中国农业科学 ›› 2011, Vol. 44 ›› Issue (2): 299-306 .

• 植物保护 • 上一篇    下一篇

成团泛菌对玉米自交系PS056致病性基因yhfK的克隆

曹慧英,张力群,朱振东,王晓鸣,李洪杰,任争光

  

  1. (中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程)
  • 收稿日期:2010-06-08 修回日期:2010-08-16 出版日期:2011-01-15 发布日期:2011-01-15
  • 通讯作者: 王晓鸣

Cloning of a Pathogenicity Gene yhfK from Pantoea agglomerans on Maize Inbred Line PS056

CAO Hui-ying, ZHANG Li-qun, ZHU Zhen-dong, WANG Xiao-ming, LI Hong-jie, REN Zheng-guang
  

  1. (中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程)
  • Received:2010-06-08 Revised:2010-08-16 Online:2011-01-15 Published:2011-01-15
  • Contact: WANG Xiao-ming

摘要:

【目的】通过克隆和鉴定成团泛菌(Pantoea agglomerans)的致病相关基因,阐明该种土壤习居细菌在玉米自交系PS056上引起细菌性干茎腐病的原因。【方法】利用转座子Tn5的随机插入突变特点,构建致病性菌株P. agglomerans XJ1突变体库;经对Tn5插入位点的侧翼扩增,确认突变体PA121的致病力相关基因;通过基因互补试验,证明yhfK与致病力的关系。【结果】通过对突变菌株的接种筛选,获得11个致病力丧失的突变体。Southern杂交和PCR扩增结果表明,Tn5在突变体PA121中为单拷贝插入,插入位点为细菌编码内膜蛋白的yhfK(Tn5 插入在yhfK的1 082 bp处)。将yhfK克隆到质粒pBBR1MCS上,并导入突变体PA121中进行互补,生物测定结果显示转化子完全恢复了正常的细菌生理功能,并能够在玉米自交系PS056上恢复致病力。RT-PCR试验证明,yhfK调控hrpA的转录。【结论】 成团泛菌XJ1中yhfK是调控其对植物致病性的基因之一,具有在玉米自交系PS056上引起细菌性干茎腐病的功能。

关键词: 成团泛菌, Tn5转座子, 玉米, 细菌性干茎腐病, yhfK

Abstract:

【Objective】 The purpose of this study was to clone and identify a pathogenicity gene of Pantoea agglomerans causing bacterial dry stalk rot on maize.【Method】 A transposon mutant library was constructed in P. agglomerans by Tn5 transposon mutagenesis. The mutants with defective pathogenicity were screened using the stalk-injecting inoculation on the susceptible maize inbred line PS056. One mutant lost pathogenicity was obtained and designated as PA121.【Result】Southern blotting analysis indicated that the mutant PA121 had a single-copy Tn5 transposon which inserted into the yhfK at the site of 1 082 bp upstream. The yhfK encodes a conserved inner membrane protein, identified by bi-directional sequencing based on Tn5 sequence. Phenotypic analysis indicated a complementary plasmid retained the pathogenicity on host PS056. The activities of extracellular enzymes, such as protease, amylase and cellulase, were not affected in mutant PA121. RT-PCR analysis demonstrated that the yhfK regulated the transcription of hrpA.【Conclusion】This study demonstrates that the yhfK affects pathogenicity of P. agglomerans, which is a critical element in the pathogenesis of P. agglomerans causing bacterial dry stalk rot on maize.

Key words: Pantoea agglomerans, Tn5 transposon, maize, bacterial dry stalk rot, yhfK