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

Abstract

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

CLC Number:

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.

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

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