中文摘要：

本研究根据转录组数据提供的基因片段设计特异引物, 利用cDNA快速末端扩增方法克隆丹参4-羟基-3-甲基-2-丁烯基焦磷酸还原酶全长cDNA （SmHDR）, GenBank 注册号JX233817, 并进行蛋白结构预测、序列多重比对和构建进化树等生物信息学分析; 然后采用实时荧光定量PCR （RT-PCR） 检测该基因在Ag＋ 诱导后的表达情况, 使用UPLC检测对应样品的丹参酮类化合物含量。获得的SmHDR全长基因由1 647个核苷酸组成, 编码463个氨基酸, 蛋白分子质量为51.88 kD, 等电点pI 5.72, 二级结构中α-螺旋结构占35.64%、β-折叠占20.30%、无规则卷曲占44.06%。序列比对和系统进化分析表明, SmHDR与其他植物HDR家族具有较高的同源性, 并与库洛胡黄连HDR两者的亲缘关系较近。RT-PCR结果显示, SmHDR受Ag＋ 诱导后表达水平在12 h时急剧上升后显著下降, 丹参酮类成分含量受Ag＋ 诱导后先缓慢上升, 在120 h时有明显提高, 两者的增加趋势呈正相关。推测SmHDR基因可能参与丹参酮类成分的生物合成, 这为进一步研究丹参酮类成分生物合成及其次生代谢调控机制奠定了基础。

英文摘要：

This study reported the obtainment of the full-length cDNA of Salvia miltiorrhiza hairy roots （Abbr： SmHDR, GenBank number： JX233817）, via extracting Salvia miltiorrhiza hairy roots total RNA, designing specific primers according to the transcriptome data and using the RACE strategy, and then analyzed it with bioinformatics approaches. On this basis, using the real-time PCR to detect SmHDR gene expression after Ag＋ induction, and testing tanshinones contents of corresponding samples by UPLC. SmHDR has 1 647 nucleotides, and an open reading frame （ORF） encoding a protein of 463 amino acid residues. The deduced protein has isoelectric point （pI） of 5.72 and a calculated molecular weight about 51.88 kD. In the secondary structure, the percentage of alpha helix, beta turn and random coil were 35.64%, 20.30% and 44.06%, respectively. Sequence alignment and phylogenetic analysis demonstrated that SmHDR had relative close relationship to the HDR of Picrorhiza kurrooa, similar to HDR from other species of plants. Real time PCR results indicated that elicitor of Ag＋ stimulated the increase of mRNA expression of SmHDR. At the same time, results of ultra performance liquid chromatography （UPLC）, used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy roots of Salvia miltiorrhiza were increased dramatically at 12 h after treated with Ag＋, and then decreased significantly. This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by Ag＋. The content of tanshinones was gradually raised, and it had an obvious increase at 120 h. The bioinformatics analysis and gene expression indicated that SmHDR might be involved in tanshinones biosynthesis, which laid the foundation for further study of secondary metabolic regulation mechanism of tanshinones.