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本研究采用RT-PCR法对地黄DXR基因进行克隆,并用生物信息学方法分析其功能结构域、系统进化等;将地黄DXR基因克隆到原核表达载体p ET-32a上,使用大肠杆菌BL21菌株进行Rg DXR蛋白的原核表达及纯化,实时荧光定量PCR方法分析Rg DXR在不同组织、不同内生菌诱导下的表达特性。获得的Rg DXR基因c DNA全长为1 425 bp,编码474个氨基酸。生物信息学分析发现Rg DXR基因编码的蛋白质具有典型的DXR功能结构域,属于植物DXR基因家族。系统进化树分析表明,地黄Rg DXR与芝麻亲缘关系最近。荧光定量PCR结果显示,栽培品种‘北京3号’根中Rg DXR表达量最高。内生真菌诱导实验表明,Rg DXR基因的转录水平与梓醇等环烯醚萜类物质的含量成正相关。本研究克隆所得的地黄Rg DXR基因属于DXR基因家族,推测其可能与地黄中环烯醚萜类物质的生物合成有关。
In this study, DXR gene was cloned by RT-PCR, and its functional domain and phylogenetic tree were analyzed by bioinformatics methods. DXR gene of Rehmannia glutinosae was cloned into prokaryotic expression vector p ET-32a and transformed into E.coli BL21 strain Prokaryotic expression and purification of Rg DXR protein were detected by real-time fluorescence quantitative PCR. The expression of Rg DXR in different tissues and endophytic bacteria was analyzed. The full length cDNA of Rg DXR gene was 1 425 bp, encoding 474 amino acids. Bioinformatics analysis revealed that the protein encoded by the Rg DXR gene has a typical DXR functional domain and belongs to the plant DXR gene family. Phylogenetic tree analysis showed that Rehmanniae Rg DXR has the closest genetic relationship with sesame. Fluorescent quantitative PCR results showed that Rg DXR expression was the highest in cultivar ’Beijing No.3’. Endophytic fungi induction experiments showed that Rg DXR gene transcription level and catalpol iridoid ethers terpineoids content is positively correlated. The Rg DXR gene of Rehmannia glutinosa rhizome isolated from this study belongs to the DXR gene family, suggesting that it may be related to the biosynthesis of iridoid terpenoids in Rehmannia glutinosa.