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

YAO Shengbo; WANG Wenzhao; LI Mingzhuo; XU Yujiao; WANG Yunsheng; LIU Yajun; GAO Liping; XIA Tao

doi:10.13305/j.cnki.jts.2015.01.007

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Journal of Tea Science ›› 2015, Vol. 35 ›› Issue (1) : 35-44. DOI: 10.13305/j.cnki.jts.2015.01.007

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,*

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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) / eukaryotic expression / expression analysis / promoter

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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 https://doi.org/10.13305/j.cnki.jts.2015.01.007

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