目的 从铁皮石斛Dendrobium officinale中克隆1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶 [1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphatereductase,HDR] 基因,并分析其在铁皮石斛不同组织中的表达差异以及不同信号分子诱导下的表达模式.方法 采用RT-PCR和RACE等方法获得铁皮石斛HDR基因(DoHDR)全长,利用DNAMAN和MEGA6.0对其他物种的HDR基因编码的氨基酸序列进行同源性分析和进化关系分析,使用实时荧光定量分析HDR基因的表达模式.结果 成功获得DoHDR基因,GenBank登录号为KC344827,全长1 658 bp,编码460个氨基酸,与其他科属植物的同源性达到80%以上.DoHDR基因在铁皮石斛叶片中表达量最高,从高到低依次是根、茎、原球茎;且受到脱落酸(abscisic acid,ABA)、水杨酸(salicylic acid,SA)信号分子的诱导.结论 从铁皮石斛中获得DoHDR基因,为进一步阐明铁皮石斛萜类化合物合成途径中该基因的重要作用奠定了理论基础.

Objective To clone the full-length cDNA encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphatereductase (HDR) gene from Dendrobium officinale (DoHDR), then to analyze the expression difference in different tissues and expression patterns of DoHDR induced by signal molecule. Methods RT-PCR and RACE technologies were used to clone the full length cDNA of DoHDR. The analyses of homologous comparison and phylogenetic tree were performed using DNAMAN and MEGA6.0 softwares, then the expression patterns of DoHDR were studied by real-time PCR. Results The DoHDR gene was successfully obtained (GenBank accession number KC344827), and the full-length cDNA was 1 658 bp, coding the protein containing 460 amino acids. DoHDR had high homology (≥ 80%) with HDR proteins from other plants. Tissue expression analysis showed that DoHDR had the highest expression in the leaves, followed by roots, stems, and protocorm. Quantitative PCR results showed that DoHDR could be induced by signal molecule such as abscisic acid (ABA) and salicylic acid (SA). Conclusion The cDNA encoding DoHDR is cloned. It is helpful for the future research on the mechanism of terpenoid biosynthesis in D. officinale.

安徽省科技攻关项目(1301032139);国家级大学生创新创业训练计划项目(201310364006)