铁皮石斛蔗糖合成酶基因的克隆及表达分析

Molecular cloning and expression analysis of sucrose synthase gene from Dendrobium officinale

目的:克隆铁皮石斛Dendrobium officinale蔗糖合成酶基因并对其表达分析,为研究铁皮石斛多糖合成与蔗糖合成酶活性关系及调控表达提供理论依据。方法:基于GenBank上已知的蔗糖合成酶基因SS同源序列,应用RT-PCR和RACE等方法,克隆铁皮石斛蔗糖合成酶基因全长,将目的片段转化大肠杆菌表达菌株BL21中,IPTG诱导表达,SDS-PAGE电泳分析。结果:成功克隆了铁皮石斛蔗糖合成酶基因,该基因cDNA全长2 424 bp,编码807个氨基酸(命名为DOSS1),登录号HQ856835,该基因氨基酸序列与兰科植物M okara yellow(AF530568)同源性达到95%,与Oncidium goldiana(AF530567)同源性达到90%,与禾本科植物的SS基因同源性在80%以上。并对其进行大肠杆菌原核表达诱导,诱导的工程菌能明显表达出一条相对分子质量约为92×103左右的蛋白质,与理论相对分子质量基本相符。结论:成功克隆了铁皮石斛蔗糖合成酶基因,并诱导出一条与理论蛋白相对分子质量相符的蛋白条带。

Objective ： Clone of sucrose synthase of Dendribium officinale and expression analysis, to provide the theory basis for research the relationship between polysaccharide synthesis of D. officinale and sucrose synthase activity. Method：  According to homologous sequence of sucrose synthase gene on GenBank, application the technology of RT-PCR and RACE, clone the full length of D. officinale. Target gene amplified with T vector was transformed into competent E. coli. BL21, IPTG induced expression, SDS-PAGE analysis. Result：  A full length cDNA encoding sucrose synthase was isolated from the D. officinale, named DOSS1 , the GenBank accession number is HQ856835, the cDNA is 2 781 bp in length containing an open reading frame of 2 424 bp encoding 807 amino acids with a predicted molecular mass of 92.3×103, the deduced amino acid sequence of D. officinale sucrose synthase shares 95% identity with Mokara yellow（AF530568）; shares 90% identity with Oncidium goldiana（AF530567）; shares more than 80%with other monocotyledonous plants. Conclusion：  Cloned the sucrose synthase gene and induced an obvious band successfully.