薯蓣皂苷糖苷酶基因的克隆及同源性分析

Cloning and Homology Analysis of Dioscin-glycosidase Gene from Absidia sp.G3d

[目的]从分子生物学水平上研究Absidia sp.G3d产薯蓣皂苷糖苷酶,对该酶基因CDS区的序列进行调取和分析。[方法]根据薯蓣皂苷糖苷酶的N端序列设计简并引物,以总RNA为模板进行RT-PCR调取cDNA,然后采用3'RACE和5'RACE技术,调取CDS区基因全序列。[结果]测序得到序列长度为1 497 bp。该基因的同源性比较分析表明,薯蓣皂苷糖苷酶基因与α-鼠李糖苷酶没有同源性,与α-淀粉酶的基因序列具有很高的同源性,达到93%以上。[结论]蛋白质结构存在差异,酶反应特性不同。

[Objective] A dioscin-glycosidase from Absidia sp.G3d was studied in molecular biology.Dioscin-glycosidase gene of CDS was cloned and analyzed.[Method] The primers were designed according to N-terminal sequence of dioscin-glycosidase.The partial cDNA of dioscin-glycosidase gene was obtained through the method of RT-PCR using total RNA as template.Moreover,a 1 497 bp full length CDS sequence of dioscin-glycosidase was obtained using RT-PCR,5＇-RACE and 3＇-RACE technique.Sequence homology analysis of dioscin-glycosidase gene was performed by BLAST and subsequently compared with GenBank data.[Result] There was no identity between dioscin-glycosidase gene and the known α-rhamnosidase gene in the Genbank.But there was a high homogeneity with alpha-amylase.[Conclusion] There was difference between the protein structures.And the reaction characteristics of the protein were different.