利用Gateway克隆技术构建丹参SmNAC1转录因子的RNAi表达载体  

Construction of RNAi vectors for SmNAC1 transcription factors of Salvia miltiorrhiza using Gateway cloning technology

作　　者：赵容[1,2] 荣齐仙[2] 刘玉忠[2] 申业[2] 黄璐琦[2] 

机构地区：[1]黑龙江中医药大学,黑龙江哈尔滨150040 [2]中国中医科学院中药资源中心,北京100700

出　　处：《中国中药杂志》2014年第9期1569-1573,共5页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81173490;81130070)

摘　　要：NAC转录因子参与植物生长发育和对生物和非生物胁迫的应答反应,利用Gateway克隆技术构建丹参NAC转录因子的RNAi植物表达载体,以便进一步研究丹参NAC转录因子的功能。根据Gateway技术要求,设计含有attB接头的引物,使用NEB的Phusion超保真聚合酶,通过PCR方法,扩增SmNAC1基因的特异性片段。通过BP重组反应,将带有attB接头的PCR产物克隆到入门载体pENTR/SD/D-TOPO上,再通过LR重组反应,利用入门载体上的SmNACi特异性基因片断克隆到植物表达载体pK7GWIWG2D上。实验结果表明Gateway载体的构建方法能够高效、快速地将目的基因克隆到表达载体上,为植物基因转化提供基础。NAC transcription factors involved in plant growth and development, as well as responses to biotic and abiotic stress. RNAi Vectors for SmNAC transcription factors of Salvia miltiorrhiza was constructed by using Gateway cloning technology, in order to further study the function of SmNAC1 transcription factor. According to Gateway cloning technology, the specific fragments of SmNAC1 containing attB adapter was amplified by PCR using ultra-fideling phusion polymerase of NEB. By the BP recombination reaction, the PCR product containing attB was transferred to an donor vector （pENTR/SD/D-TOPO） . Finally, SmNACi specific gene was cloned into pK7GWIWG2D plant expression vectors by LR recombination reaction. Experimental results showed that Gateway cloning technology provide a rapid and highly efficient way to clone the interested gene.

关 键 词：RNAI Gateway载体 BP和LR反应 

分 类 号：R341[医药卫生—基础医学]