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CLC number: S571.1

On-line Access: 2015-02-02

Received: 2014-07-08

Revision Accepted: 2014-10-23

Crosschecked: 2015-01-08

Cited: 1

Clicked: 4534

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Zhi Lin

http://orcid.org/0000-0001-6738-6663

Yue Zhang

http://orcid.org/0000-0001-5793-6318

Hai-peng Lv

http://orcid.org/0000-0002-4644-4323

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Journal of Zhejiang University SCIENCE B 2015 Vol.16 No.2 P.103-112

http://doi.org/10.1631/jzus.B1400193


Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity


Author(s):  Yue Zhang, Hai-peng Lv, Cheng-ying Ma, Li Guo, Jun-feng Tan, Qun-hua Peng, Zhi Lin

Affiliation(s):  Key Laboratory of Tea Biology and Resource Utilization of Ministry of Agriculture, Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China

Corresponding email(s):   lvhaipeng@tricaas.com, linz@tricaas.com

Key Words:  Tea (Camellia sinensis), O-methyltransferase, CsCCoAOMT, Prokaryotic expression, Catalytic activity, Methylated epigallocatechin-3-O-gallate (EGCG)


Yue Zhang, Hai-peng Lv, Cheng-ying Ma, Li Guo, Jun-feng Tan, Qun-hua Peng, Zhi Lin. Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity[J]. Journal of Zhejiang University Science B, 2015, 16(2): 103-112.

@article{title="Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity",
author="Yue Zhang, Hai-peng Lv, Cheng-ying Ma, Li Guo, Jun-feng Tan, Qun-hua Peng, Zhi Lin",
journal="Journal of Zhejiang University Science B",
volume="16",
number="2",
pages="103-112",
year="2015",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1400193"
}

%0 Journal Article
%T Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity
%A Yue Zhang
%A Hai-peng Lv
%A Cheng-ying Ma
%A Li Guo
%A Jun-feng Tan
%A Qun-hua Peng
%A Zhi Lin
%J Journal of Zhejiang University SCIENCE B
%V 16
%N 2
%P 103-112
%@ 1673-1581
%D 2015
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1400193

TY - JOUR
T1 - Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity
A1 - Yue Zhang
A1 - Hai-peng Lv
A1 - Cheng-ying Ma
A1 - Li Guo
A1 - Jun-feng Tan
A1 - Qun-hua Peng
A1 - Zhi Lin
J0 - Journal of Zhejiang University Science B
VL - 16
IS - 2
SP - 103
EP - 112
%@ 1673-1581
Y1 - 2015
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1400193


Abstract: 
Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) present in leaves of Camellia sinensis has many beneficial biological activities for human health. However, EGCG3"Me occurs naturally in tea leaves in extremely limited quantities. Finding an enzyme from C. sinensis to catalyze the synthesis of EGCG3"Me is an alternative method to make up for the scarcity of EGCG3"Me in natural situations. In the present study, a complementary DNA (cDNA) encoding region and genomic DNA of the caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) gene were isolated from C. sinensis (designated CsCCoAOMT). Nucleotide sequence analysis of CsCCoAOMT revealed an open reading frame of 738 bp that encodes a polypeptide with a predicted molecular weight of 28 kDa, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The full-length DNA sequence (2678 bp) contained five exons and four introns. The deduced amino acid sequence of CsCCoAOMT shared 92% identity with CCoAOMTs from Codonopsis lanceolata and Betula luminifera. The catalytic activity of CsCCoAOMT was analyzed. Three monomethylated epigallocatechin-3-O-gallate (EGCG) compounds (EGCG4"Me, EGCG3"Me, and EGCG3'Me) were produced by CsCCoAOMT with Km in the micromolar range. Real-time polymerase chain reaction (RT-PCR) experiments indicated that the CsCCoAOMT transcript was present at low levels during the early stages of leaf maturity (the first leaf and bud on a shoot) but the relative expression was augmented at advanced stages of leaf maturity (the third or fourth leaf on a shoot), which accorded well with changes in EGCG3"Me content in fresh leaves. Hence, we concluded that CsCCoAOMT catalyzes the syntheses of methylated EGCGs.

茶树中咖啡酰辅酶A氧甲基转移酶的基因克隆及其催化活性研究

目的:从茶树中克隆一种可以催化表没食子儿茶素没食子酸酯(EGCG)生成甲基化EGCG的酶——咖啡酰辅酶A氧甲基转移酶(CCoAOMT),实现甲基化EGCG的酶学合成,为甲基化EGCG的进一步开发利用提供理论依据和技术指导。
创新点:本研究首次从茶树中克隆了一条CCoAOMT基因组序列;分析了CCoAOMT基因在不同茶树品种和不同成熟度茶鲜叶中的基因表达规律;证明了CCoAOMT具有催化合成甲基化EGCG的生物活性。
方法:采用聚合酶链式反应(PCR)和序列分析获得CCoAOMT的编码序列和基因组序列;采用高效液相色谱-四级杆-飞行时间串联质谱技术(HPLC-QTOF-MS)分析酶促反应生成的甲基化EGCG产物(图4);采用实时荧光定量PCR分析CCoAOMT基因的表达差异(图5)。
结论:本研究从茶树中克隆了CCoAOMT基因的编码序列(738bp)和基因组序列(2678bp),明确了该基因具有4个内含子和5个外显子;揭示了CCoAOMT可以催化EGCG生成EGCG4"Me、EGCG3"Me和EGCG3’Me等多种甲基化产物;证明了CCoAOMT具有催化生成甲基化EGCG的活性;并发现该基因的表达量高低与茶鲜叶的成熟度呈正相关关系。

关键词:茶树;咖啡酰辅酶A氧甲基转移酶(CCoAOMT);催化活性;甲基化表没食子儿茶素没食子酸酯(EGCG)

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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