金铁锁β-香树素合酶cDNA的克隆、原核表达和功能鉴定

Cloning, prokaryotic expression, and functional identification of β-amyrin synthase cDNA of Psammosilene tunicoides

目的克隆金铁锁Psammosilene tunicoides三萜皂苷生物合成途径中的关建酶基因——β-香树素合酶全长cDNA,并对其进行表达和分析,为研究其在三萜皂苷合成途径的关键作用及次生代谢产物工程技术在金铁锁上的应用奠定基础。方法应用RT-PCR和RACE等方法,克隆金铁锁β-香树素合成酶基因全长,将目的片段转化大肠杆菌表达菌株BL21中,IPTG诱导表达,以2,3-氧化鲨烯为底物进行体外酶促,采用HPLC法鉴定产物。结果克隆得到金铁锁β-香树素合成酶基因,全长2 882 bp,ORF长2 284 bp,编码760个氨基酸。表达产物具有催化2,3-氧化鲨烯生成β-香树素合酶的活性。结论克隆所得的全长cDNA通过大肠杆菌表达β-香树素合酶,并能催化2,3-氧化鲨烯生成β-香树素,为金铁锁次生代谢工程研究提供了重要的基础。

Objective To clone, express, and characterize the full-length cDNA of β-amyrin synthase provided an important basis for the study on the key role in the biosynthetic pathway of triterpenoid saponins and secondary metabolism engineering applied to Psammosilene tunicoides. Methods The full-length cDNA fragment ofP. tunicoides was isolated by the method of RT-PCR and rapid amplification of cDNA ends （RACE）. The fragment was transformed into Escherichia coli expression strain BL21, which was induced by IPTG, and the crude recombinant enzyme was purified from E. coli cell. In the presence of 2, 3-oxidosqualene and other substances, 2, 3-oxidosqualene was converted into β-amyrin efficiently. The catalytic product ofP. tunicoides β-amyrin synthase was detected by high-performance liquid chromatography （HPLC） and identified as β-amyrin. Results The full-length cDNA fragment of P. tunicoides is 2 882 bp and contains an open reading frame of 2 284 bp nucleotides, which codes for 760 amino acids. Conclusion The full-length cDNA can produce β-amyrin synthase by prokaryotic expression. The expression product has the catalytic activity of 2, 3-oxidosqualene into β-amyrin, which would provide an important basis for the secondary metabolism engineering to P. tunicoides.