辣椒γ-生育酚甲基转移酶(γ-TMT)基因克隆和生物信息学分析
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摘要
维生素E包括生育酚和生育三烯酚,是在光合生物中合成的一种脂溶性抗氧化物质。γ-生育酚甲基转移酶(Gamma-tocopherol methyl-transferase,γ-TMT)是决定植物中维生素E成分和活性的关键酶之一,它分别将δ-生育酚和γ-生育酚催化转变为β-生育酚和α-生育酚,其中α-生育酚的生物活性最高。本实验采用RACE方法从贵州商业辣椒-‘黔椒4号’中克隆出γ-TMT,基因序列全长1 110 bp。利用相关软件对其进行生物信息学分析,结果显示,该基因编码369个氨基酸,是一种不稳定的亲水性非分泌蛋白,并且该蛋白没有信号肽,也没有明显的跨膜结构域,主要在叶绿体基质中行使功能。这些数据为今后利用基因工程研究γ-TMT对辣椒维生素E含量及抗性的影响提供了帮助。
Vitamin E, including tocopherol and tocotrienol, is a lipid-soluble antioxidant synthesized in photosynthetic organisms. Gamma-tocopherol methyl-transferase(γ-TMT) is one of the key enzymes to determine the component and activity of vitamin E in plants, It converts δ-tocopherol and gamma-tocopherol into beta-tocopherol and alpha-tocopherol, with the highest bioactivity of alpha-tocopherol. In this experiment, γ-TMT was cloned from Guizhou commercial chili pepper('Qianjiao No. 4') by RACE method, and the full length of gene sequence was 1 110 bp. The results of bioinformatics analysis by related software showed that the gene encoded369 amino acids and was an unstable hydrophilic non-secretory protein. Moreover, the protein had no signal peptide and no obvious transmembrane domain. It mainly functioned in the chloroplast matrix. These data will be helpful for the future use of genetic engineering to study the effect of γ-TMT on vitamin E content and resistance of pepper.
Vitamin E, including tocopherol and tocotrienol, is a lipid-soluble antioxidant synthesized in photosynthetic organisms. Gamma-tocopherol methyl-transferase(γ-TMT) is one of the key enzymes to determine the component and activity of vitamin E in plants, It converts δ-tocopherol and gamma-tocopherol into beta-tocopherol and alpha-tocopherol, with the highest bioactivity of alpha-tocopherol. In this experiment, γ-TMT was cloned from Guizhou commercial chili pepper('Qianjiao No. 4') by RACE method, and the full length of gene sequence was 1 110 bp. The results of bioinformatics analysis by related software showed that the gene encoded369 amino acids and was an unstable hydrophilic non-secretory protein. Moreover, the protein had no signal peptide and no obvious transmembrane domain. It mainly functioned in the chloroplast matrix. These data will be helpful for the future use of genetic engineering to study the effect of γ-TMT on vitamin E content and resistance of pepper.
引文
Chai Y.Q.,Zhang Y.H.,Han N.,and Zhu M.Y.,2014,Advances in plant vitamin E gene engineering,Zhongguo Shengwu Gongcheng(Chinese Journal of Bioengineering),34(11):100-106(柴玉琼,张玉红,韩凝,朱睦元,2014,植物维生素E基因工程研究进展,中国生物工程杂志,34(11):100-106)
Eckardt N.A.,2003,Vitamin e-defective mutants of Arabidopsis tell tales of convergent evolution,Plant Cell,15(10):2233-2235
Ferro M.,Brugière S.,Salvi D.,Seigneurin-Berny D.,Court M.,Moyet L.,Ramus C.,Miras S.,Mellal M.,Le Gall S.,Kieffer-Jaquinod S.,Bruley C.,Garin J.,Joyard J.,Masselon C.,and Rolland N.,2010,AT_CHLORO:a comprehensive chloroplast proteome database with subplastidial localization and information on envelope proteins,Mol.Cell.Porteomics,9(6):1063-1084
Li S.Z.,Wan Y.S.,and Liu F.Z.,2012,Cloning and bioinformatic analysis ofγ-tocopherol methyltransferase gene(γ-TMT)in peanut,Zuowu Xuebao(Acta Agronomica Sinica),38(10):1856-1863(李拴柱,万勇善,刘凤珍,2012,花生γ-生育酚甲基转移酶基因(γ-TMT)的克隆及序列分析,作物学报,38(10):1856-1863)
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In Silico cloning bioinformatics analysis of KNOXgene in sugarcane(Scknl),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Scknl)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Ochoa A.N.,and Ramire M.R.,2001,In vitro chili pepper biotechnology,In vitro Cell.Dev.,37(6):701-729
Shintani D.,and Dellapenna D.,1998,Elevating the vitamin Econtent of plants through metabolic engineering,Science,282(5396):2098-2100
Shintani D.K.,Cheng Z.,and DellaPenna D.,2002,The role of2-methyl-6-phytylbenzoquinone methylt ransferase in determining tocopherol composition in Synechocytis sp.PCC-6803,FEBS Lett.,511(1-3):1-5
Van Eenennaam A.L.,Lincoln K.,Durrett T.P.,Valentin H.E.,Shewmaker C.K.,Thorne G.M.,Jiang J.,Baszis S.R.,Levering C.K.,Aasen E.D.,Hao M.,Stein J.C.,Norris S.R.,and Last R.L.,2003,Engineering vitamin E content:from Arabidopsis mutant to soy oil,Plant Cell,15(12):3007-3019
Wahyuni Y.,Ballester A.R.,Sudarmonowati E.,Bino R.J.,and Bovy A.G.,2011,Metabolite biodiversity in pepper(Capsicum)fruits of thirty-two diverse accessions:Variation in health-related compounds and implications for breeding,Phytochemistry,72(11-12):1358-1370
Wahyuni Y.,Ballester A.R.,Sudarmonowati E.,Bino R.J.,and Bovy A.G.,2013,Secondary Metabolites of Capsicum Species and Their Importance in the Human Diet,Journal of Natural Products,76(4):783-793
Ye C.Y.,Liu N.B.,and Zheng S.P.,2009,Cloning of key enzyme genes involved in vitamin E biosynthesis and acquirement of transgenic plants,Xiandai Shipin Keji(Modern Food Science and Technology),25(9):1020-1023(叶从勇,刘南波,郑穗平,2009,维生素E合成途径关键酶基因的克隆及转基因植株的构建,现代食品科技,25(9):1020-1023)
Zou L.P.,and Gao H.P.,2009,Cloning and sequence analysis ofγ-tocopherol methyltransferase(γ-TMT)cDNA from cotton(Gossypium hirsutum),Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),25(3):490-493(邹礼平,高和平,2009,棉花γ-生育酚甲基转移酶基因全长cD-NA的克隆与序列分析,江苏农业学报,25(3):490-493)
Eckardt N.A.,2003,Vitamin e-defective mutants of Arabidopsis tell tales of convergent evolution,Plant Cell,15(10):2233-2235
Ferro M.,Brugière S.,Salvi D.,Seigneurin-Berny D.,Court M.,Moyet L.,Ramus C.,Miras S.,Mellal M.,Le Gall S.,Kieffer-Jaquinod S.,Bruley C.,Garin J.,Joyard J.,Masselon C.,and Rolland N.,2010,AT_CHLORO:a comprehensive chloroplast proteome database with subplastidial localization and information on envelope proteins,Mol.Cell.Porteomics,9(6):1063-1084
Li S.Z.,Wan Y.S.,and Liu F.Z.,2012,Cloning and bioinformatic analysis ofγ-tocopherol methyltransferase gene(γ-TMT)in peanut,Zuowu Xuebao(Acta Agronomica Sinica),38(10):1856-1863(李拴柱,万勇善,刘凤珍,2012,花生γ-生育酚甲基转移酶基因(γ-TMT)的克隆及序列分析,作物学报,38(10):1856-1863)
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In Silico cloning bioinformatics analysis of KNOXgene in sugarcane(Scknl),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Scknl)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Ochoa A.N.,and Ramire M.R.,2001,In vitro chili pepper biotechnology,In vitro Cell.Dev.,37(6):701-729
Shintani D.,and Dellapenna D.,1998,Elevating the vitamin Econtent of plants through metabolic engineering,Science,282(5396):2098-2100
Shintani D.K.,Cheng Z.,and DellaPenna D.,2002,The role of2-methyl-6-phytylbenzoquinone methylt ransferase in determining tocopherol composition in Synechocytis sp.PCC-6803,FEBS Lett.,511(1-3):1-5
Van Eenennaam A.L.,Lincoln K.,Durrett T.P.,Valentin H.E.,Shewmaker C.K.,Thorne G.M.,Jiang J.,Baszis S.R.,Levering C.K.,Aasen E.D.,Hao M.,Stein J.C.,Norris S.R.,and Last R.L.,2003,Engineering vitamin E content:from Arabidopsis mutant to soy oil,Plant Cell,15(12):3007-3019
Wahyuni Y.,Ballester A.R.,Sudarmonowati E.,Bino R.J.,and Bovy A.G.,2011,Metabolite biodiversity in pepper(Capsicum)fruits of thirty-two diverse accessions:Variation in health-related compounds and implications for breeding,Phytochemistry,72(11-12):1358-1370
Wahyuni Y.,Ballester A.R.,Sudarmonowati E.,Bino R.J.,and Bovy A.G.,2013,Secondary Metabolites of Capsicum Species and Their Importance in the Human Diet,Journal of Natural Products,76(4):783-793
Ye C.Y.,Liu N.B.,and Zheng S.P.,2009,Cloning of key enzyme genes involved in vitamin E biosynthesis and acquirement of transgenic plants,Xiandai Shipin Keji(Modern Food Science and Technology),25(9):1020-1023(叶从勇,刘南波,郑穗平,2009,维生素E合成途径关键酶基因的克隆及转基因植株的构建,现代食品科技,25(9):1020-1023)
Zou L.P.,and Gao H.P.,2009,Cloning and sequence analysis ofγ-tocopherol methyltransferase(γ-TMT)cDNA from cotton(Gossypium hirsutum),Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),25(3):490-493(邹礼平,高和平,2009,棉花γ-生育酚甲基转移酶基因全长cD-NA的克隆与序列分析,江苏农业学报,25(3):490-493)