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  • 张岳平,邹学校.新模式植物短柄二叶草SGT1 和RAR1 基因沉默和蛋白纯化载体构建[J].植物遗传资源学报,2011,12(1):138-144.    [点击复制]
  • .Construction of Effective High-Throughout Inducible RNAi and Tap-tag Vector for SGT1 and RAR1 Genes in the New Model Plant Brachypodium distachyon[J].植物遗传资源学报,2011,12(1):138-144.   [点击复制]
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新模式植物短柄二叶草SGT1 和RAR1 基因沉默和蛋白纯化载体构建
张岳平,邹学校
0
(中南大学 研究生院隆平分院;湖南省农业科学院)
摘要:
短柄二叶草(Brachypodium distachyon)作为一种新型的模式植物,已成为当前研究热点。其具有基因组小、生长周期短、生存环境简单和容易人工转化等重要生理和遗传特性,被认为是一种潜力巨大的人类研究能源和粮食作物的重要模式植物。2010年国际短柄二叶草协会(IBI)顺利完成其全基因组测序。SGT1和RAR1基因是高度保守的并与植物抗病功能紧密相关的两个重要基因。本文采用Gateway 克隆技术对此二基因进行了高通效基因沉默表达载体的构建(ihpRNA)。阳性克隆载体经PCR、酶切和测序鉴定。为进一步研究SGT1 和RAR1 互作蛋白及其功能,采用该克隆技术将此二基因cDNA全长克隆至串联亲和纯化(Tandem affinity purification, TAP)蛋白表达载体pEarleyGate205中,为纯化SGT1和RAR1及其互作蛋白提供了基础。正确的阳性克隆载体经农杆菌介导转化至短柄二叶草Bd21获得转化株,以期进一步研究SGT1和RAR1基因及蛋白互作对于短柄二叶草抗病功能的影响。这些数据将在另文中发表
关键词:  短柄二叶草  SGT1和 RAR1  基因沉默  Gateway克隆  串联亲和纯化
DOI:
基金项目:国家“863”计划项目(2006AA10Z1A6-3);国家“十一五”重大科技攻关项目“国家粮食丰产科技工程”(2006BA520A01)
Construction of Effective High-Throughout Inducible RNAi and Tap-tag Vector for SGT1 and RAR1 Genes in the New Model Plant Brachypodium distachyon
()
Abstract:
Brachypodium distachyon which characters a small genome size, a short lifecycle, simple cultivation condition, and high transformation efficient is one of most popular model plants recently. The International Brachypodium Initiative (IBI) just sequenced the whole genome in February 2010. The SGT1 and RAR1 genes are pretty important and well conserved genes related the plant resistant disease function. RNA interference (RNAi) is an excellent and major method to study the genes’ function of plants nowadays. In this paper, we used the new powerful cloning system called Gateway Cloning to generate the high-throughout inducible RNAi gene silence vectors for these two important disease related genes in Brachypodium distachyon. Gateway Cloning system is a time-saving, easy to operating, and high efficient molecular cloning technology. It concluded two steps which called BP reaction and LR reaction respectively. At the same time we also used the Gateway Cloning to generate the protein TAP-tag fusion vectors which can be used for the study of the purification the SGT1 and RAR1 proteins and their interactive proteins in this new model plant Brachypodium distachyon. Finally we used the Agrobacterium-mediated transformation system (ATMT) to transform these RNAi and TAP-tag fusion vectors into Brachypodium distachyon genetype Bd21, and got the RNAi TAP-tag fusion plant transformants which can be used for further studying the function of these two genes related disease resistance in Brachypodium distachyon in future.
Key words:  Brachypodium distachyon  SGT1 and RAR1  Gene silence  TAP  Gateway cloning

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