滇重楼鲨烯环氧酶基因的克隆及原核表达研究

发布时间：2018-03-07 22:32

本文选题：滇重楼　切入点：鲨烯环氧酶　出处：《中草药》2017年09期 　论文类型：期刊论文

【摘要】：目的克隆滇重楼Paris polyphylla var.yunnanensis三萜皂苷生物合成途径中的关键酶鲨烯环氧酶(squalene epoxidase,SE)基因,并进行原核表达。并用Real-time PCR法检测cDNA样本中SE1基因和SE2基因的相对量。方法以滇重楼根为材料提取总RNA并反转录为cDNA。以cDNA为模板,根据转录组数据中的2组SE基因全长序列设计特异性引物,对SE基因进行克隆后转入到pEASY-T1 Simple Cloning Vector中,经测序鉴定正确后,构建pEASY-E1-SE表达载体,利用Art Media protein Expression/Amp+培养基自动诱导表达。Real-time PCR法检测cDNA样本中SE1和SE2基因的相对量。结果获得了2条滇重楼SE基因,命名为ppSE1和ppSE2。ppSE1的全长为1 932 bp,开放阅读框(ORF)为1 578 bp,编码525个AA;ppSE2的全长为1 828 bp,ORF长为1 548 bp,编码515个氨基酸。荧光定量PCR结果显示ppSE1基因和ppSE2基因在茎和叶中的表达具有显著差异,ppSE1的表达在叶中最为显著。酶切和测序结果表明,原核表达载体pEASY-E1-SE构建成功;SDS-PAGE分析显示,在BL21(DE3)表达感受态细胞中成功诱导表达了SE基因的2种融合蛋白。结论克隆了滇重楼的SE基因,获得了在体外具有生物学活性的SE蛋白。ppSE1基因和ppSE2基因在滇重楼中具有不同的表达模式,在滇重楼次生代谢产物的合成中起着不同的作用。
[Abstract]:Objective to clone the key enzyme squalene epoxidaseSEE in the biosynthesis pathway of Paris polyphylla var.yunnanensis triterpenoid saponins. The relative amounts of SE1 gene and SE2 gene in cDNA samples were detected by Real-time PCR method. Methods Total RNA was extracted from root of Rhizoma Polygonum yunnanensis and reversed to cDNA. CDNA was used as template. According to the full length sequence of two sets of SE gene in transcriptional data, specific primers were designed and cloned into pEASY-T1 Simple Cloning Vector. After sequencing, the pEASY-E1-SE expression vector was constructed. The relative quantities of SE1 and SE2 genes in cDNA samples were detected by means of Art Media protein Expression/Amp medium. The results showed that two SE genes were obtained. The total length of ppSE1 and ppSE2.ppSE1 was 1 932 BP, the open reading frame was 1 578 BP, the length of ppSE1 and ppSE2 gene was 1 548 BP and 1 548 BP, respectively. The results of fluorescence quantitative PCR showed that ppSE1 gene and ppSE2 gene were in stem and leaf. The expression of PppSE1 was the most significant in the leaves. The results of restriction endonuclease digestion and sequencing showed that, SDS-PAGE analysis of prokaryotic expression vector pEASY-E1-SE showed that two kinds of fusion proteins of SE gene were successfully induced and expressed in BL21DE3 cells. Conclusion the SE gene of Rhizoma Dianopsis was cloned. Different expression patterns of SE protein, ppSE1 gene and ppSE2 gene were obtained in vitro and played different roles in the synthesis of secondary metabolites.
【作者单位】：云南中医学院药学院;
【基金】：国家自然科学基金资助项目(81160502) 云南省自然科学基金资助项目(2011FZ153) 云南省教育厅自然科学基金资助项目(2013J040);云南省教育厅自然科学基金资助项目(2014J063)
【分类号】：S567.239

【相似文献】