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棉花学报 ›› 2011, Vol. 23 ›› Issue (1): 64-68.

• 研究与进展 • 上一篇    下一篇

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

高 峰,彭 姗,彭晓玲,李 晖,李国英   

  1. 1. 石河子大学绿洲农作物病害防控重点实验室,石河子大学,新疆 石河子 832003;2.中国农业大学植物病理系,北京 100193
  • 收稿日期:2010-06-11 出版日期:2011-01-15 发布日期:2011-01-15
  • 通讯作者: 李国英,教授,lgy_agr@shzu.edu.cn
  • 作者简介:高 峰(1976-),男,博士研究生,gf_agr@shzu.edu.cn
  • 基金资助:

    农业部基础性工作专项(SB2007FY027)

Identification of the Pathogenicity-related Gene DVK1 in Verticillium dahliae

GAO Feng1, PENG Shan1, PENG Xiao-ling1, LI Hui2, LI Guo-ying1*   

  1. 1. The Key Laboratory of Prevention and Control for Oasis Crop Disease, Shihezi University, Shihezi, Xinjiang 832003, China; 2. Department of Plant Pathology, China Agricultural University, Beijing 100193, China
  • Received:2010-06-11 Online:2011-01-15 Published:2011-01-15

摘要: 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。

关键词: T-DNA插入; 突变体文库; 基因克隆; 棉花黄萎病; 功能鉴定

Abstract: 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.

Key words: T-DNA insertional; mutants library; gene clone; Verticillium dahliae; functional identification

中图分类号: 
  • Q785