油菜乙酰羟基酸合酶基因 BnAHAS1的克隆及其重组蛋白质的原核表达

摘要

利用RT-PCR技术从甘蓝型油菜宁油16号( Brassica napus L.)中克隆到乙酰羟基酸合酶基因BnA-HAS1的cDNA序列,该序列含有1个1968 bp的开放阅读框,编码蛋白质655个氨基酸,分子量约为7.1×104,预测等电点为6.16。将该基因克隆到原核表达载体pCold II中,经酶切和测序鉴定后,将正确的重组质粒pCold II-BnA-HAS1导入大肠杆菌 BL21(DE3)。在15℃下以终浓度为1 mmol/L的IPTG诱导12 h后,获得预期大小的重组蛋白质His-BnAHAS1,并用Western blot确定此重组蛋白质为目的蛋白质。%The acetohydroxyacid synthase (AHAS) gene cDNA, named BnAHAS1, was cloned from a rapeseed cul-tivar Ningyou16(Brassica napus L. )by RT-PCR. The open reading frame of BnAHAS1 was 1 968 bp in length, which enco-ded a 655 amino acids sequence with the molecular mass of approximately 7. 1×104 and the isoelectric point of 6. 16. The BnAHAS1 gene was inserted into the prokaryotic expression vector pCold II. After confirmation by digestion and sequencing, the recombinant plasmid pCold II-BnAHAS1 was transformed into Escherichia. coli strain BL21(DE3). The recombinant protein with the predicted molecular weight was successfully induced to express using 1 mmol/ L of isopropyl-b-D-thiogalactopyranoside (IPTG) at 15 ℃ for 12 h and was detected and confirmed by Western blot analysis.