茶树二氢黄酮醇4-还原酶基因的克隆、表达及功能分析

doi:10.13305/j.cnki.jts.2013.03.010

茶叶科学 ›› 2013, Vol. 33 ›› Issue (3): 193-201.doi: 10.13305/j.cnki.jts.2013.03.010

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茶树二氢黄酮醇4-还原酶基因的克隆、表达及功能分析

王云生^1,2, 许玉娇¹, 胡晓婧¹, 蒋晓岚², 杨琴¹, 李伟伟¹, 刘亚军¹, 高丽萍^1,*, 夏涛^2,*

1. 安徽农业大学生命科学学院,安徽合肥 230036;
2. 安徽农业大学教育部茶叶生物化学与生物技术重点实验室,安徽合肥 230036

收稿日期:2012-12-14 修回日期:2013-01-25 出版日期:2013-06-30 发布日期:2019-09-04
通讯作者: *
作者简介:王云生（1977— ）,男,安徽临泉人,博士,主要从事茶树次生代谢与分子生物学研究。
基金资助:
国家自然科学基金（NO. 31000314、NO. 31170647、NO.31170282、NO.31270730）

Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)

WANG Yun-sheng^1,2, XU Yu-jiao¹, HU Xiao-jing¹, JIANG Xiao-lan², YANG Qing¹, LI Wei-wei¹, LIU Ya-jun¹, GAO Li-ping^1,*, XIA Tao^2,*

1. School of Biology Science, Anhui Agricultural University, Hefei 230036, China;
2. Key Lab of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei 230036, China

Received:2012-12-14 Revised:2013-01-25 Online:2013-06-30 Published:2019-09-04

摘要/Abstract

摘要： 茶树二氢黄酮醇4-还原酶（dihydroflavonol 4-reductase, DFR）是儿茶素合成途径中的关键酶。本研究采用RT-PCR技术,获得了茶树二氢黄酮醇4-还原酶基因（CsDFR）的开放阅读框,它编码含347个氨基酸的蛋白质,推测分子量为38.69βkD,等电点为6.02。成功地将该基因重组到表达载体SUMO上,并在大肠杆菌BL21中进行原核表达;优化了原核表达中诱导时间、诱导温度、IPTG浓度;纯化出目的蛋白。利用HPLC-MS方法对重组蛋白进行了体外酶活检测,结果表明目的蛋白具有DFR酶活性,可催化DHQ和DHM的还原反应。

关键词: 茶树, 二氢黄酮醇4-还原酶, 原核表达, 酶活性

Abstract: Dihydroflavonol 4-reductase (DFR) is a key enzyme in the biosynthesis of catechins in tea plant. However, the functions and the zymologic properties of DFR were not deeply identified in recent researches. The open reading frame of DFR gene, which encoding a 347 amino acids protein, was cloned from tea plant (Camellia sinensis) by RT-PCR. The deduced protein molecular weight was 38.69βkD and its theoretical isoelectric point was 6.02. The gene was cloned into the expression vector SUMO for expression in prokaryotic cells. The SDS-PAGE results showed that the dihydroflavonol 4-reductase peoteins was expressed in Escherichia coli BL21. The optimal inducing conditions including time, temperature and IPTG concentration were studied. The deduced protein was purified and its activity was detected by HPLC-MS method. The results indicated that purified protein showed the DFR activity, catalyzed the reduction reaction of DHQ and DHM. The research provides a valuable foundation for better understanding the substrate specificity and enzymatic properties of CsDFR.

Key words: tea plant (Camellia sinensis), dihydroflavonol 4-reductase, prokaryotic expression, enzyme activity

中图分类号:

王云生, 许玉娇, 胡晓婧, 蒋晓岚, 杨琴, 李伟伟, 刘亚军, 高丽萍, 夏涛. 茶树二氢黄酮醇4-还原酶基因的克隆、表达及功能分析[J]. 茶叶科学, 2013, 33(3): 193-201. doi: 10.13305/j.cnki.jts.2013.03.010.

WANG Yun-sheng, XU Yu-jiao, HU Xiao-jing, JIANG Xiao-lan, YANG Qing, LI Wei-wei, LIU Ya-jun, GAO Li-ping, XIA Tao. Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2013, 33(3): 193-201. doi: 10.13305/j.cnki.jts.2013.03.010.

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茶树二氢黄酮醇4-还原酶基因的克隆、表达及功能分析

Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)

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