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本研究根据金龙胆草转录组数据库筛选获得了皂苷合成途径的一个关键酶基因——鲨烯环氧酶(SQE)。以筛选获得的基因序列,设计带有限制性内切酶Bam HⅠ和XhoⅠ位点的特异引物,通过RT-PCR方法对SQE基因进行克隆,并利用限制性内切酶和T4连接酶的共同作用,构建p ET30b(+)-SQE原核表达载体,导入大肠杆菌BL21(DE3)宿主细胞中,当OD600达到0.6时加入终浓度为1 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,最终以SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)检测蛋白的表达情况。结果表明本研究成功克隆了金龙胆草鲨烯环氧酶基因(Cb SQE),该基因开放阅读框为1 575 bp,编码525个氨基酸。系统发育树分析显示Cb SQE与毛曼陀罗及绞股蓝SQE基因亲缘关系比较近;酶切及测序结果表明,成功构建了Cb SQE基因原核表达载体;SDS-PAGE显示成功诱导表达了Cb SQE融合蛋白,蛋白分子量为57 k D,与理论预测结果相符合。本研究结果为解析金龙胆草的皂苷合成途径提供了一定的理论依据。
In this study, a key enzyme of squalane epoxidase (SQE), a key enzyme of saponin biosynthesis pathway, was screened from the GenBank database. By screening the obtained gene sequences, specific primers with Bam HI and Xho I restriction endonucleases were designed. The SQE gene was cloned by RT-PCR and combined with restriction endonuclease and T4 ligase The prokaryotic expression vector p ET30b (+) - SQE was constructed and introduced into E. coli BL21 (DE3) host cells. When the OD600 reached 0.6, isopropyl-β-D-thiogalactam The expression of IPTG was induced by SDS-PAGE (SDS-PAGE). The results showed that this study successfully cloned the genistein squalene epoxidase gene (Cb SQE), the gene open reading frame of 1 575 bp encoding 525 amino acids. Phylogenetic tree analysis showed that Cb SQE was closely related to the gerberas and Gynostemma pentaphyllum SQE genes. The results of restriction enzyme digestion and sequencing showed that the prokaryotic expression vector of Cb SQE gene was successfully constructed. SDS-PAGE showed that the Cb SQE fusion protein , The molecular weight of 57 kD, consistent with the theoretical prediction. The results of this study provide a theoretical basis for the analysis of saponin biosynthetic pathway.