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作物学报 ›› 2011, Vol. 37 ›› Issue (11): 2106-2110.doi: 10.3724/SP.J.1006.2011.02106

• 研究简报 • 上一篇    下一篇

利用BSMV-VIGS技术快速分析小麦TNBL1基因的抗黄矮病功能

赵丹1,2，赵继荣1，黄茜1,2，李宁1，黄占景1,2，张增艳1,*   

  1. 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 / 农业部作物遗传育种重点开放实验室, 北京 100081; 2河北师范大学生命科学院, 河北石家庄 050016
  • 收稿日期:2011-02-18 修回日期:2011-06-25 出版日期:2011-11-12 网络出版日期:2011-09-06
  • 通讯作者: 张增艳, E-mail: zhangzy@mail.caas.net.cn, Tel: 010-82108781
  • 基金资助:

    本研究由国家高技术发展计划(863计划)重点项目(2006AA10A104)资助。

Functional Analysis of TNBL1 Gene in Wheat Defense Response to Barley yellow dwarf virus Using BSMV-VIGS Technique

ZHAO Dan1,2,ZHAO Ji-Rong1,HUANG Xi1,2,LI Ning1,HUANG Zhan-Jing2,ZHANG Zeng-Yan1,*   

  1. 1 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences / Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 College of Life Science, Hebei Normal University, Shijiazhuang 050016, China
  • Received:2011-02-18 Revised:2011-06-25 Published:2011-11-12 Published online:2011-09-06
  • Contact: 张增艳, E-mail: zhangzy@mail.caas.net.cn, Tel: 010-82108781

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被引次数

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摘要： 小麦黄矮病是由蚜虫介导的大麦黄矮病毒(Barley yellow dwarf virus, BYDV)侵染引起的小麦重要病害之一。利用cDNA-AFLP分析，筛选出在抗黄矮病小麦易位系YW642中特异表达的长度为292 bp的 cDNA片段，以此片段为启始序列，利用RACE和RT-PCR技术克隆出该基因的全长cDNA序列，推导该基因编码1个NBS-LRR蛋白，将其命名为TNBL1。本研究利用大麦条纹花叶病毒(BSMV)诱导的基因沉默(virus-inducing gene silencing, VIGS)技术，快速分析TNBL1是否参与小麦抗黄矮病反应。通过PCR添加酶切位点、定向酶切与连接，将TNBL1特异的292bp片段反向整合到BSMV-γ链的多克隆位点上，获得重组载体BSMV-γ: TNBL1as，体外转录BSMV-VIGS载体的3个组分(BSMV-TNBL1as、BSMV-α和BSMV-β)，等量混合、摩擦接种到抗黄矮病的小麦易位系YW642幼苗叶片上，使YW642中TNBL1基因沉默，然后接种BYDV病原进行黄矮病抗性鉴定。结果表明，TNBL1基因沉默后的YW642对BYDV敏感、显现感病症状，其体内BYDV含量较未发生基因沉默的YW642中的明显增加，证明TNBL1基因是正向调控小麦抗BYDV反应的1个重要基因。

关键词: 小麦, 中间偃麦草, 黄矮病, 病毒诱导的基因沉默, NBS-LRR

Abstract: Barley yellow dwarf virus (BYDV), transmitted by at least 25 species of aphids, causes one of the most serious virus diseases of wheat worldwide. Through cDNA-AFLP analysis, we identified a cDNA fragment with 292bp expressing in the BYDV-resistant wheat-Thinopyrun intermedium translocation line YW642, but not in susceptible wheat Zhong8601. The full-length cDNA sequence of the gene, namely TNBL1, was cloned by RACE and RT-PCR methods, which encodes a putative NBS-LRR protein. This study focused on the functional analysis of TNBL1 in wheat defense to BYDV infection using Barley stripe mosaic virus (BSMV)-based virus-inducing gene silencing method. After the specific fragment of TNBL1 was addedwith 2 restriction-enzyme sequences byPCR, and digested and ligased with the digested BSMV-γ, the recombinant BSMV-γ:TNBL1as construct was obtained. The three components of the BSMV-VIGS vectors, BSMV-TNBL1as、BSMV-α and BSMV-β were transcribed in vitro, and mixed with equal quantity and inoculated onto the first and second leaves of the resistant line YW642 seedlings at the two-leaf stage. As a result, the TNBL1 expression was obviously repressed (silenced) in YW642 treated by BSMV:TNBL1. These seedlings were further inoculated with BYDV aphids. The BYDV content was much higher in the TNBL1-silenced YW642 plants than that in the control YW642 plants without BSMV:TNBL1 treatment. Furthermore, the TNBL1-silenced YW642 plants were susceptible to BYDV infection with the viral symptom. These results indicated that the TNBL1 gene is an important gene positively involved in wheat defense response to BYDV infection.

Key words: Wheat, Barley yellow dwarf virus, Thinopyrum intermedium, Virus-induced gene silencing, NBS-LRR

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