铁皮石斛磷酸烯醇式丙酮酸羧化酶基因的克隆及表达分析

Cloning and expression analysis on phosphoenolpyruvate carboxylase gene in Dendrobium officinale

目的克隆铁皮石斛Dendrobium officinale磷酸烯醇式丙酮酸羧化酶(pepc)基因,并对其在F型和H型铁皮石斛中的表达进行分析,为研究铁皮石斛pepc基因的结构、功能提供依据。方法基于GenBank上已知的pepc基因的同源序列设计引物,应用RT-PCR和RACE等方法,克隆铁皮石斛pepc基因全长。采用荧光定量PCR方法研究pepc基因在2种铁皮石斛中的表达。结果成功克隆了铁皮石斛pepc基因,登录号为JF423930,该基因全长为3 560 bp,其中cds序列为2 895 bp,与兰科植物的同源性为80%左右,与其他科属植物的同源性达到70%以上,编码964个氨基酸。pepc基因在F型铁皮石斛中的表达量为H型的5.55倍。结论成功克隆了铁皮石斛pepc基因,且F型铁皮石斛pepc基因的表达量高于H型。

Objective To study the structure and function of phosphoenolpyruvate carboxylase（pepc） gene in Dendrobium officinale,pepc was cloned and gene sequence was analyzed.Expression of pepc gene in H and F type of D.officinale was analyzed.Methods Primers were designed by analyzing the homologous sequence of pepc gene in GenBank,and complete pepc sequence in D.officinale was cloned by RT-PCR and RACE.Expression of pepc gene was detected by fluorescent quantitative PCR in two kinds of D.officinale.Results The complete sequence（3 560 bp） of pepc gene in D.officinale was cloned（Genebank accession number： JF423930）,including 2 895 bp cds sequence,and shared 80% identity with orchid plants and 70% identity with other plants.The pepc cDNA encoded a precursor protein of 964 amino acids.Expression of pepc gene of F type D.officinale was 5.55 times as that of H type.Conclusion The complete sequence of pepc gene in D.officinale is cloned,and expression of pepc in F type D.officinale is higher than H type.