苎麻香豆酸–3–羟化酶基因的原核表达

Prokaryotic expression of coumarate 3–hydroxylase gene in ramie

以湘苎3号为材料,采用RT–PCR克隆,获得苎麻香豆酸–3–羟化酶(Bn C3H)基因的开放阅读框(ORF)序列,测序正确后将该基因片段连接到原核表达载体p ET–22b中,将构建成功的重组子p ET–22b–C3H转入BL21并诱导表达。结果显示:Bn C3H基因ORF区大小为1 536 bp,编码511个氨基酸,推测蛋白相对分子质量为57 800,理论等电点为8.61,编码的蛋白质为亲水性蛋白质;重组子在IPTG浓度为1 mmol/L时,融合蛋白的表达量在诱导2 h后达到最高,经IPTG诱导和SDS–PAGE检测,获得的融合蛋白与预期蛋白相符。

The gene BnC3H cloned from ramie and its recombinant proteins expressed in Escherichia coli were analyzed to reveal the enzymatic characteristics and the metabolic mechanism of regulating the lignin in ramie.The open reading frame(ORF)sequence of BnC3H gene was obtained by RT–PCR,and then the cloned genes of BnC3H were inserted into vector pET–22b.The recombinant plasmids pET–22b–C3H were expressed in the prokaryotic expression system after its transformation into E.coli BL21.The result showed that the BnC3H ORF sequence was 1 536 bp and encodes 511 amino acids.The relative molecular weight is 57 800.The optimum concentration of IPTG was 1 mmol/L,results of SDS–PAGE showed that the specific fusion protein was successfully induced by IPTG.