石蒜儿茶酚氧位甲基转移酶基因克隆与原核表达

采用同源克隆与RACE相结合的方法,首次从石蒜叶片中克隆到可能与加兰他敏生物合成相关的儿茶酚氧位甲基转移酶基因,命名为LrCOMT。核酸序列分析显示,该cDNA全长1 246bp,开放阅读框1 101bp,编码366氨基酸。氨基酸序列分析与比对发现,LrCOMT编码的氨基酸序列具有COMT蛋白的特征区域,且与鸢尾、玉米、烟草等植物COMT的同源性大于60%。将LrCOMT基因连接到原核表达载体pET-29a上,转化大肠杆菌BL21(DE3)的原核表达结果显示,重组蛋白受IPTG诱导表达,分子量大小约为40kD,与已报道的其他植物COMT大小基本一致。

Cloning and Prokaryotic Expression of LrCOMT from Lycoris radiata

A full-length cDNA of COMT,which is closely related to the biosynthesis of galanthamine in Amaryllidacea plants,was first successfully cloned from Lycoris radiata by the method of homology cloning and RACE,and was named as LrCOMT.The nucleotide analysis showed that the cDNA sequence of LrCOMT gene had 1 246 bp,containing a 1 101 bp open reading frame,which encodes 366 amino acid residues.Amino sequence analysis demonstrated that the deduced amino sequence of LrCOMT has many typical features of COMTs,and shared more than 60% indentity with COMTs from Iris hollandica,Zea mays,Nicotiana tabacum and other plants.Subsequently,the LrCOMT was ligated into pET-29a vector,and transferred into E.coli strain BL21 for heterologous expression.And the result demonstrated that the recombinant protein was induced by IPTG,and its molecular weight is about 40 kD,which is consistent with other COMTs from other plants.