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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。
A fragment of the chalcone synthase gene (SmCHS) was obtained from the Saussurea medusa Maxim.cDNA library of Saussurea medusa and the complete cDNA of chalcone synthase gene was obtained by RT-PCR. Sequence analysis showed that SmCHS was 1 313 bp in length with an open reading frame of 1 170 bp encoding a protein of 389 amino acids. The molecular weight of the expressed protein was 43 kDa. The prokaryotic expression plasmid pET28a (+) - SmCHS was constructed and the recombinant plasmid was transformed into E. coli BL21 (DE3) to obtain the expression strain. After induced by IPTG, the expressed products were analyzed by SDS-PAGE. The results showed that the expressed fusion protein existed in partially soluble form. The Ni-NTA pre-packed column was used to affinity purify the fusion protein, and the purified protein was tested for enzyme activity. The results showed that the fusion protein has chalcone synthase activity and can catalyze the substrates 4-coumaroyl-CoA and malonyl Coenzyme A condensation product Naringen Chalcone.