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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(Do HDR)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Do HDR基因,Gen Bank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。Do HDR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Do HDR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 1-hydroxy-2-methyl-2- (E) -butenyl-4-pyrophosphate reductase from Dendrobium officinale, 4-diphosphatereductase, HDR] genes were detected and analyzed for their expression differences in different tissues of Dendrobium candidum and expression patterns induced by different signaling molecules. Methods The full-length HDR gene (Do HDR) of Dendrobium candidum was obtained by RT-PCR and RACE. Homology analysis and phylogenetic analysis were performed on the amino acid sequences of HDR genes encoded by other species using DNAMAN and MEGA 6.0. Real-time fluorescence Quantitative analysis of HDR gene expression patterns. Results The Do HDR gene was successfully obtained. The GenBank accession number is KC344827, which is 1 658 bp in length and encodes a protein of 460 amino acids. Its homology with other genera is above 80%. The expression of Do HDR gene was the highest in leaves of Dendrobium officinale, followed by roots, stems and protocorms in descending order, and was induced by abscisic acid (ABA) and salicylic acid (SA) signaling molecules. Conclusion The Do HDR gene was obtained from Dendrobium candidum, which laid a theoretical foundation for further elucidation of the important role of this gene in the synthesis pathway of terpenoids from Dendrobium officinale.