一个新的忽地笑O-甲基转移酶基因的克隆与原核表达

别庆玲, 徐晟, 傅江燕, 夏冰, 汪仁*
江苏省中国科学院植物研究所, 南京210014

通信作者:汪仁;E-mail: jswangren@aliyun.com;Tel: 025-84347111

摘 要:

采用RACE技术克隆得到一个新的忽地笑O-甲基转移酶(OMT)基因, 命名为LaOMTLaOMT的cDNA序列为1 379 bp, 开放阅读框1 077 bp, 预测编码蛋白包含358个氨基酸。序列比对分析发现, LaOMT编码的蛋白序列具有依赖于S-腺苷-L-甲硫氨酸(SAM)的高度保守的结构域, 与葡萄、大豆等其他植物OMTs蛋白的相似性为50%左右。将LaOMT基因连接到原核表达载体pET28a上, 转化大肠杆菌Rosetta (DE3)的SDS-PAGE电泳结果显示, 重组蛋白受异丙基硫代半乳糖苷(IPTG)诱导表达, 分子量大小约为45 kDa, 与已报导的其他植物的OMTs蛋白大小基本一致。此外, 半定量RT-PCR分析结果表明, LaOMT在忽地笑的叶、鳞茎、根和花中均有表达, 其中花中的表达量最低。

关键词:忽地笑; O-甲基转移酶; 加兰他敏; 原核表达

收稿:2013-12-24   修定:2014-04-10

资助:国家自然科学基金(31270339和31301798)和江苏省农业自主创新资金项目[CX(11)1016]。

Cloning and Prokaryotic Expression of a New O-Methyltransferase Gene in Lycoris aurea (L’Her) Herb.

BIE Qing-Ling, XU Sheng, FU Jiang-Yan, XIA Bing, WANG Ren*
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China

Corresponding author: WANG Ren; E-mail: jswangren@aliyun.com; Tel: 025-84347111

Abstract:

In this study, a full-length cDNA sequence of O-methyltransferase was obtained from Lycoris aurea with the RACE method, which was named as LaOMT. The full-length cDNA of LaOMT is 1 379 bp with a 1 077 bp open reading frame, encoding a deduced polypeptide of 358 amino acids. Multiple sequence alignment showed that the deduced protein of LaOMT contains S-adenosyl-L-methionine (SAM)-dependent conserved motifs and shares more than 50% identity with O-methyltransferases (OMTs) from Vitis vinifera, Glycine max and other plants. Subsequently, the LaOMT was ligated into pET28a vector, and transferred into Escherichia coli strain Rosetta (DE3) for heterologous expression. The recombinant protein was induced by isopropyl- β-D-thiogalactopyranoside (IPTG) and its molecular weight is about 45 kDa, which is consistent with OMTs from other plants. Additionally, semi-quantitative RT-PCR analysis indicated that LaOMT was ubiquitously expressed in leaves, bulbs, roots and flowers.

Key words: Lycoris aurea; O-methyltransferase (OMT); galanthamine; prokaryotic expression

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