川西獐牙菜SmSLS2基因的克隆、生物信息学及表达分析

Cloning,Bioinformatics and Expression of SmSLS2 Gene in Swertia mussotii

裂环马钱子苷合成酶(SLS)是环烯醚萜类化合物合成途径的限速酶。该研究根据川西獐牙菜转录组信息获得SmSLS2基因序列,对该基因进行克隆、生物信息分析、原核表达载体构建及蛋白体外诱导。结果表明SmSLS2 cDNA全长1 566 bp(GenBank编号MH535904),编码521个氨基酸,推测其蛋白相对分子质量为59. 79kDa,等电点为8. 92。结构域分析表明SmSLS2含一个跨膜结构域,SmSLS2的gDNA全长为2 576 bp,含5个外显子和4个内含子。SmSLS2蛋白与其他植物中SLS蛋白具有高度的相似性。进一步将SmSLS2构建到原核表达载体pET-28a-sumo上,转入大肠杆菌BL21(DE3)中,在37℃、0. 5 mmol·L^(-1)IPTG条件下进行原核表达,诱导出与预测蛋白大小一致的目的蛋白。该研究成功从川西獐牙菜中克隆了SmSLS2,为进一步阐明该基因在环烯醚萜合成途径中的作用和通过基因工程手段改良环烯醚萜类化合物产量提供了理论基础。

Secologanin synthase(SLS) is the key enzyme of secoiridoid pathway.We obtained the SmSLS2 gene full-length sequence according to the transcriptome of Swertia mussotii.We cloned the gene, analyzed the bioinformation of SmSLS2 , constructed the vectors and performed the gene expression.The results showed that SmSLS2 cDNA complete sequence had a length of 1 566 bp(GenBank: MH535904), encoding 521 amino acid residues, and the pI of SmSLS2 was 8.92.Domain analysis results showed that SmSLS2 had one transmembrane domain, and the protein secondary and tertiary structures were analyzed and forecasted.The gDNA sequence of SmSLS2 was 2 576 bp, contained five exons and four introns.The SmSLS2 protein shared high identity with other SLS proteins of plants.Prokaryotic expression vector pET-28a-sumo-SmSLS2 was constructed and transformed into Escherichia coli BL21(DE3) for expression under 37℃, and induced by 0.5 mmol·L^-1 IPTG.The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size.SmSLS2 gene was successfully cloned from S.mussotii , and it will provide a foundation for further functional research on SmSLS2 protein and increasing the product of iridoid compound by genetic engineering in S.mussotii.