获得了一类茶树氧甲基转移酶基因cDNA全长并构建了该基因的原核表达载体。以从茶树叶片中提取的总RNA为模板,结合RT-PCR与RACE克隆技术获得氧甲基转移酶（O-methyltransferase）基因cDNA全长1β280βbp,其开放阅读框为1β068βbp,编码355个氨基酸,推测的蛋白分子量为39.1βkD,理论等电点为5.68。其氨基酸序列与葡萄和蓖麻氧甲基转移酶基因相似性分别为73%、71%。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后诱导重组蛋白的表达,经IPTG诱导,SDS-PAGE检测到1条与预测融合蛋白分子量相符的外源蛋白。
A full length cDNA of O-methyltransferase gene was obtained from Camellia sinensis and the prokaryotic expression vector for this gene was constructed. Based on total RNA from tea leaves, a O-methyltransferase cDNA sequence of tea was obtained by RT-PCR and RACE. The whole cDNA sequence 1β280βbp which contains an ORF of 1β068βbp and encodes 355 amino acids. The putative protein of this gene had an isoelectric point of 5.68 and a calculated molecular weight of 39.1βkD. The amino acid sequence of tea O-methyltransferase showed 73%, 71% identity with that of Vitis vinifera and Ricinus communis respectively. The coding sequence had been cloned into pET-28a and transformed into the host BL21. Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG.
[1] 田铃, 嵇保中, 刘曙雯, 等. 甲基转移酶的功能与分类[J]. 生命的化学, 2007, 27(5): 425-427.
[2] 李波, 倪志勇, 王娟, 等. 木质素生物合成关键酶咖啡酸-O-甲基转移酶基因(COMT)的研究进展[J]. 分子植物育种, 2010, 8(1): 117-124.
[3] 张传丽, 仲月明, 沈丹红, 等. 植物类黄酮O-甲基转移酶研究进展[J]. 西北植物学报, 2012, 32(6): 1274-1281.
[4] Boudet AM, Lapierre C, Grima-Pettenati J.Bio-chemistry and molecular biology of lignifications[J]. New Phytologist, 1995, 129: 203-236.
[5] Li L, Popko JL, Zhang XH, et al. A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine[J]. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94: 5461-5466.
[6] Li L, Zhou Y, Cheng X, et al. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100: 4939-4944.
[7] Muzac I, Wang J, Anzellotti D, et al. Functional Expression of an Arabidopsis cDNA Clone Encoding a Flavonol 3′-O-Methyltransferase and Characterization of the Gene Product[J]. Archives of Biochemistry and Biophysics, 2000, 375(2): 385-388
[8] French CJ, Elder M, Leggett F, et al. Flavonoids inhibit infectivity of tobacco mosaic virus[J]. Canadian Journal of Plant Pathology, 1991, 13(1): 1-6.
[9] Long SR.Rhizobium-legume nodulation: life together in the underground[J]. Cell, 1989, 56(2): 203-214.
[10] Yang H, Ahn JH, Ibrahim RK, et a1. The three-dimensional structure of Arabidopsis thaliana O-methyltransferase predicted by homology-based modeling[J]. Journal Molecular Graph Model, 2004, 23(1): 77-87.
[11] Maeda-Yamamoto M, Nagai H, Asai K, et al. Changes in epigallocatechin-3-O-(3-O-methyl gallate and strictinin contents of tea (Camellia sinensis L.) cultivar 'Benifuki' in various degrees of maturity and leaf order[J]. Food Science and Technology Research, 2004, 10(2): 186-190.
[12] Kelavkar UP, Nixon JB, Cohen C, et al. Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis[J]. Carcinogenesis, 2001, 22(11): 1765-1773.
[13] Kurita I, Maeda YM, Tachibana H, et al. Antihypertensive effect of Benifuuki tea containing O-methylated EGCG[J]. Journal of Agricultural and Food Chemistry, 2010, 58(3): 1903-1908.
[14] Wu SQ, Watanabe N, Mita S, et al. Two O-Methyltransferases isolated from flower petals of Rosa chinensis var. spontanea involved in scent biosynthesis[J]. Journal of Bioscience and Bioengineering, 2003, (96): 119-128.
[15] Joshi CP, Chiang VL.Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferase[J]. Plant Molecular Biology, 1998, 37: 663-674.
[16] Bong-Gyu Kim, Su Hyun Sung, Youhoon Chong, et al. Plant Flavonoid O-Methyltransferases: Substrate Specificity and Application[J]. J Plant Biology, 2010, 53: 321-329.
[17] Schmitt D, Pakusch AE, Matern U.Molecular cloning, induction and taxonomic distribution of caffeoyl-CoA 3-O-methyltransferase, an enzyme involved in disease resistance[J]. Biological Chemistry, 1991, 266: 17416-17423.
[18] 马成英, 吕海鹏, 林智, 等. 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.
[19] 李小霞, 余有本. 茶树咖啡酸氧甲基转移酶基因的克隆及原核表达[J]. 西北农业学报, 2011, 20(5): 139-143.
[20] Masanobu K, Daiki H, Yoshihisa T, et al. Cloning of a novel O-methyltransferase from Camellia sinensis and synthesis of O-methylated EGCG and evaluation of their bioactivity[J]. Journey of Agricultural and Food Chemistry, 2010, 58(12): 7196-7201.
[21] 费冬梅, 林智, 吕海鹏, 等. EGCG-O-甲基转移酶(EOMT)在重组大肠杆菌中的表达条件研究[J]. 茶叶科学, 201l, 31(4): 333-340.
[22] Kces RE, Quattrocchio F, Mol JNM.The flavonoid biosynthetic pathway in plants: function and evolution[J]. BioEssays, 1994, 16(2): 123-132.
[23] Gauthier A, Patrick JG, Ibrahim R K, et al. Characterization of two cDNA clones which encode O-methyltransferases for the methylation of both flavonoid and phenylpropanoid compounds[J]. Archives of Biochemistry and Biophysics, 1998, 351(2): 243-249.
[24] Frick S, Kutchan TM.Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis[J]. Plant J, 1999, 17: 329-339.
[25] Schroder G, Wehinger E, Schroder J.Predicting the substrates of cloned plant O-methyltransferases[J]. Phytochemistry, 2002, 59: 1-8.
[26] Wang J, Pichersky E.Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases[J]. Archives of Biochemistry and Biophysics, 1999, 368: 172-180.
[27] Zubieta C, He XZ,Dixon RA, et al. Structures of two natural product methyhransferases reveal the basis for substrate specificity in plant O-methyltransferases[J]. Nature Structural Biology, 2001, 8: 271-279.
浙公网安备 33019902000101号 
