茶树多酚氧化酶基因的生物信息学分析及原核表达

Bioinformatic Analysis and Prokaryotic Expression of Polyphenol Oxidase Gene in Tea Plant(Camellia sinensis)

通过PCR方法扩增出迎霜品种茶多酚氧化酶(PPO)的编码区序列,并将其登录于GenBank,注册号为GQ214317。生物信息学分析表明,该基因编码区全长1 800 bp,编码599个氨基酸,分子式为C3008H4677N813O900S18,存在两个铜离子结合域,没有跨膜区;进化树分析表明,茶与毛叶茶亲缘关系最近。成功构建了原核表达载体pET-ppo,并转化E.coli BL21(DE3),实现了PPO的原核表达。SDS-PAGE电泳检测结果表明,在71 KD处有一条特异蛋白条带,与预测大小相符。

The polyphenol oxidase（PPO,GenBank accession No.DQ812086） gene was amplified by PCR.Bioinformatics analysis indicated that the cds of PPO DNA sequence is 1 800 bp,which encoded a protein of 599 amino acid residues（molecular formula： C3008H4677N813O900S18） and predicted that it had two Cu-band functional domains with no transmembrane.The phylogeny analysis showed that it has a most close phylogenetic relationship with Camellia nitidissima.The PPO was cloned into pET28a vector to construct recombination prokaryotic expression vector pET-ppo.After transformed to E1coli BL21 and induced by isopropyl-β-D-thiogalactopyranoside（IPTG）,recombinant protein about 71 kD was expressed in pET28a system and separated by SDS-PAGE electrophoresis.