浙贝母环阿屯醇合成酶基因5`-RACE快速扩增及序列分析  

Rapid Cloning and Sequencing Analysis of 5` RACE of Cycloartenol Synthase Gene from Fritillaria thunbergii Miq.

作　　者：朱振洪[1] 葛立军[1] 王丽丽[1] 李雪玲[1] 

机构地区：[1]浙江中医药大学生物工程学院,杭州310053

出　　处：《浙江中医药大学学报》2012年第5期558-562,共5页Journal of Zhejiang Chinese Medical University

基　　金：浙江省自然科学基金资助项目(Y2101282)~~

摘　　要：[目的]克隆浙贝母环阿屯醇合成酶(Cycloartenol synthase,CAS)5`末端cDNA序列。[方法]利用简并引物及RT-PCR方法获得CAS的核心基因,应用cDNA末端快速扩增(Rapid-amplification of cDNA ends,RACE)技术快速克隆5`末端片段,采用NCBI网站上的Protein Blast软件进行氨基酸同源性比对。[结果]从浙贝母叶片中成功地克隆出一条长691bp的CAS基因5`末端cDNA片段,含起始密码子及163bp的5`延伸区。对5`末端的150个氨基酸同源性分析表明,该片段与C.asiatica,G.glabra,A.thaliana,W.somnifera的CAS同源性分别达72%,66%,71%,71%。[结论]上述结果表明所克隆的片段为浙贝母CAS的5`末端序列,为下一步CAS全长基因的克隆及CAS在浙贝母次生代谢中的功能研究打下基础。[Objective] To clone the 5-terminal cDNA sequence of Cycloartenol synthase（CAS） in Fritillaria thunbergii Miq. [Methods] Core genes of CAS were obtained by RT-PCR using degenerate primers. 5 terminal cDNA fragment was cloned by SMART-RACE method, and sequence alignment was done by Protein-Blast software on NCBI website. [Results] A 691 bp long cDNA fragment, containing initiation codon and 163 bp 5 extension, was successfully cloned from Fritillaria thunbergii leaves. Protein sequence analysis showed that amino acid homology respectively reached 72%, 66%, 71%,71% with CAS of C.asiatica, G.glabra,A.thaliana and W.somnifera. [Conclusion]These results implied that the cloned gene should be 5 terminal fragment of CAS. It was a good foundation for full-length gene cloning of CAS and function studies of secndary metabolism of CAS in Fritillaria thunbergii Miq.

关 键 词：浙贝母 CAS 5`-RACE 克隆 序列分析 

分 类 号：R282.71[医药卫生—中药学]