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目的:克隆滇重楼鲨烯合酶(squalene synthase,SQS)基因(PpSQS),对该基因进行序列分析及原核表达。方法:采用同源克隆和RACE技术获得PpSQS的cDNA,并对其进行生物信息学分析;构建原核表达载体pET-30b(+)-PpSQS,在大肠杆菌Escherich coli BL21(DE3)中进行诱导表达。结果:PpSQS基因cDNA全长1 498 bp,包含1个1 212 bp的开放阅读框(ORF),可编码403个氨基酸;PpSQS理论标准分子质量为46.36 kDa,等电点为pI 6.83;SDS-PAGE分析表明,经1 mmol.L-1IPTG诱导后,重组PpSQS蛋白在大肠杆菌中获得表达。结论:首次获得了滇重楼PpSQS基因cDNA全长序列,该基因编码产物具有植物SQS同源蛋白的典型特征,实现重组PpSQS在大肠杆菌中的表达。
OBJECTIVE: To clone squalene synthase (SQS) gene (PsSQS) from Yunnan relic, and analyze the sequence and prokaryotic expression of the gene. Methods: The cDNA of PpSQS was obtained by homologous cloning and RACE, and its bioinformatics analysis was carried out. The prokaryotic expression vector pET-30b (+) - PpSQS was constructed and induced in Escherichia coli BL21 (DE3). RESULTS: The cDNA of PpSQS gene was 1 498 bp in length and contained a 1 212 bp open reading frame (ORF) encoding 403 amino acids. The theoretical molecular weight of PpSQS was 46.36 kDa and the isoelectric point was pI 6.83. SDS-PAGE The analysis showed that after induced by 1 mmol.L-1IPTG, the recombinant PpSQS protein was expressed in E.coli. CONCLUSION: The full-length cDNA of PpSQS gene was obtained for the first time in Yunnan. The gene encoding the product possesses the typical characteristics of plant SQS homologous protein, and the recombinant PpSQS can be expressed in E. coli.