岗梅磺酸基转移酶基因的克隆与体外表达

Cloning and in vitro expression of sulfotransferase genes from Ilex asprella

目的药用植物岗梅Ilex asprella含有多种活性磺化三萜类成分,克隆岗梅中的磺酸基转移酶编码(sulfotransferase,ST)基因,以了解其独特的磺化机制。方法利用RT-PCR技术,由岗梅叶片RNA扩增获得了2个可能的磺酸基转移酶基因IaST1和IaST2,利用生物信息学工具对其进行分析,并将其转化至大肠杆菌E. coli进行原核表达。结果序列分析显示,IaST1与IaST2各包含1个开放阅读框,分别长1002bp和993bp,编码333和330个氨基酸,理论相对分子质量为55500和54700。二者均具有ST的5个保守结构氨基酸序列,与黄顶菊Flaveriabidentis的黄酮醇C-3磺酸基转移酶亲缘关系较近。SDS-PAGE显示在E. coli中成功表达了IaST1和IaST2蛋白。结论首次从岗梅中克隆了ST基因,为后续阐明其功能及其在岗梅三萜磺化中的作用奠定了基础。

Objective The medicinal plant Ilex asprella contains various biologically active sulfonated triterpenoids and triterpenoid saponins.The objective of this study was to clone sulfotransferases(ST)from I.asprella and facilitate the elucidation of the sulfonation mechanism therein.Methods The physicochemical properties,secondary structure and tertiary structure of two ST candidates of IaST1 and IaST2 were forecasted and analyzed using related software.IaST1 and IaST2 were cloned by RT-PCR and expressed in Escherichia coli.Results The open reading frame(ORF)of IaST1 was 1 002 bp long and encoded a protein of 333 amino acids with the calculated molecular weight of 55 500,while the ORF of IaST2 was 993 bp long and encoded a protein of 330 amino acids with the calculated molecular weight of 54 700.Both contain the five highly conserved domains of ST.Phylogenetic analysis revealed that IaST1 and IaST2 were genetically closely related and clustered together with flavonol C-3 ST of Flaveria bidentis.Conclusion This is the first report on the cloning of STs from I.asprella,providing an important basis for further investigations into their functions and roles in the biosynthesis of sulfonated triterpenoids in I.asprella.