水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

Cloning, expression and charaterization of chalcone synthase from Saussurea medusa

摘要:

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段，然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp，其开放阅读框为1 170 bp，编码389个氨基酸，预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS，重组质粒转化大肠杆菌BL21(DE3)，获得表达菌株。经IPTG诱导表达后，对表达产物进行SDS-PAGE分析，结果显示，表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化，对纯化蛋白进行酶活检测，结果表明融合蛋白具有查尔酮合酶活性，可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。

关键词:水母雪莲，查尔酮合酶，原核表达，纯化，酶活检测

Abstract:

A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1 313 bp, containing an open reading frame (1 170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.

Key words:Saussurea medusa, chalcone synthase, prokaryotic expression, purification, enzymatic activity assay