茶树氧甲基转移酶基因的克隆及原核表达

doi:10.13305/j.cnki.jts.2013.06.014

茶叶科学 ›› 2013, Vol. 33 ›› Issue (6): 532-540.doi: 10.13305/j.cnki.jts.2013.06.014

茶树氧甲基转移酶基因的克隆及原核表达

马成英, 施江, 吕海鹏, 张悦, 谭俊峰, 郭丽, 彭群华, 林智^*

中国农业科学院茶叶研究所/茶叶加工工程研究中心,浙江杭州 310008

收稿日期:2013-02-18 修回日期:2013-05-02 出版日期:2013-12-30 发布日期:2019-09-04
通讯作者: *linz@mail.tricaas.com
作者简介:马成英（1985— ）,女,山东淄博人,博士研究生,主要从事分子生物学研究。
基金资助:
国家自然科学基金项目（30972404）、现代农业产业技术体系建设专项资金资助（CARS-23）、中央级公益性科研院所基本科研业务费专项（2012zl053）

Cloning and Prokaryotic Expression of O-methyltransferase from Camellia sinensis

MA Cheng-ying, SHI Jiang, LV Hai-peng, ZHANG Yue, TAN Jun-feng, GUO Li, PENG Qun-hua, LIN Zhi^*

Engineering Research Center for Tea Processing; Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China

Received:2013-02-18 Revised:2013-05-02 Online:2013-12-30 Published:2019-09-04

摘要/Abstract

摘要： 获得了一类茶树氧甲基转移酶基因cDNA全长并构建了该基因的原核表达载体。以从茶树叶片中提取的总RNA为模板,结合RT-PCR与RACE克隆技术获得氧甲基转移酶（O-methyltransferase）基因cDNA全长1β280βbp,其开放阅读框为1β068βbp,编码355个氨基酸,推测的蛋白分子量为39.1βkD,理论等电点为5.68。其氨基酸序列与葡萄和蓖麻氧甲基转移酶基因相似性分别为73%、71%。将该基因片段连接到原核表达载体pET-28a中,转化大肠杆菌BL21后诱导重组蛋白的表达,经IPTG诱导,SDS-PAGE检测到1条与预测融合蛋白分子量相符的外源蛋白。

关键词: 茶树, 氧甲基转移酶, 克隆, 原核表达

Abstract: A full length cDNA of O-methyltransferase gene was obtained from Camellia sinensis and the prokaryotic expression vector for this gene was constructed. Based on total RNA from tea leaves, a O-methyltransferase cDNA sequence of tea was obtained by RT-PCR and RACE. The whole cDNA sequence 1β280βbp which contains an ORF of 1β068βbp and encodes 355 amino acids. The putative protein of this gene had an isoelectric point of 5.68 and a calculated molecular weight of 39.1βkD. The amino acid sequence of tea O-methyltransferase showed 73%, 71% identity with that of Vitis vinifera and Ricinus communis respectively. The coding sequence had been cloned into pET-28a and transformed into the host BL21. Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG.

Key words: tea, O-methyltransferase, cloning, prokaryotic expression

中图分类号:

S571.1
Q555

马成英, 施江, 吕海鹏, 张悦, 谭俊峰, 郭丽, 彭群华, 林智. 茶树氧甲基转移酶基因的克隆及原核表达[J]. 茶叶科学, 2013, 33(6): 532-540. doi: 10.13305/j.cnki.jts.2013.06.014.

MA Cheng-ying, SHI Jiang, LV Hai-peng, ZHANG Yue, TAN Jun-feng, GUO Li, PENG Qun-hua, LIN Zhi. Cloning and Prokaryotic Expression of O-methyltransferase from Camellia sinensis[J]. Journal of Tea Science, 2013, 33(6): 532-540. doi: 10.13305/j.cnki.jts.2013.06.014.

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茶树氧甲基转移酶基因的克隆及原核表达

Cloning and Prokaryotic Expression of O-methyltransferase from Camellia sinensis

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