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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 clone Dendrobium officinale phosphoenolpyruvate carboxylase (pepc) gene from Dendrobium officinale and analyze its expression in F and H Dendrobium officinale so as to provide basis for studying the structure and function of Dendrobium officinale pepc gene. Methods Primers were designed based on the homologous sequences of known pepc genes in GenBank. The full-length cDNA of pepc gene was cloned by RT-PCR and RACE. The expression of pepc gene in two species of Dendrobium officinale was studied by fluorescence quantitative PCR. Results The pepc gene of Dendrobium candidum was successfully cloned. The accession number was JF423930. The full-length cDNA was 3 560 bp in length, of which the cds sequence was 2 895 bp. The homology with Orchidaceae was about 80% Homology over 70%, encoding 964 amino acids. The expression level of pepc gene in F-type Dendrobium candidum is 5.55 times of that of H type. Conclusion The pepc gene of Dendrobium candidum was successfully cloned, and the expression of pepc gene of Dendrobium candidum was higher than that of H type.