摘要
克隆并鉴定何首乌中二苯乙烯合成酶基因FmSTS2,并研究其在何首乌各组织的表达与二苯乙烯苷表达的关系,为研究二苯乙烯合成酶基因在二苯乙烯苷合成及其调控中的功能奠定基础。采用互补cDNA末端快速扩增技术克隆目的片段,得到一种二苯乙烯合成酶基因FmSTS2(GeneBank登录号:JX914503),其互补cDNA全长1 644 bp。将FmSTS2连接pET-28a原核表达载体并转化BL21大肠杆菌构建原核表达系统,Ni-NTA亲和树脂纯化可溶性蛋白后以丙二酰-COA和香豆酰-COA为底物进行催化反应,HPLC鉴定反应产物为含有二苯乙烯母核的白藜芦醇,证明FmSTS2是一种二苯乙烯合成酶基因。HPLC测定何首乌各组织二苯乙烯苷含量;RT-PCR检测何首乌的块根,老藤,茎,叶中表达FmSTS2的程度依次降低,与二苯乙烯苷在各个组织的含量高低一致。
In order to provide information fi^r further research on the functions of stilbene synthase（STS） in the biosynthesis path- way and metabolic regulatiun of Stilbene Glycoside of Fallopia multiflora,the full-length eDNA of a new gene designated as FroSTS2 （JX914503） was cloned using RACE from Fallopia multiflora and its eDNA full-length is 1 644 bp. The FroSTS2 gene was trans- formed into prokaryotic expression eells E. coli BL21 and sueeessfully expressed in the form of soluble proteins. Soluhle protein was purified by Ni-NTA chelating sepharose ＇affinity chromatography. It catalyzed malonyl-CoA and eoumaroyl-CoA, of whieh enzymatie product was identified to be resw,ratrol by HPLC. RT-PCR was performed for FmSTS2 gene expression in different tissues of Fallopia multiflora and showed that rhizome expresss FroSTS2 most,old stem expresss FmSTS2 more and the young stem and leaf express FroSTS2 least. The content of stilhene glycoside in different F. multiflora tissues was determined by HPLC. The expression of FroSTS2 gene was correspondent to the expression of stilbenes in different tissues of Fallopia multiflora.
出处
《药物生物技术》
CAS
2013年第2期124-127,共4页
Pharmaceutical Biotechnology
基金
广东省自然科学基金(No.10151001002000012)
关键词
FmSTS2
二苯乙烯苷
原核表达
活性鉴定
基因表达
FmSTS2.Stilbene glyeoside,Prokaryotie expression,Aetivity Identification,Gene expression
