滇重楼鲨烯环氧酶基因的克隆及原核表达研究

Cloning and expression of squalene epoxidase from Paris polyphylla var. yunnanensis

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 and express a full-length cDNA encoding squalene epoxidase which is key to the biosynthetic pathway of triterpenoid saponins in Paris polyphylla var. yunnanensis. Using real-time PCR method to detect the relative content of SE1 gene and SE2 gene from the cDNA sample. Methods Total RNA was extracted from the roots of P. polyphylla var. yunnanensis and transcription was reversed. Using the reversed transcription of cDNA as template, specific primer was designed according to the transcriptome data about two groups of SE gene sequence from the Hi Seq2500 sequencing platform, and then SE gene was cloned. The production was inserted to pEASY-T1 Simple Cloning Vector, and pEASY-E1-SE expression vector was built after sequencing appraisal right. The Art Media protein Expression/Amp＋ medium was used to induce expression automatically. Using real-time PCR method to detect the relative contents of SE1 gene and SE2 gene from the cDNA sample. Results Two SE genes of P. polyphylla var. yunnanensis were obtained, which were named as ppSE1 and ppSE2, repectively. cDNA was 1 598 bp of ppSE1 and 1 509 bp of ppSE2, respectively. The full length for ppSE1 was 1 932 bp, length of ORF was 1 578 bp, coding 525 AA; The full length for ppSE2 was 1 828 bp, length of ORF was 1 548 bp, coding 515 AA. Fluorescence quantitative PCR results showed that the expression of ppSE1 and ppSE2 genes had significant differences in stem and leaf, and the expression of ppSE1 was most pronounced in the leaf. Enzyme digestion and sequencing results showed that the prokaryotic expression vector pEASY-E1-SE was built successfully. SDS-PAGE analysis showed that two fusion proteins of SE genes were induced expression successfully in BL21（DE3） express competent cells. Conclusion The SE gene of P. polyphylla var. yunnanensis has been cloned, and the SE protein with biological activity in vitro has been obtained. The ppSE1 and ppSE2 genes have different expression patterns in P. polyphylla var. yunnanensis, and play a different role in the synthesis of secondary metabolites.