茶树肉桂酸4-羟基化酶基因的克隆及表达分析

doi:10.13305/j.cnki.jts.2015.01.007

茶叶科学 ›› 2015, Vol. 35 ›› Issue (1): 35-44.doi: 10.13305/j.cnki.jts.2015.01.007

茶树肉桂酸4-羟基化酶基因的克隆及表达分析

姚胜波¹, 王文钊¹, 李明卓¹, 许玉娇², 王云生², 刘亚军², 高丽萍^2,*, 夏涛^1,*

1. 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;
2. 安徽农业大学生命科学学院,安徽合肥 230036

收稿日期:2014-07-14 修回日期:2014-08-09 出版日期:2015-02-15 发布日期:2019-08-23
通讯作者: *
作者简介:姚胜波,男,硕士,主要从事茶树次生代谢及生物化学研究。
基金资助:
国家自然科学基金（31170647、31170282、31270730、31200229）、安徽省自然科学基金（1408085QC51）

The Gene Cloning and Expression Analysis of C4H in Tea Plant (Camellia sinensis)

YAO Shengbo¹, WANG Wenzhao¹, LI Mingzhuo¹, XU Yujiao², WANG Yunsheng², LIU Yajun², GAO Liping^2,*, XIA Tao^1,*

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

Received:2014-07-14 Revised:2014-08-09 Online:2015-02-15 Published:2019-08-23

摘要/Abstract

摘要： 肉桂酸4-羟基化酶（Cinnamate 4-hydroxylase, C4H）是茶树苯丙烷代谢途径中的关键酶,能够影响木质素和类黄酮等次级代谢物的生物合成。本文采用5′-RACE技术,克隆了茶树肉桂酸4-羟基化酶基因（CsC4H）的全长序列,其中开放阅读框长1β518βbp,编码505个氨基酸,推测蛋白分子量为58.15βkD,理论等电点为9.29。利用基因组步移技术克隆得到该基因上游1β840βbp的启动子序列。该启动子区域除了分布有TAAT-box和CAAT-box等基本转录元件外,还存在多个诱导型和组织特异型的顺式作用元件。实时荧光定量PCR结果表明该基因在芽、叶、茎、根中都有表达。将该基因重组至表达载体pYES-DEST52上,并在酿酒酵母WAT11中进行真核表达。利用LC-MS方法检测酶反应产物,结果表明目的蛋白能够催化肉桂酸生成p-香豆酸。

关键词: 茶树, 肉桂酸4-羟基化酶, 启动子, 表达分析, 真核表达

Abstract: Cinnamate 4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway in tea plant. The gene can influence the biosynthesis of secondary metabolites such as lignin and flavonoids. The cDNA full-length of C4H gene was cloned from tea plant by rapid amplification of cDNA ends with a 1β518βbp open reading frame encoding a protein of 505 amino acids. The deduced protein molecular weight was 58.15βkD and its theoretical isoelectric point was 9.29. A 1β840βbp promoter sequence was isolated by genome walking method. The promoter region not only has the basic transcriptional elements of TATA-box and CAAT-box, but also has many potential inducible and tissue-specific cis-acting elements. Quantitative RT-PCR analysis showed that the CsC4H gene expressed in bud, leaf, stem and root. The gene was cloned into the expression vector pYES-DEST52 for eukaryotic expression in Saccharomyces cerevisiae WAT11. The enzyme reaction products were detected by LC-MS method. The results indicated that cinnamic acid was para-hydroxylated by target proteins to generate p-coumaric acid.

Key words: Camellia sinensis, cinnamate 4-hydroxylase(C4H), promoter, expression analysis, eukaryotic expression

中图分类号:

姚胜波, 王文钊, 李明卓, 许玉娇, 王云生, 刘亚军, 高丽萍, 夏涛. 茶树肉桂酸4-羟基化酶基因的克隆及表达分析[J]. 茶叶科学, 2015, 35(1): 35-44. doi: 10.13305/j.cnki.jts.2015.01.007.

YAO Shengbo, WANG Wenzhao, LI Mingzhuo, XU Yujiao, WANG Yunsheng, LIU Yajun, GAO Liping, XIA Tao. The Gene Cloning and Expression Analysis of C4H in Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2015, 35(1): 35-44. doi: 10.13305/j.cnki.jts.2015.01.007.

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茶树肉桂酸4-羟基化酶基因的克隆及表达分析

The Gene Cloning and Expression Analysis of C4H in Tea Plant (Camellia sinensis)

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