中国农业科学 ›› 2010, Vol. 43 ›› Issue (14): 2944-2952 .doi: 10.3864/j.issn.0578-1752.2010.14.013

• 园艺 • 上一篇    下一篇

白粉菌诱导的华东葡萄环化cDNA文库中抗病基因筛选

张军科,杜敬,李爽,朱自果,王跃进

  

  1. (西北农林科技大学园艺学院/农业部西北园艺植物种质资源与利用重点开放实验室)
  • 收稿日期:2010-04-19 修回日期:2010-05-24 出版日期:2010-07-15 发布日期:2010-07-15

Resistance Genes Screening in Cycled Uncinular necator Induced cDNA Library of Vitis pseudoreticulata

ZHANG Jun-ke, DU Jing, LI Shuang, ZHU Zi-guo, WANG Yue-jin
  

  1. (西北农林科技大学园艺学院/农业部西北园艺植物种质资源与利用重点开放实验室)
  • Received:2010-04-19 Revised:2010-05-24 Online:2010-07-15 Published:2010-07-15

摘要:

【目的】从环化的中国华东葡萄白河35-1经白粉菌接种诱导后的叶片所构建的cDNA文库中,筛选抗病相关基因序列。【方法】根据植物抗病基因在氨基酸水平上的保守结构域NBS的P-loop区和LRR设计特异引物,采用三维文库筛选法,对环化的葡萄白粉菌接种诱导后的叶片cDNA文库进行PCR筛选。【结果】共建组池24个,每个组池中含有单菌落768个,包含18 432个克隆。从中筛选出具有抗病基因保守序列的阳性克隆585个,其中具有NBS保守序列引物筛选出330个,LRR保守序列引物筛选出255个。对获得的阳性克隆中的插入片段进行了测序,经生物信息学分析去除重复序列后,获得422条具有抗病基因保守结构域的基因序列,其中具有NBS保守序列的250条,具有LRR保守序列的172条,共有NBS和LRR保守序列的25条。【结论】经翻译分析,其中8条编码产物包含NBS的P-loop结构域(GW787739—787742、GW787746、GW787747、GW787749、 GW787758),5条编码产物包含LRR结构(GW787743、GW787744、GW787750、GW787751、GW787753),1条华东葡萄特有基因(GW787754)。从中选出具有不同抗病结构域的10条基因进行病源侵染过程中的基因实时定量表达分析,发现其中5条抗病相关基因的表达可被白粉菌接种所诱导,可能与白粉病侵染过程有关。

关键词: 华东葡萄, cDNA文库, 文库筛选, 抗病基因, 定量PCR

Abstract:

【Objective】 This research aimed to screen resistance genes from cycled Chinese wild grape Vitis pseudoreticulata cv. Baihe35-1 cDNA library which was constructed from leaves induced by grape powdery mildew (Uncinular necator) inoculation. 【Method】Specific oligonucleotide primers were designed according to P-loop region of conservative NBS and LRR sequence of resistance genes and the universal sequence primers on cloning vector. According to the method of three dimensional library, colony PCR was employed to screen the circled cDNA library. 【Result】 Twenty-four super pools, each contains 768 colonies, were built, obtaining 18 432 colonies, and 585 positive colonies were obtained, in which 330 by NBS primers and 255 by LRR primers. The sequencing results were assembled into 422 contig sequences that 250 and 172 sequences from NBS and LRR primer screening respectively, including 25 sequences from the both. 【Conclusion】 There were 8 sequences (GW787758, GW787739—GW787742, GW787746, GW787747, GW787749) belong to P-loop superfamily and 5 sequences (GW787743, GW787744, GW787751, GW787753, GW787750) with LRR and a specific gene in Vitis pseudoreticulata (GW787754). Ten sequences which have different resistance domains were chosen for real-time quantitative PCR to check the expression profile in the process of white powdery mildew invasion. Five showed significant variation during Uncinular necator induction and supposed to be a pathogenesis related gene.

Key words: Vitis pseudoreticulata, cDNA library, library screening, disease resistance gene, real-time quantitative PCR