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该研究根据丹参的转录组数据提供的基因片段设计引物,采用逆转录聚合酶链式反应方法,从白花丹参中克隆鲨烯合酶SQS2的全长cDNA序列,命名为SmSQS2,GenBank注册号KM244731。序列长度为1 597 bp,包含1 245 bp的开放阅读框,推测编码414个氨基酸,含有115 bp的5′UTR和237 bp的3′UTR。利用生物信息学软件对获得的序列进行同源性分析,得出白花丹参SQS2的与紫花丹参SQS2的序列无明显差异。原核表达分析结果表明SmSQS2在大肠杆菌中能表达出与预测蛋白大小相当的目标蛋白,对影响蛋白表达的4个因素,即诱导温度、诱导时间、IPTG浓度和诱导时宿主菌的吸光度(A_(600))进行了优化,得出SmSQS2蛋白表达的最佳条件为:IPTG终浓度0.2 mmol·L~(-1),宿主菌的吸光度(A_(600))为0.6,温度30℃,诱导时间20 h。这为进一步研究白花丹参鲨烯合酶SQS2在丹参萜类和甾醇类生物合成途径中的作用提供了理论依据。
In this study, primers were designed based on the gene fragments provided by transcriptome of Salvia miltiorrhiza, and the full-length cDNA sequence of squalene synthase SQS2 from Salvia miltiorrhiza was named as SmSQS2 and GenBank accession number KM244731 by reverse transcription-polymerase chain reaction. The sequence was 1 597 bp in length and contained an open reading frame of 1 245 bp. It was presumed to encode 414 amino acids with a 115 bp 5’UTR and a 237 bp 3 ’UTR. The bioinformatics software was used to analyze the homology of the obtained sequences. The results showed that there was no significant difference between the sequences of Salvia miltiorrhiza SQS2 and Salvia miltiorrhiza SQS2. The results of prokaryotic expression analysis showed that SmSQS2 could express the target protein in Escherichia coli with the same size as the predicted protein. The four factors affecting the protein expression were induction temperature, induction time, IPTG concentration and absorbance of host bacteria (A_ 600), the optimal conditions for the expression of SmSQS2 protein were as follows: the final concentration of IPTG was 0.2 mmol·L -1, the absorbance of host bacteria (A_ (600)) was 0.6, the temperature was 30 ℃, the induction time 20 h. This provides a theoretical basis for further study on the role of Scutellarin Squalene synthase SQS2 in the pathway of the synthesis of danshensu and sterols.