北柴胡UGT基因的克隆及其过量表达和RNAi转基因载体的构建  

Cloning of UGT gene of Bupleurum chinense and construction of over expressing and RNAi transgenic vectors

作　　者：隋春[1,2] 徐洁森[1,2] 赵立子[1,2] 魏建和[1,2] 徐艳红[1,2] 孙鹏[1,2] 

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所,北京100193 [2]中草药物质基础与资源利用教育部重点实验室,北京100193

出　　处：《中国中药杂志》2012年第5期558-563,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81072994);北京市自然科学基金项目(5102033);中医药行业科研专项(201107011)

摘　　要：目的:克隆北柴胡中可能参与柴胡皂苷生物合成的UGT基因,构建其过表达和抑制表达的转基因载体,为通过转基因验证其功能奠定基础。方法:在454高通量测序已获得部分cDNA序列基础上,通过RACE和LD-PCR方法扩增全长cDNA克隆。设计含有酶切位点的PCR引物,PCR扩增UGT基因的ORF和部分片段后,酶切,分别插入到pCAMBIA-SUPER1 300和pHANNIBAL中。重组的pHANNIBAL经Not I酶切后插入到pART27中。最终以构建出过表达和抑制表达的转基因载体。结果:扩增到了北柴胡1个UGT基因全长cDNA克隆,构建了这一基因的过表达和抑制表达转基因载体。结论:通过UGT基因的全长cDNA克隆和转基因载体构建,为后续开展转基因研究,验证其生物功能奠定了基础。Objective： To clone the full-length cDNA of a uridine diphosphate glycosyltransferase（UGT） gene which may be involved in saikosaponin biosynthesis of Bupleurum chinense,and construct the transgenic vectors for over expression and RNAi of the cloned UGT.These works will provide foundation for further its function analysis by transgene study.Method： RAGE and LD-PCR were used to clone the full-length cDNA of the UGT,on the basis of its partial cDNA sequence obtained from our previous 454-sequenced dataset.The ORF and partial sequences of the UGT were PCR cloned using primers with corresponding restriction enzymes cutting sites.The PCR products were digested with corresponding restriction enzymes and then were inserted into pCAMBIA-SUPER 1 300 and pHANNIBAL.The recombinant pHANNIBAL were digested with Not I and then were inserted into a binary vector,pART27.Finally,the transgenic vectors for over expression and RNAi of the cloned UGT were constructed.Result： The full-length cDNA of a UGT were cloned from B.chinense.The recombinant vectors for over expression and RNAi of the UGT were obtained.Conclusion： Our works on full-length cDNA cloning and transgenic vectors construction provide a substantial foundation for follow-up bio-function analysis of the UGT through transgenic research.

关 键 词：北柴胡 UGT基因 转基因载体 

分 类 号：S567.79[农业科学—中草药栽培]