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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。
Objective To clone the squalene epoxidase (SE) gene, which is the key enzyme in biosynthesis pathway of triterpene saponin from Paris polyphylla var. Yunnanensis, and express it in prokaryotic cells. Real-time PCR was used to detect the relative amounts of SE1 and SE2 in cDNA samples. Methods The total RNA was extracted from the roots of D. orientalis and reverse transcribed into cDNA. Using the cDNA as a template, specific primers were designed based on the full-length sequence of two SE genes in the transcriptome data. The SE gene was cloned and transformed into pEASY-T1 Simple Cloning Vector. After sequencing, the pEASY-E1 -SE expression vector, using Art Media protein Expression / Amp + medium automatically induced expression. The relative amount of SE1 and SE2 genes in cDNA samples was detected by Real-time PCR. Results Two SEB genes were obtained, named ppSE1 and ppSE2. The full length of ppSE1 was 1 932 bp and the open reading frame (ORF) was 1 578 bp encoding 525 AA. The full length of ppSE1 was 1 828 bp with an ORF of 1 548 bp encoding 515 amino acids. Fluorescent quantitative PCR results showed that the expression of ppSE1 gene and ppSE2 gene in stems and leaves were significantly different, and the expression of ppSE1 was the most significant in leaves. The results of enzyme digestion and sequencing showed that the prokaryotic expression vector pEASY-E1-SE was constructed successfully. SDS-PAGE analysis showed that the fusion protein of SE gene was expressed successfully in BL21 (DE3) competent cells. Conclusion The SE gene was cloned and the SE protein was obtained in vitro. The ppSE1 gene and the ppSE2 gene have different expression patterns in D. reticulata, which play different roles in the synthesis of secondary metabolites of D. reticulata.