薹菜CYP79B_5基因的克隆及原核表达

Molecular cloning and prokaryotic expression of CYP79B_5 from tai-tsai

以薹菜品种京研薹菜叶片cDNA为模板,采用RT-PCR技术,获得了编码薹菜CYP79B5基因全序列1 847 bp,包含有1 620 bp的开放阅读框,编码540个氨基酸。以薹菜基因组DNA为模板,获得了2 112 bp的目的片段。经比对发现其氨基酸序列与油菜、白芥、拟南芥等CYP79B5编码的氨基酸序列具有较高同源性,与油菜同源性达100%。经过生物信息学分析,发现所推导的氨基酸序列具有明显的CYP蛋白信号域,且可以在SWISS-MODEL数据库中搜索到与之相近的三维结构。扩增CYP79B5完整的编码区序列,构建pET-24α(+)重组质粒转化大肠杆菌Rosetta(DE3),IPTG诱导融合蛋白高效表达,SDS-PAGE电泳检测发现在相对分子质量64 000处有一特异条带,与预期的目的产物蛋白带大小一致。

CYP79B5 gene was cloned from Jingyan tai-tsai by RT-PCR.The full-length cDNA of CYP79B5 was 1 847 bp long,with a 1 620 bp open reading frame（ORF）encoding 540 amino acids.2 112 bp fragment was obtained by PCR amplification using genome DNA as template.Blast results showed that the deduced amino acid sequence had high homology to Brassica napus,Sinapis alba and Arabidopsis thaliana.It had 100% identity with that of B.napus.Bioinformatics analysis showed that there was a significant protein signal domain in the deduced amino acid sequence,and a similar 3D structure could be found in the SWISS-MODEL database.The complete coding sequence was amplified by PCR technique.Then the recombinant plasmid pET-24α（＋）was constructed,and transformed to Escherichia coli Rosetta（DE3）for expression induced by IPTG.SDS-PAGE analysis revealed that there was a specific band at 64 000,which conformed to the expected molecular weight of the recombinant protein.