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)

作　　者：姚胜波 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] 

机构地区：[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

出　　处：《茶叶科学》2015年第1期35-44,共10页Journal of Tea Science

基　　金：国家自然科学基金(31170647;31170282;31270730;31200229);安徽省自然科学基金(1408085QC51)

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(Cs C4H)的全长序列,其中开放阅读框长1 518 bp,编码505个氨基酸,推测蛋白分子量为58.15 k D,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1 840 bp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p YES-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。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 1 518 bp open reading frame encoding a protein of 505 amino acids. The deduced protein molecular weight was 58.15 kD and its theoretical isoelectric point was 9.29. A 1 840 bp 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.

关 键 词：茶树 肉桂酸4-羟基化酶 启动子 表达分析 真核表达 

分 类 号：S571.1[农业科学—茶叶生产加工] Q946.5[农业科学—作物学]