利用BSMV-VIGS技术快速分析小麦TNBL1基因的抗黄矮病功能  

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

作　　者：赵丹[1,2] 赵继荣[1] 黄茜[1,2] 李宁[1] 刘艳[3] 黄占景[1,2] 张增艳[1] 

机构地区：[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 [3]中国农业科学院植物保护研究所,北京100093

出　　处：《作物学报》2011年第11期2106-2110,共5页Acta Agronomica Sinica

基　　金：国家高技术发展计划(863计划)重点项目(2006AA10A104)资助

摘　　要：小麦黄矮病是由蚜虫介导的大麦黄矮病毒(Barley yellow dwarfvirus,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292bp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。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 added with 2 restriction-enzyme sequences by PCR, and digested and ligased with the digested BSMV-γ, the recombinant BSMV-γ：TNBL1as construct was obtained. The three com-ponents 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.

关 键 词：小麦 中间偃麦草 黄矮病 病毒诱导的基因沉默 NBS-LRR 

分 类 号：S512.1[农业科学—作物学]