茶树类黄酮O-甲基转移酶基因的克隆及原核表达分析

doi:10.3864/j.issn.0578-1752.2013.02.012

摘要/Abstract

摘要： 【目的】克隆茶树（Camellia sinensis）的一个类黄酮O-甲基转移酶基因，并对其进行生物信息学分析，构建该基因的原核表达载体，在大肠杆菌中诱导表达融合蛋白，为进一步研究其酶学特性和调控茶树中甲基化类黄酮物质的代谢机理奠定基础。【方法】采用同源克隆法，以从茶树叶片中提取的总RNA为模板，结合RT-PCR与RACE克隆技术获得该基因cDNA全长，测序正确后将该基因片段连接到原核表达载体pET-28a中，转化大肠杆菌BL21并诱导表达。【结果】该基因cDNA全长为1 246 bp，其开放阅读框为1 077 bp，编码358个氨基酸，推测的蛋白分子量为40.2 kD，理论等电点为5.62；其核苷酸编码序列与葡萄和毛果杨的类黄酮O-甲基转移酶基因相似性分别为80%和81%；经IPTG诱导和SDS-PAGE检测，所构建的原核表达载体表达的融合蛋白与预期蛋白相符合。【结论】利用RT-PCR与RACE克隆技术从茶树叶片中克隆得到了一个类黄酮O-甲基转移酶基因，成功构建了原核表达载体，并使其在大肠杆菌中得到了高效表达。

关键词: 茶树 , 类黄酮O-甲基转移酶 , 克隆 , 原核表达

Abstract: 【Objective】Cloning, sequence analysis of the CsFOMT genes from Camellia sinensis and expression of recombinant proteins in Escherichia coli were conducted to further research the enzymatic characteristics and the metabolic mechanism of regulating the flavonoid in tea plant. 【Method】 The CsFOMT cDNA fragments were amplified from Camellia sinensis by reverse transcription PCR (RT-PCR) and RACE, and then the cloned genes of CsFOMT were inserted into vector pET-28a. The recombinant plasmids pET-28a-CsFOMT were expressed in the prokaryotic expression system after its transformation into E. coli BL21. 【Result】 The whole cDNA sequence was 1 246 bp which contains an ORF of 1 077 bp and encodes 358 amino acids. The putative protein of the gene had an isoelectric point of 5.62 and a calculated molecular weight of 40.2 kD. The coding nucleotide sequence of tea showed 80% and 81% identity with that of Vitis vinifera and Populus trichocarpa, respectively. Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG. 【Conclusion】In this study, a full length cDNA of flavonoid O-methyltransferase gene was obtained from Camellia sinensis by RT-PCR and RACE，and the prokaryotic expression vector for this gene was constructed, and then successfully induced to express by IPTG in BL21.

Key words: Camellia sinensis , flavonoid O-methyhransferase , cloning , prokaryotic expression

马成英, 吕海鹏, 林智, 张悦, 郭丽, 谭俊峰. 茶树类黄酮O-甲基转移酶基因的克隆及原核表达分析[J]. 中国农业科学, 2013, 46(2): 325-333.

MA Cheng-Ying, 吕Hai-Peng , LIN Zhi, ZHANG Yue, GUO Li, TAN Jun-Feng. Cloning and Prokaryotic Expression of Flavonoid O-methyltransferase from Camellia sinensis[J]. Scientia Agricultura Sinica, 2013, 46(2): 325-333.

0
/ 收藏文章 0 / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.02.012

https://www.chinaagrisci.com/CN/Y2013/V46/I2/325

参考文献

[1]Punyasiri P A N, Abeysinghe I S B, Kumar V, Treutter D, Duy D, Gosch C, Martens S, Forkmann G, Fischer T C. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways. Archives of Biochemistry and Biophysics, 2004, 431: 22-30.

[2]乔小燕, 马春雷, 陈亮. 植物类黄酮生物合成途径及重要基因的调控. 天然产物研究与开发, 2009, 21:354-360.

Qiao X Y, Ma C L, Chen L. Plant flavonoid biosynthesis pathway and regulation of its important genes. Natural Product Research and Development, 2009, 21: 354-360.(in Chinese)

[3]Kces R E, Quattrocchio F, Mol J N M. The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays, 1994, 16(2):123-132.

[4]Caltagirone S, Rossi C, Poggi A, Ranelletti F O, Natali P G, Brunetti M, Aiello F B, Piantelli M. Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential. International Journal of Cancer, 2000, 87(4): 595-600.

[5]Carlo G D, Mascolo N, Lzzo A A, Capasso F. Flavonoids: Old and new aspects of a class of natural therapeutic drugs. Life Science, 1999, 65(4): 337-353.

[6]Harborne J B, Williams C A. Advances in flavonoid research since 1992. Phytochemistry, 2000, 55(6): 481-504.

[7]Shenouda N S, Zhou C, Browning J D, Ansell P J, Sakla M S, Lubahn D B, MacDonald R S. Phytoestrogens in common herbs regulate prostate cancer cell growth in vitro. Nutrition and Cancer, 2004, 49(2): 200-208.

[8]Zhu G X, Li C Z, Cao Z G. Inhibitory effect of flavonoid baicalin on degranulation of human polymorphonuclear leukocytes induced by interleukin-8: potential role in periodontal disease. Journal of Ethnopharmacology, 2007, 109(2): 325-330.

[9]Kim B G, Kim D H, Hur H G, Lim J, Lim Y, Ahn J H. O-Methyltransferases from Arabidopsis thaliana. Agricultural Chemistry and Biotechnology, 2005, 48(3): 113-119.

[10]Muzac I, Wang J, Anzellotti D, Zhang H, Ibrahim R K. Functional expression of an Arabidopsis cDNA clone encoding a flavonol 3′-O-methyltransferase and characterization of the gene product. Archives of Biochemistry and Biophysics, 2000, 375(2): 385-388.

[10]Maeda-Yamamoto M, Nagai H, Asai K, Moriwaki S, Horie H, Kohata K, Tachibana H, Miyase T, Sano M. Changes in epigallocatechin-3- O-(3-O-methylgallate and strictinin contents of tea (Camellia sinensis L.) cultivar Benifuki in various degrees of maturity and leaf order. Food Science and Technology Research, 2004, 10(2): 186-190.

[12]Kelavkar U P, Nixon J B, Cohen C, Dillehay D, Eling T E, Badr K F. Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis. Carcinogenesis, 2001, 22(11), 1765-1773.

[13]Kurita I, Maeda Y M, Tachibana H., Kamei M. Antihypertensive effect of Benifuuki tea containing O-methylated EGCG. Journal of Agricultural and Food Chemistry, 2010, 58(3): 1903-1908.

[14]Christensen A B, Gregersen P L, Olsen C E, Collinge D B. A flavonoid 7-O-methyltransferase is expressed in barley leaves in response to pathogen attack. Plant Molecular Biology, 1998, 36(2): 219-227.

[15]Kim B G, Lee Y, Hur H G, Lim Y, Ahn J H. Flavonoid 3′-O-methyltransferase from rice: cDNA cloning, characterization and functional expression. Phytochemistry, 2006, 67(4): 387-394.

[16]Kim B G, Lee H J, Park Y, Lim Y, Ahn J H. Characterization of an O-methyltransferase from soybean. Plant Physiology and Biochemistry, 2006, 44(4): 236-241.

[17]Cacacea S, Schrodera G, Wehingera E, Strack D, Schmidt J, Schroder J. A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations. Phytochemistry, 2003, 62(2): 127-137.

[18]Willitsa M G, Giovannib M, Prataa R T N, Kramer C M, Luca V D, Steffens J C, Graser G. Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites. Phytochemistry, 2004, 65(1): 31-41.

[19]Seguin J, Muzac I, Ibrahim R K. Purification and immunological characterization of a recombinant trimethyl- flavonol 3′-O-methyltransferase. Phytochemistry, 1998, 49(2): 319-325.

[20]Kim B G, Kim H, Hur H G, Lim Y, Ahn J H. Regioselectivity of 7-O-methyltransferase of poplar to flavones. Journal of Biotechnology, 2006, 126(2): 241-247.

[21]Zhou J M, Gold N D, Martin V J J, Wollenweber E, Ibrahim RK. Sequential O-methylation of tricetin by a single gene product in wheat. Biochimica et Biophysica Acta (BBA)-General Subjects, 2006, 1760(7): 1115-1124.

[22]Deavours B E, Liu C J, Naoumkina M A, Tang Y H, Farag M A, Sumner L W, Noel J P, Dixon R A. Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula. Plant Molecular Biology, 2006, 62(4-5):715-733.

[23]田铃, 嵇保中, 刘曙雯, 金凤, 高洁. 甲基转移酶的功能与分类. 生命的化学, 2007, 27(5): 425-427.

Tian L, Ji B Z, Liu S W, Jin F, Gao J. Function and classification of methyltransferase. Chemistry of Life, 2007, 27(5): 425-427.(in Chinese)

[24]French C J, Elder M, Leggett F, Ibrahim R K, Towers G H N. Flavonoids inhibit infectivity of tobacco mosaic virus. Canadian Journal of Plant Pathology, 1991, 13(1): 1-6.

[25]董昌金, 赵斌. 类黄酮物质apigenin和daidzein诱导AM真菌侵染十字花科植物芥菜. 科学通报, 2004, 49(10): 953-960.

Dong C J, Zhao B. Chinese Science Bulletin, 2004, 49(10): 953-960. (in Chinese)

[26]Gauthier A, Gulick P J, Ibrahim R K. cDNA cloning and characterization of a 3′/5′-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum. Plant Molecular Biology, 1996, 32(6): 1163-1169.

[27]Tuskan G A, Difazio S, Bohlmann J, et al. The genome of black cottonwood, Populus trichocarpa (Torr.&Gray). Science, 2006, 313(5793): 1596-1604

[28]Kirita M, Honma D, Tanaka Y, Usui S, Shoji T, Sami M, Yokota T, Tagashira M, Muranaka A, Uchiyama M, Kanda T, Maeda- Yamamoyo M. Cloning of a novel O-Methyltransferase from Camellia sinensis and synthesis of O-methylated EGCG and evaluation of their bioactivity. Journal of Agricultural and Food Chemistry, 2010, 58(12): 7196-7201.

[29]Esposito D, Chatterjee D K. Enhancement of soluble protein expression through the use of fusion tags. Current Opinion in Biotechnology, 2006, 17(4): 353-358.

[30]Waugh D S. Making the most of affinity tags. Trends in Biotechnology, 2005, 23(6): 316-320.

Metrics

Viewed

Full text

345

HTML			PDF

Just accepted	Online first	Issue	Just accepted	Online first	Issue
0	0	0	0	0	345

From	Others	local

Times	163	182
Rate	47%	53%

Abstract

230

Just accepted	Online first	Issue

0	0	230

From	Others	local

Times	218	12
Rate	95%	5%

Cited

Web of Science	Crossref	ScienceDirect	Search for Citations in Google Scholar >>


This page requires you have already subscribed to WoS.

Shared

[1]	古丽旦,刘洋,李方向,成卫宁. 小麦吸浆虫小热激蛋白基因Hsp21.9的克隆及在滞育过程与温度胁迫下的表达特性[J]. 中国农业科学, 2023, 56(1): 79-89.
[2]	林馨颖,王鹏杰,杨如兴,郑玉成,陈潇敏,张磊,邵淑贤,叶乃兴. 高茶氨酸茶树新品系‘福黄1号’黄化变异机理[J]. 中国农业科学, 2022, 55(9): 1831-1845.
[3]	蒋琪琪,许建建,苏越,张琦,曹鹏,宋晨虎,李中安,宋震. 柑橘黄化花叶病毒侵染性克隆构建及应用[J]. 中国农业科学, 2022, 55(24): 4840-4850.
[4]	李雨泽,朱嘉伟,林蔚,蓝茉莹,夏黎明,张艺粒,罗聪,黄桂香,何新华. 香水柠檬RHF2A的克隆与互作蛋白的筛选[J]. 中国农业科学, 2022, 55(24): 4912-4926.
[5]	张冯禧,肖琦,朱家平,尹力鸿,赵霞玲,严明帅,徐晋花,温立斌,牛家强,何孔旺. 非洲猪瘟病毒P30蛋白单克隆抗体制备、鉴定及阻断ELISA方法的建立[J]. 中国农业科学, 2022, 55(16): 3256-3266.
[6]	魏天,王成宇,王凤杰,李忠鹏,张芳毓,张守峰,扈荣良,吕礼良,王永志. 非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位[J]. 中国农业科学, 2022, 55(15): 3062-3070.
[7]	李正刚,汤亚飞,佘小漫,于琳,蓝国兵,何自福. 侵染萝卜的油菜花叶病毒广东分离物分子特征及其致病性分析[J]. 中国农业科学, 2022, 55(14): 2752-2761.
[8]	段雅如,高美玲,郭宇,梁晓雪,刘秀杰,徐洪国,刘继秀,高越,栾非时. 西瓜果形基因图位克隆及分子标记开发[J]. 中国农业科学, 2022, 55(14): 2812-2824.
[9]	渠成,王然,李峰奇,罗晨. 烟粉虱味觉受体基因BtabGR1和BtabGR2的克隆与表达模式分析[J]. 中国农业科学, 2022, 55(13): 2552-2561.
[10]	张莉,张楠,江虎强,吴帆,李红亮. 中华蜜蜂NPC2基因家族克隆及表达模式分析[J]. 中国农业科学, 2022, 55(12): 2461-2471.
[11]	郭丰辉,丁勇,纪磊,李贤松,李西良,侯向阳. 羊草克隆后代对刈割处理的响应[J]. 中国农业科学, 2022, 55(11): 2257-2264.
[12]	郭永春, 王鹏杰, 金珊, 侯炳豪, 王淑燕, 赵峰, 叶乃兴. 基于WGCNA鉴定茶树响应草甘膦相关的基因共表达模块[J]. 中国农业科学, 2022, 55(1): 152-166.
[13]	樊晓静, 于文涛, 蔡春平, 林浥, 王泽涵, 房婉萍, 张见明, 叶乃兴. 利用SNP标记构建茶树品种资源分子身份证[J]. 中国农业科学, 2021, 54(8): 1751-1760.
[14]	徐翔,解屹,宋丽云,申莉莉,李莹,王勇,刘明宏,刘东阳,王小彦,赵存孝,王凤龙,杨金广. 高效靶向降解烟草花叶病毒核酸的dsRNA筛选与大量制备[J]. 中国农业科学, 2021, 54(6): 1143-1153.
[15]	梁雨欣,吴建祥,李小宇,张春雨,侯吉超,周雪平,王永志. 马铃薯Y病毒衣壳蛋白抗原表位分析及其快速ELISA检测方法的建立[J]. 中国农业科学, 2021, 54(6): 1154-1162.