中间偃麦草RAR1基因的分离及其在小麦背景中的功能分析  

Isolation of RAR1 Gene from Thinopyrum intermedium and Analysis of Its Function in Wheat Background

作　　者：廖勇[1,2] 张增艳[1] 杜丽璞[1] 徐惠君[1] 姚乌兰[1] 石建业[1] 任正隆[3] 辛志勇[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 [2]四川农业大学农学院,雅安625000 [3]四川农业大学农学院

出　　处：《中国农业科学》2007年第8期1667-1674,共8页Scientia Agricultura Sinica

基　　金：国家"863"项目(2006AA10A104)

摘　　要：【目的】RAR1是大麦抗白粉病基因和多种植物抗病(resistance,R)基因介导的抗病信号途径中的重要信号元件。为明确RAR1在小麦抗白粉病反应中的作用,为小麦广谱抗病分子育种提供基因资源,开展了本研究。【方法】采用生物信息学技术、RT-PCR和RACE方法分离克隆中间偃麦草RAR1基因,通过酶切和连接构建该基因的单子叶高效表达载体,采用基因枪法转化小麦,对转基因小麦植株进行分子检测、表达分析和抗病鉴定。【结果】分离克隆出中间偃麦草RAR1基因,其编码蛋白具有典型的RAR1功能结构域;构建了中间偃麦草RAR1基因的单子叶高效表达载体pUBI∷RAR1;用基因枪法轰击小麦推广品种扬麦12幼胚愈伤组织1545个,获得152株再生植株,通过PCR检测出阳性植株18株,转化率为1.17%;对T1、T2代转基因材料进行PCR检测、RT-PCR分析和白粉病抗病鉴定,结果表明转入的RAR1基因能够在小麦背景中遗传,RAR1基因的超量表达可提高小麦对白粉病菌的抗性、拓宽小麦的抗病谱。【结论】分离出中间偃麦草RAR1基因,RAR1基因在小麦中超量表达可提高小麦对白粉病菌的抗性、拓宽其抗病谱,可以作为小麦白粉病广谱抗性分子育种的潜在的重要基因资源。[ Objective ]The study established the foundation to understand the role of RAR1 gene in wheat resistance to diseases especially powdery mildew, as RAR1 is a required component in some signaling pathways mediated by disease resistance （R） genes in many plant species. [Method] RT-PCR and RACE methods were used to isolate the full-length cDNA sequence of the RAR1 gene from Thinopyrum intermedium, a wheat wild-relative with multi-resistance to various pathogens. The gene expression vector pUBI： ：RAR1 was constructed and then transformed into a wheat variety ‘Yangmai 12＇ by the biolistic particle method. The regenerated transgenic plants were detected through PCR, RT-PCR and test of the resistance to powdery mildew. [Result] The full-length cDNA of the RAR1 gene was isolated from Thinopyrum intermedium, named TiRAR1, which encodes the RAR1 protein possessing 2 CHORD domains and 1 CCCH domain, The sequence of the TiRAR1 protein shows high homology with the sequences of RAR1 proteins in barley and rice. The expression vector of TiRAR1 gene, pUBI：：RAR1, was correctly constructed, and transformed into the wheat variety ‘Yangmai 12＇. 1545 immature embryos of ＇Yangmai 12＇ were bombarded by the particle containing pUBI： ：RAR 1. From the regenerated 154 plants, 18 positive transgenic individuals were identified by PCR assay in the To generation with a transformation frequency of 1.17%. The results of PCR and RT-PCR assay showed that the RAR1 gene could beinheritable from To generation to Tt one, then from Tt generation to T2 one, and could be transcribed and expressed. The resistance test showed that the RAR1 transgenic individuals in Tt and T2 generations showed high resistance to powdery mildew in their all lifecycle, suggesting that the overexpression of RAR1 enhanced the resistance to powdery mildew. [Conclusion] The full-length cDNA of the RAR1 gene in Thinopyrum intermedium was isolated and transformed into a wheat variety ＇Yangmal 12＇. The RAR1 gene could confer high resistance to powde

关 键 词：RAR1 分离 小麦 转基因 PCR检测 抗病性 

分 类 号：S512.1[农业科学—作物学]