應用GatewayTM技術構建煙草抗真菌病相關基因的RNAi載體

趙百英，李鳳霞，楊愛國，孫玉合，蘇振剛，楊帆，劉慧，王元英

摘要：根據(jù)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絨毛狀煙草（Nicotiana tomentosiformis）全基因組上獲得6個完整可靠的抗真菌病候選基因。在這6個基因的非保守區(qū)設計特異引物，以普通煙草品種紅花大金元反轉錄cDNA為模板克隆抗真菌病相關基因的干擾片段，利用GatewayTM克隆技術構建上述候選基因的RNAi載體，經(jīng)酶切和測序證明載體構建正確，為進一步鑒定這6個候選基因的具體抗病性，并為將其應用于煙草抗病新品種的選育工作奠定了基礎。

關鍵詞：煙草（Nicotiana）；GatewayTM技術；RNAi載體；R基因；相似性；結構域

中圖分類號：S572 文獻標識碼：A 文章編號：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