茶树多酚氧化酶基因的单核苷酸多态性分析
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摘要
多酚氧化酶(PPO)是一类含铜氧化还原酶,是红茶加工中品质形成的关键酶。本研究对44个茶树品种进行测序,研究PPO基因m RNA编码区的单核苷酸多态性(SNP),结果表明:共发现50个SNP位点,核苷酸多态性指数pi为0.014 06,基因的单倍型多样性(HD)为0.955,说明PPO基因具有较高的遗传多样性;转换类型有(A?G, C?T),颠换类型有(A?T, G?C, G→T, A→C),其中18个SNPs属于同义突变,32个外显子SNPs属于错义突变,有6个SNPs发生在核酸内切酶酶切位点。表明PPO基因存在多个等位基因,而且新等位基因的产生对PPO基因的表达有重要影响,对茶树新品种选育和早期鉴定具有十分重要的意义,为进一步研究茶树分子育种提供理论依据。
Polyphenol oxidase(PPO) is a type of copper-containing oxidoreductase, which is the key enzyme in the formation of black tea quality during its processing. In this study, 44 tea cultivars were sequenced to study single nucleotide polymorphisms(SNPs) in PPO gene mRNA coding region. The results showed that a total of 50 SNPs were discovered. The nucleotide polymorphism index pi was 0.014 06 and the gene haplotype diversity(HD)was 0.955, indicating that PPO gene had a high level in genetic diversity. Transition types included(A?G, C?T),and transversion types included(A?T, G?C, G→T, A→C). Among them, 32 SNPs were missense mutations,and 18 SNPs were synonymous mutations. 6 SNPs arose at restriction site of endonuclease. These results indicated that there were multiple alleles in the PPO gene, and the generation of new alleles had an important impact on the expression of PPO genes, which would be of great significance for the early identification and breeding of tea varieties and could provide a theoretical basis for further studies on molecular breeding of tea plants.
Polyphenol oxidase(PPO) is a type of copper-containing oxidoreductase, which is the key enzyme in the formation of black tea quality during its processing. In this study, 44 tea cultivars were sequenced to study single nucleotide polymorphisms(SNPs) in PPO gene mRNA coding region. The results showed that a total of 50 SNPs were discovered. The nucleotide polymorphism index pi was 0.014 06 and the gene haplotype diversity(HD)was 0.955, indicating that PPO gene had a high level in genetic diversity. Transition types included(A?G, C?T),and transversion types included(A?T, G?C, G→T, A→C). Among them, 32 SNPs were missense mutations,and 18 SNPs were synonymous mutations. 6 SNPs arose at restriction site of endonuclease. These results indicated that there were multiple alleles in the PPO gene, and the generation of new alleles had an important impact on the expression of PPO genes, which would be of great significance for the early identification and breeding of tea varieties and could provide a theoretical basis for further studies on molecular breeding of tea plants.
引文
Chen C.H.,Qi C.H.,and Wang Y.X.,2011,Study on polyphenol oxidase activities of tea cultivars grown in Sichuan,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),39(1):51-53(陈昌辉,齐桂年,汪艳霞,2011,四川主栽茶树品种的多酚氧化酶活性,贵州农业科学,39(1):51-53)
Chen D.,and Wu D.J.,2008,Approaches for SNP genotyping,Shengwu Jishu Tongbao(Biotechnology Bulletin),(2):93-104(陈冬,吴登俊,2008,单核苷酸多态性检测方法的研究进展,生物技术通报,(2):93-104)
Du W.N.,Sun H.X.,and Fang F.D.,2000,The research development of single nucleotide polymorphism,Zhongguo Yixue Kexueyuan Xuebao(Acta Academiae Medicinae Sinicae),22(4):392-394(杜玮南,孙红霞,方福德,2000,单核苷酸多态性的研究进展,中国医学科学院学报,22(4):392-394)
Fu Y.X.,and Liu T.X.,2015,The effect of polyphenol oxidase on the pile-fermentation process and quality of pu-erh tea,Xiandai Shipin Keji(Modern Food Science and Technology),31(3):197-201(付赢萱,刘通讯,2015,多酚氧化酶对普洱茶渥堆发酵过程中品质变化的影响,现代食品科技,31(3):197-201)
Huang J.A.,Huang Y.H.,Luo J.W.,Li J.X.,Gong Z.H.,and Liu Z.H.,2007,Identification of single nucleotide polymorphisms(SNPs)in polyphenol oxidase gene in tea plant(Camellia sinensis),Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Sciences)),33(4):454-485(黄建安,黄意欢,罗军武,李佳贤,龚志华,刘仲华,2007,茶树多酚氧化酶基因的SNP分析,湖南农业大学学报(自然科学版),33(4):454-485)
Huo J.H.,Chen S.P.,Lu Q.,Zhang J.,Bai L.,and Ma A.Q.,2017,Mutations of NOS3 gene in coronary artery spasm,Shaanxi Yixue Zazhi(Shaanxi Medical Journal),46(11):1498-1500(霍建华,陈姝萍,卢群,张俊,白玲,马爱群,2017,冠状动脉痉挛相关基因NOS3同义突变位点鉴定研究,陕西医学杂志,46(11):1498-1500)
Jin J.Q.,Yao M.Z.,Ma C.L.,and Ma J.Q.,2016,Natural allelic variations of TCS1 play a crucial role in caffeine biosynthesis of tea plant and its related species,Plant Physiol.Biochem.,100:18-26
Li J.L.,Wang S.,Yu J.,Wang L.,and Zhou S.M.,2013,A modified CTAB protocol for plant DNA extraction,Zhiwu Xuebao(Chinese Bulletin of Botany),48(1):72-78(李金璐,王硕,于婧,王玲,周世良,2013,一种改良的植物DNA提取方法,植物学报,48(1):72-78)
Liang C.M.,Deng J.X.,Li J.N.,Wei F.Y.,and Li Z.Y.,2017,Clone and bioinformatic analysis of PGC-1αin Guangxi Nandan-Yao,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(12):5113-5115(梁彩梅,邓继贤,黎剑能,韦凤英,黎卓炎,2017,广西南丹瑶鸡PGC-1α基因的克隆与生物信息学分析,基因组学与应用生物学,36(12):5113-5115)
Liu B.,2003,The SNPs of MHC classⅡin 15 pig breeds and their association with immune traits,Dissertation for Ph.D.,Huazhong Agricultural University,Supervisor:Li K.,pp.42(刘榜,2003,15个猪品种MHCⅡ类区4个基因的SNPs分析及与免疫性状的关联,博士学位论文,华中农业大学,导师:李奎,pp.42)
Nie X.M.,2017,Identification and characterization of the HLAnew alleles and preparation of their phage-display monoclonal antibodies,Dissertation for Ph.D.,Shandong University,Supervisor:Zhao Y.R.,pp.25-66(聂向民,2017,HLA新等位基因的确认,序列分析及其噬菌体展示单克隆抗体的制备,博士学位论文,山东大学,导师:赵跃然,pp.25-66)
Palle S.R.,Seeve C.M,Eckert A.J,Wegrzym J.L.,and Neala D.B.,2013,Association of loblolly pine xylem development gene expression with single-nucleotide polymorphisms,Tree Physiol.,33(7):763-774
Shi R.M.,Qiang H.,Zhang Y.M.,Ma A.Q.,and Gao J.,2013,Site-directed mutagenesis and protein expression of SCN5Agene associated with congenital Long QT syndrome,Zhongguo Dangdai Erke Zazhi(Chinese Journal of Contemporary Pediatrics),15(3):223-226(史瑞明,强华,张艳敏,马爱群,高洁,2013,先天性QT间期延长综合征相关基因SCN5A定点突变及蛋白表达研究,中国当代儿科杂志,15(3):223-226)
Sun M.F.,Zhang J.,and Guo G.Y.,2014,Features of polyphenol oxidase in baihaozao fresh tea leaves,Guizhou Nongye Kaxue(Guizhou Agricultural Sciences),42(6):140-143(孙慕芳,张洁,郭桂义,2014,白毫早鲜叶茶多酚氧化酶酶学特性,贵州农业科学,42(6):140-143)
Teng J.,Feng J.Y.,Xiong S.,Qin Y.,Liu K.Y.,and Gong Z.H.,2014,Research progress on polyphenol oxidase and its isozyme of tea,Chaye Tongxun(Tea Communication),41(2):10-13(滕杰,丰金玉,熊硕,秦昱,刘昆言,龚志华,2014,茶叶多酚氧化酶及其同工酶的研究进展,茶叶通讯,41(2):10-13)
Wang L.,Lin Z.,Lv H.P.,Tan J.F.,and Guo L.,2010,Research progress in affecting factors of tea aroma,Shipin Kexue(Food Science),31(15):293-298(王力,林智,吕海鹏,谭俊峰,郭丽,2010,茶叶香气影响因子的研究进展,食品科学,31(15):293-298)
Wang L.Y.,Zhang C.C.,Cheng H.,and Wei K.,2012,Characterization of EST-derived SNPs and development of SNP-markers in tea(Camellia sinensis),Chaye Kexue(Journal of Tea Science),32(4):369-376(王丽鸳,张成才,成浩,韦康,2012,茶树EST-SNP分布特征及标记开发,茶叶科学,32(4):369-376)
Wu M.Y.,Huang Y.J.,Yao Y.N.,and Huang Y.Y.,2017,Characterization of crude polyphenol oxidase isolated from Camellia sinensis var.assamica cv.Mengku.Zhejiang Daxue Xuebao(Journal of Zhejiang University(Agricul ture and Life Sciences)),43(5):579-588(伍梦瑶,黄莹捷,姚燕妮,黄友谊,2017,勐库大叶种茶树多酚氧化酶粗酶的酶学性质,浙江大学学报(农业与生命科学版),43(5):579-588)
Zhang L.Q.,Wei K.,Wang L.Y.,Cheng H.,Liu B.Y.,and Gong W.Y.,2014,The structure and single nucleotide polymorphism analysis of chalcone synthase genes in tea plant(Camellia sinenesis),Zhongguo Nongye Kexue(Scientia Agricultura Sinica),47(1):133-144(张丽群,韦康,王丽鸳,成浩,刘本英,龚武云,2014,茶树CHS基因结构及编码区单核苷酸多态性分析,中国农业科学,47(1):133-144)
Zhou Z.H.,Zhang D.,Ni H.,and Li H.H.,2017,Bioinformatic screening of natural polyphenol oxidass for thesflavins synthesis,Huanan Shifan Daxue Xuebao(Journal of South China Normal University(Natural Science Edition)),49(3):59-67(周仲华,张达,倪贺,李海航,2017,生物信息学筛选催化茶黄素合成的多酚氧化酶,华南师范大学学报(自然科学版),49(3):59-67)
Zou Y.P.,and Ge S.,2003,A novel molecular marker-SNPs and its application,Shengwu Duoyangxing(Biodiversity Science),11(5):370-382(邹喻苹,葛颂,2003,新一代分子标记-SNPs及其应用,生物多样性,11(5):370-382)
Chen D.,and Wu D.J.,2008,Approaches for SNP genotyping,Shengwu Jishu Tongbao(Biotechnology Bulletin),(2):93-104(陈冬,吴登俊,2008,单核苷酸多态性检测方法的研究进展,生物技术通报,(2):93-104)
Du W.N.,Sun H.X.,and Fang F.D.,2000,The research development of single nucleotide polymorphism,Zhongguo Yixue Kexueyuan Xuebao(Acta Academiae Medicinae Sinicae),22(4):392-394(杜玮南,孙红霞,方福德,2000,单核苷酸多态性的研究进展,中国医学科学院学报,22(4):392-394)
Fu Y.X.,and Liu T.X.,2015,The effect of polyphenol oxidase on the pile-fermentation process and quality of pu-erh tea,Xiandai Shipin Keji(Modern Food Science and Technology),31(3):197-201(付赢萱,刘通讯,2015,多酚氧化酶对普洱茶渥堆发酵过程中品质变化的影响,现代食品科技,31(3):197-201)
Huang J.A.,Huang Y.H.,Luo J.W.,Li J.X.,Gong Z.H.,and Liu Z.H.,2007,Identification of single nucleotide polymorphisms(SNPs)in polyphenol oxidase gene in tea plant(Camellia sinensis),Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Sciences)),33(4):454-485(黄建安,黄意欢,罗军武,李佳贤,龚志华,刘仲华,2007,茶树多酚氧化酶基因的SNP分析,湖南农业大学学报(自然科学版),33(4):454-485)
Huo J.H.,Chen S.P.,Lu Q.,Zhang J.,Bai L.,and Ma A.Q.,2017,Mutations of NOS3 gene in coronary artery spasm,Shaanxi Yixue Zazhi(Shaanxi Medical Journal),46(11):1498-1500(霍建华,陈姝萍,卢群,张俊,白玲,马爱群,2017,冠状动脉痉挛相关基因NOS3同义突变位点鉴定研究,陕西医学杂志,46(11):1498-1500)
Jin J.Q.,Yao M.Z.,Ma C.L.,and Ma J.Q.,2016,Natural allelic variations of TCS1 play a crucial role in caffeine biosynthesis of tea plant and its related species,Plant Physiol.Biochem.,100:18-26
Li J.L.,Wang S.,Yu J.,Wang L.,and Zhou S.M.,2013,A modified CTAB protocol for plant DNA extraction,Zhiwu Xuebao(Chinese Bulletin of Botany),48(1):72-78(李金璐,王硕,于婧,王玲,周世良,2013,一种改良的植物DNA提取方法,植物学报,48(1):72-78)
Liang C.M.,Deng J.X.,Li J.N.,Wei F.Y.,and Li Z.Y.,2017,Clone and bioinformatic analysis of PGC-1αin Guangxi Nandan-Yao,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(12):5113-5115(梁彩梅,邓继贤,黎剑能,韦凤英,黎卓炎,2017,广西南丹瑶鸡PGC-1α基因的克隆与生物信息学分析,基因组学与应用生物学,36(12):5113-5115)
Liu B.,2003,The SNPs of MHC classⅡin 15 pig breeds and their association with immune traits,Dissertation for Ph.D.,Huazhong Agricultural University,Supervisor:Li K.,pp.42(刘榜,2003,15个猪品种MHCⅡ类区4个基因的SNPs分析及与免疫性状的关联,博士学位论文,华中农业大学,导师:李奎,pp.42)
Nie X.M.,2017,Identification and characterization of the HLAnew alleles and preparation of their phage-display monoclonal antibodies,Dissertation for Ph.D.,Shandong University,Supervisor:Zhao Y.R.,pp.25-66(聂向民,2017,HLA新等位基因的确认,序列分析及其噬菌体展示单克隆抗体的制备,博士学位论文,山东大学,导师:赵跃然,pp.25-66)
Palle S.R.,Seeve C.M,Eckert A.J,Wegrzym J.L.,and Neala D.B.,2013,Association of loblolly pine xylem development gene expression with single-nucleotide polymorphisms,Tree Physiol.,33(7):763-774
Shi R.M.,Qiang H.,Zhang Y.M.,Ma A.Q.,and Gao J.,2013,Site-directed mutagenesis and protein expression of SCN5Agene associated with congenital Long QT syndrome,Zhongguo Dangdai Erke Zazhi(Chinese Journal of Contemporary Pediatrics),15(3):223-226(史瑞明,强华,张艳敏,马爱群,高洁,2013,先天性QT间期延长综合征相关基因SCN5A定点突变及蛋白表达研究,中国当代儿科杂志,15(3):223-226)
Sun M.F.,Zhang J.,and Guo G.Y.,2014,Features of polyphenol oxidase in baihaozao fresh tea leaves,Guizhou Nongye Kaxue(Guizhou Agricultural Sciences),42(6):140-143(孙慕芳,张洁,郭桂义,2014,白毫早鲜叶茶多酚氧化酶酶学特性,贵州农业科学,42(6):140-143)
Teng J.,Feng J.Y.,Xiong S.,Qin Y.,Liu K.Y.,and Gong Z.H.,2014,Research progress on polyphenol oxidase and its isozyme of tea,Chaye Tongxun(Tea Communication),41(2):10-13(滕杰,丰金玉,熊硕,秦昱,刘昆言,龚志华,2014,茶叶多酚氧化酶及其同工酶的研究进展,茶叶通讯,41(2):10-13)
Wang L.,Lin Z.,Lv H.P.,Tan J.F.,and Guo L.,2010,Research progress in affecting factors of tea aroma,Shipin Kexue(Food Science),31(15):293-298(王力,林智,吕海鹏,谭俊峰,郭丽,2010,茶叶香气影响因子的研究进展,食品科学,31(15):293-298)
Wang L.Y.,Zhang C.C.,Cheng H.,and Wei K.,2012,Characterization of EST-derived SNPs and development of SNP-markers in tea(Camellia sinensis),Chaye Kexue(Journal of Tea Science),32(4):369-376(王丽鸳,张成才,成浩,韦康,2012,茶树EST-SNP分布特征及标记开发,茶叶科学,32(4):369-376)
Wu M.Y.,Huang Y.J.,Yao Y.N.,and Huang Y.Y.,2017,Characterization of crude polyphenol oxidase isolated from Camellia sinensis var.assamica cv.Mengku.Zhejiang Daxue Xuebao(Journal of Zhejiang University(Agricul ture and Life Sciences)),43(5):579-588(伍梦瑶,黄莹捷,姚燕妮,黄友谊,2017,勐库大叶种茶树多酚氧化酶粗酶的酶学性质,浙江大学学报(农业与生命科学版),43(5):579-588)
Zhang L.Q.,Wei K.,Wang L.Y.,Cheng H.,Liu B.Y.,and Gong W.Y.,2014,The structure and single nucleotide polymorphism analysis of chalcone synthase genes in tea plant(Camellia sinenesis),Zhongguo Nongye Kexue(Scientia Agricultura Sinica),47(1):133-144(张丽群,韦康,王丽鸳,成浩,刘本英,龚武云,2014,茶树CHS基因结构及编码区单核苷酸多态性分析,中国农业科学,47(1):133-144)
Zhou Z.H.,Zhang D.,Ni H.,and Li H.H.,2017,Bioinformatic screening of natural polyphenol oxidass for thesflavins synthesis,Huanan Shifan Daxue Xuebao(Journal of South China Normal University(Natural Science Edition)),49(3):59-67(周仲华,张达,倪贺,李海航,2017,生物信息学筛选催化茶黄素合成的多酚氧化酶,华南师范大学学报(自然科学版),49(3):59-67)
Zou Y.P.,and Ge S.,2003,A novel molecular marker-SNPs and its application,Shengwu Duoyangxing(Biodiversity Science),11(5):370-382(邹喻苹,葛颂,2003,新一代分子标记-SNPs及其应用,生物多样性,11(5):370-382)