通过PCR方法扩增出迎霜品种茶多酚氧化酶（PPO）的编码区序列,并将其登录于GenBank,注册号为GQ214317。生物信息学分析表明,该基因编码区全长1800bp,编码599个氨基酸,分子式为C3008H4677N813O900S18,存在两个铜离子结合域,没有跨膜区;进化树分析表明,茶与毛叶茶亲缘关系最近。成功构建了原核表达载体pET-ppo,并转化E. coli BL21（DE3）,实现了PPO的原核表达。SDS-PAGE电泳检测结果表明,在71KD处有一条特异蛋白条带,与预测大小相符。
The polyphenol oxidase (PPO, GenBank accession No. DQ812086) gene was amplified by PCR. Bioinformatics analysis indicated that the cds of PPO DNA sequence is 1800bp, which encoded a protein of 599 amino acid residues (molecular formula: C3008H4677N813O900S18) and predicted that it had two Cu-band functional domains with no transmembrane. The phylogeny analysis showed that it has a most close phylogenetic relationship with Camellia nitidissima. The PPO was cloned into pET28a vector to construct recombination prokaryotic expression vector pET-ppo. After transformed to E1coli BL21 and induced by isopropyl-β-D-thiogalactopyranoside(IPTG), recombinant protein about 71kD was expressed in pET28a system and separated by SDS-PAGE electrophoresis.
[1] 宛晓春. 茶树生物化学[M]. 北京:中国农业出版社, 2003: 30-31.
[2] Nathalie Wuyts, Dirk De Waele, Rony Swennen.Extraction and partial characterization of polyphenol oxidase from banana (Musa acuminata Grande naine) roots[J]. Plant Physiology and Biochemistry, 2006, 44(5/6): 308-314.
[3] 屠幼英, 方青, 梁惠玲, 等. 固定化酶膜催化茶多酚形成茶黄素反应条件优选[J]. 茶叶科学, 2004, 24(2): 129-134.
[4] Yoshino K, Hara Y, Sano M, et al. Antioxidative effects of black tea theaflavinsand thearubigin on Lipid peroxidation of rat liver homogenates induced by tert-buty hydroperoxide[J]. Biol Pharm Bμll, 1994, 17(1): 146-149.
[5] Sano M, Takashi Y, Yoshino K, et al. Effect of Lipid Peroxidation on Liver and Kidney: a comparison of green and black tea feeding[J]. Biol Pharm Bμll, 1995, 18(7): 100-108.
[6] Miller NJ, Castelluccio C, Tijburg L, et al. The antiodant properties of theaflavins and their gallate esters radical scavengers or metal chelators[J]. FEBS Letter, 1996, 392(1): 40-44.
[7] Roberts E A H, Cartwright R A, Oldschool Miss M. The phenolic substances of manufactured tea. I. —Fractionation and paper chromatography of water-soluble substances[J]. J Sci Food Agric, 1957, 8(2): 72-80.
[8] Roberts E A H. The phenolic substances of manufactured tea. II. — Their origin as enzymic oxidation products in fermentation[J]. J Sci Food Agric, 1958, 9(4): 212-216.
[9] Roberts E A H. The phenolic substances of manufactured tea. IX. —the spectrophotometric evaluation of tea liquors[J]. J Sci Food Agric, 1963, 14(10): 689-700.
[10] 萧伟祥, 钟瑾, 萧慧. 红茶色素形成机理与制取[J]. 茶叶科学, 1997, 17(1): 1-8.
[11] 李立祥, 萧伟祥. 茶黄素制取与分离研究进展[J]. 中国茶叶加工, 2000(4): 29-33.
[12] 宛晓春, 李大祥, 夏涛. 茶色素及其药理学功能[J]. 天然产物研究与开发, 2001, 13(4): 65-70.
[13] 赵东, 刘祖生, 奚彪. 茶树多酚氧化酶基因的克隆及其序列比较[J]. 茶叶科学, 2001, 21(2): 94-98.
[14] Raizada J, Kumar S, Ahuja PS.Cloning and characterizationof polyphenol oxidase gene in Camellia sinensis (L.) O.kuntze. http://www.ncbi.nlm.nih.gov, Accessing No.AY659975.
[15] 倪穗, 田敏, 李纪元, 等. 红山茶高质量基因组DNA的提取[J]. 宁波大学学报, 2007, 202(2): 163-167.
[16] Cary J W, Lax A R, Flurkey W H.Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase[J]. Plant Mol Biol, 1992, 20(2): 245-253.
[17] 冯思玲. 生物信息学技术研究[J]. 信息技术, 2009(5): 20-22.
[18] 刘智珺. 生物信息学中数据库技术的应用[J]. 计算机与数字工程, 2009(5): 157-159.
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