 张宇瑶[1] 黎广卫 LI Qiao;WANG Ya-qin;HE Hong;ZHANG Yong;ZHANG Yu-yao;LI Guang-wei(School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Research Center of Chinese Herbal Resource Science and Engineering,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]广州中医药大学中药学院,广东广州510006 [2]广州中医药大学中药资源科学与工程研究中心,广东广州510006

出　　处：《中草药》2021年第10期3068-3074,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(81373901);广东省自然科学基金面上项目(2019A1515012151)。

摘　　要：目的对广藿香青枯菌菌株PRS-84的致病相关基因RsgidA进行克隆及生物信息学分析。方法以广藿香青枯病菌Tn5转座子插入的低致病力突变株PRS-84-4-7为材料,对广藿香进行致病性实验;以Tn5转座子序列为基础,对突变株PRS-84-4-7的基因组进行反向PCR扩增,克隆转座子插入位点的侧翼序列;根据序列的同源基因设计特异性引物,对野生菌株PRS-84的基因组进行PCR扩增,对克隆获得的目标基因序列进行生物信息学分析。结果低致病力突变株PRS-84-4-7对广藿香的致病性不明显。成功获得了低致病力突变株转座子插入位点的侧翼序列,该序列与gidA基因的相似度最高,将插入位点基因命名为广藿香青枯病菌RsgidA基因。从野生菌株PRS-84中克隆了致病相关基因RsgidA,全长1881bp,编码626个氨基酸。系统进化分析显示,RsgidA基因编码蛋白与植物病原中丁香假单胞菌Pseudomonassyringae的GidA蛋白亲缘关系较近。结论从广藿香青枯病菌菌株PRS-84中成功克隆了致病相关基因RsgidA,为了解青枯病菌的致病机制、研究新的防治广藿香青枯病的方法奠定了基础。Objective To clone the pathogenicity-related gene RsgidA in Ralstonia solanacearum isolated from Guanghuoxiang(Pogostemon cablin,patchouli)and perform bioinformatic analysis.Methods The wild-type R.solanacearum strain PRS-84 was isolated from patchouli plants infected with bacterial wilt and its Tn5 transposon insertion mutant PRS-84-4-7 attenuated in virulence was constructed and screened in our previous study.Pathogenicity assays of wild-type strain PRS-84 and low virulence mutant PRS-84-4-7 were performed on patchouli plants.The flanking fragment that is adjacent to Tn5 transposon insertion site in mutant PRS-84-4-7 was amplified by inverse PCR.The full-length sequence of the Tn5 transposon insertion-site gene in mutant PRS-84-4-7 was amplified from the genome of wild-type strain PRS-84 by PCR based on primers designed according to the homologous gene sequences,and the nucleotide and protein sequences of the gene were analyzed.Results Pathogenicity assays showed that mutant PRS-84-4-7 was significantly less virulent than its wild-type parent strain PRS-84 on patchouli plants.The flanking sequence analysis showed that mutant PRS-84-4-7 had a transposon insertion in a pathogenicity-related gene which was named RsgidA due to its high identicality to gidA gene.RsgidA gene in wild-type strain PRS-84 was cloned,with 1881 bp long encoding 626 amino acids.Phylogenetic analysis showed that RsGidA protein has the nearest phylogenetic relationship to the GidA protein from Pseudomonas syringae among different plant pathogen strains.Conclusion The pathogenicity-related gene RsgidA was cloned successfully from R.solanacearum strain PRS-84.This paper might facilitate the study of pathogenicity mechanism of R.solanacearum on patchouli and develop new control methods against patchouli bacterial wilt.

关 键 词：广藿香 青枯菌 RsgidA基因 克隆 生物信息学分析 

分 类 号：R282.12[医药卫生—中药学]