应用GatewayTM技术构建烟草抗真菌病相关基因的RNAi载体

赵百英，李凤霞，杨爱国，孙玉合，苏振刚，杨帆，刘慧，王元英

摘要：根据抗病基因（Resistance genes，R基因）的功能保守域，利用生物信息分析方法在绒毛状烟草（Nicotiana tomentosiformis）全基因组上获得6个完整可靠的抗真菌病候选基因。在这6个基因的非保守区设计特异引物，以普通烟草品种红花大金元反转录cDNA为模板克隆抗真菌病相关基因的干扰片段，利用GatewayTM克隆技术构建上述候选基因的RNAi载体，经酶切和测序证明载体构建正确，为进一步鉴定这6个候选基因的具体抗病性，并为将其应用于烟草抗病新品种的选育工作奠定了基础。

关键词：烟草（Nicotiana）；GatewayTM技术；RNAi载体；R基因；相似性；结构域

中图分类号：S572 文献标识码：A 文章编号：0439-8114（2013）05-1190-06

Construction of Tobacco RNAi Vectors for Fungi Resistance-related Genes Using GatewayTM Technology

ZHAO Bai-ying1，LI Feng-xia1，YANG Ai-guo1，SUN Yu-he1，SU Zhen-gang2，YANG Fan1，

LIU Hui1，WANG Yuan-Ying1

（1.Tobacco Research Institute， Chinese Academy of Agricultural Sciences /Key Laboratory of Tobacco Genetic Improvement and Biotechnology， Qingdao 266101，Shandong，China； 2.Hongta Tobacco （Group） Co.， Ltd.， Yuxi 653100，Yunnan， China）

Abstract： According to the conserved domain of resistance genes（R gene）， six disease resistance candidate genes were obtained from the genome of Nicotiana tomentosiformis by bioinformatics. Specific primers was designed according to the non-conservative regions of these six genes， and applied for cloning of interference clips of fungus-resistant genes using reverse transcriptase cDNA of N. tobacum cv. Honghuadajinyuan as templates. Then the RNAi vectors of these six candidate genes were constructed through GatewayTM cloning techniques. According to restriction endonuclease digestion and sequencing， the RNAi vectors was correctly constructed， which lay a foundation for further identification of the six candidate genes and applying in breeding of tobacco disease resistant varieties.

Key words： tobacco（Nicotiana）； GatewayTM technology； RNAi vectors； resistance genes； identity； domain