小麦γ-TMT基因的克隆与表达分析
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摘要
γ-生育酚甲基转移酶(γ-TMT)是维生素E合成途径的关键酶之一。该研究采用RT-PCR方法,从小麦品种‘中国春’中克隆得到小麦γ-TMT基因的3个同源拷贝,其CDs(coding sequence)序列相似性为99.12%;其中Ta-γ-TMT-6A和Ta-γ-TMT-6B编码区长度为1 101 bp,编码366个氨基酸,而Ta-γ-TMT-6D因发生无义突变,编码365个氨基酸。生物信息学分析表明,3个蛋白分子量分别为39.58、39.53和39.50 kDa,理论等电点分别为6.67、6.41和7.08,都属于亲水性不稳定非分泌型蛋白。系统进化分析表明,小麦Ta-γ-TMT蛋白与糜子Pm-γ-TMT的亲缘关系相对较近。实时定量PCR分析表明,小麦Ta-γ-TMT基因在根、茎、叶、颖壳、种子中均有表达,在叶片中表达量最高;ABA、NaCl、PEG均能诱导Ta-γ-TMT基因的上调表达。该研究结果为进一步探讨小麦γ-TMT基因的功能奠定了基础。
Gamma-tocopherol methyltransferase(γ-TMT) is one of the key enzymes in the vitamin E synthesis pathway. In this study, we cloned three homologous copies of the γ-tocopherol methyltransferase gene(γ-TMT) from the wheat cultivar-Chinese Spring using RT-PCR(reverse transcription PCR). The similarity of three sequence CDs(coding sequence) was 99.12%. Ta-γ-TMT-6A and Ta-γ-TMT-6B coding sequences both contain 1 101 bp and encode 366 amino acids, while Ta-γ-TMT-6D encodes only 365 amino acids due to nonsense mutations. The results of bioinformatics analysis showed that the molecular weights of the three proteins were 39.58, 39.53 and 39.50 kDa, respectively, and the theoretical isoelectric points were 6.67, 6.41 and 7.08. They all belonged to hydrophilic unstable non-secretory proteins. Phylogenetic tree analysis indicated that Ta-γ-TMT protein shared high similarity to γ-TMT protein from Panicum miliaceum and closest genetic relationship. Real-time quantitative PCR analysis showed that the Ta-γ-TMT gene was expressed in roots, stems, leaves, glumes and seeds, and highly expressed in leaves. Meanwhile, expression of the Ta-γ-TMT gene was up-regulated induced by ABA, NaCl, PEG treatments. This result will provide the foundation for further study of the function of the Ta-γ-TMT gene in wheat.
Gamma-tocopherol methyltransferase(γ-TMT) is one of the key enzymes in the vitamin E synthesis pathway. In this study, we cloned three homologous copies of the γ-tocopherol methyltransferase gene(γ-TMT) from the wheat cultivar-Chinese Spring using RT-PCR(reverse transcription PCR). The similarity of three sequence CDs(coding sequence) was 99.12%. Ta-γ-TMT-6A and Ta-γ-TMT-6B coding sequences both contain 1 101 bp and encode 366 amino acids, while Ta-γ-TMT-6D encodes only 365 amino acids due to nonsense mutations. The results of bioinformatics analysis showed that the molecular weights of the three proteins were 39.58, 39.53 and 39.50 kDa, respectively, and the theoretical isoelectric points were 6.67, 6.41 and 7.08. They all belonged to hydrophilic unstable non-secretory proteins. Phylogenetic tree analysis indicated that Ta-γ-TMT protein shared high similarity to γ-TMT protein from Panicum miliaceum and closest genetic relationship. Real-time quantitative PCR analysis showed that the Ta-γ-TMT gene was expressed in roots, stems, leaves, glumes and seeds, and highly expressed in leaves. Meanwhile, expression of the Ta-γ-TMT gene was up-regulated induced by ABA, NaCl, PEG treatments. This result will provide the foundation for further study of the function of the Ta-γ-TMT gene in wheat.
引文
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[18] DELLAPENNA D.A decade of progress in understanding vitamin E synthesis in plants[J].Journal of Plant Physiology,2005,162(7):729-737.
[19] CROWELL E F,MCGRATH J M,DOUCHES D S.Accumulation of vitamin E in potato (Solanum tuberosum) tubers[J].Transgenic Research,2008,17(2):205-217.
[20] 贾会丽,王学敏,高洪文,等.紫花苜蓿γ-生育酚甲基转移酶(γ-TMT)基因的克隆与逆境下的表达分析[J].草业学报,2012,21(6):198-206.JIA H L,WANG X M,GAO H W,et al.Cloning the gene of γ-tocopherol methyltransferase(γ-TMT) from alfalfa and expression analysis in adverse situations[J].Acta Prataculturae Sinica,2012,21(6):198-206.
[21] GHIMIRE B K,SEONG E S,CHAN O L,et al.Improved antioxidant activity in transgenic Perilla frutescens plants via overexpression of the γ-tocopherol methyltransferase (γ-tmt) gene[J].Protoplasma,2015,252(5):1 285-1 290.
[22] ALI-REZA A,MOHAMAD H,et al.Specific roles of alpha-and gamma-tocopherol in abiotic stress responses of transgenic tobacco[J].Plant Physiology,2007,143(4):1 720-1 738.
[23] YUSUF M A,KUMAR D,RAJWANSHI R,et al.Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress:physiological and chlorophyll a fluorescence measurements[J].BBA-Bioenergetics,2010,1 797(8):1 428-1 438.
[24] SHUANGXIA J,HENRY D.Expression of γ-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stresses by reducing reactive oxygen species[J].Plant Biotechnology Journal,2015,12(9):1 274-1 285.
[25] JIANG J,JIA H,FENG G,et al.Overexpression of Medicago sativa TMT elevates the α-tocopherol content in Arabidopsis seeds,alfalfa leaves,and delays dark-induced leaf senescence[J].Plant Science An International Journal of Experimental Plant Biology,2016,249:93-104.
[26] CHANG Y J,KIM Y H,KIM H S,et al.Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco[J].Plant Physiology & Biochemistry,2016,106:118-128.
[27] 姜霁珊.紫花苜蓿维生素E合成基因γ-TMT、HPT和HPPD分离、遗传转化及功能分析[D].北京:中国农业科学院,2016.
[28] ZHANG G Y,LIU R R,XU G,et al.Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase[J].Transgenic Research,2013,22(1):89-99.
[29] MUTHUKRISHNAN A,KONDETI S,JEEVARAJ T,et al.Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars[J].Applied Biochemistry & Biotechnology,2014,172(4):1 763-1 776.
[2] 欧阳青,蔡文启.天然维生素E的生物合成途径[J].植物生理学报,2003,39(5):501-507.OUYANG Q,CAI W Q.Biosynthesis pathway of nature vitamin E[J].Plant Physiology Communications,2003,39(5):501-507.
[3] BRIGELIUS-FLOHé R,TRABER M G.Vitamin E:function and metabolism[J].Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology,1999,13(10):1 145-1 155.
[4] KEY T J,APPLEBY P N,TRAVIS R C,et al.Carotenoids,retinol,tocopherols,and prostate cancer risk:pooled analysis of 15 studies[J].American Journal of Clinical Nutrition,2015,102(5):1 142-1 157.
[5] RASHID S,QAMAR K,TASSADUQ I.Role of vitamin E in preventing arteriohyalinization in kidneys of streptozotocin induced diabetic mice[J].Journal of the Pakistan Medical Association,2015,65(10):1 085-1 088.
[6] RIPOLL,GONZALEZ-CALVO,et al.Effects of finishing period length with vitamin E supplementation and alfalfa grazing on carcass color and the evolution of meat color and the lipid oxidation of light lambs[J].Meat Science,2013,93(4):906-913.
[7] 李拴柱,万勇善,刘风珍.花生γ-生育酚甲基转移酶基因(γ-TMT)的克隆及序列分析[J].作物学报,2012,38(10):1 856-1 863.LI S Z,WAN Y S,LIU F Z.Cloning and bioinformatic analysis of γ-tocopherol methyltransferase gene (γ-TMT) in peanut[J].Acta Agronomica Sinica,2012,38(10):1 856-1 863.
[8] CARETTO S,NISI R,PARADISO A,et al.Tocopherol production in plant cell cultures[J].Molecular Nutrition & Food Research,2010,54(5):726-730.
[9] ZINGG J M.Vitamin E:an overview of major research directions[J].Molecular Aspects of Medicine,2007,28(5):400-422.
[10] SHINTANI D,DELLAPENNA D.Elevating the vitamin E content of plants through metabolic engineering[J].Science,1998,282(5 396):2 098-2 100.
[11] TEWARI K,KUMAR V,KUMAR A,et al.Molecular cloning and functional analysis of the promoter of gamma-tocopherol methyltransferase (gamma-TMT) gene of soybean (Glycine max)[J].Biotech,2018,8(8):325.
[12] 邹礼平.玉米γ-生育酚甲基转移酶基因的克隆与分析[J].安徽农业科学,2011,39(33):20 316-20 317.ZOU L P.Cloning and sequence analysis of γ-tocopherol methyltransferase (γ-TMT) cDNA from maize (Zea mays) [J].Journal of Anhui Agricultural Sciences,2011,39(33):20 316-20 317.
[13] 刘婷,唐月异,胡东青,等.花生栽培种γ-TMT基因的克隆和序列分析[J].花生学报,2018,47(2):34-40.LIU T,TANG Y Y,HU D Q,et al.Cloning and sequence analysis of γ-tocopherol methyltransferase (γ-TMT) genes from the cultivated peanut[J].Journal of Peanut Science,2018,47(2):34-40.
[14] 刘燕,赵毅,吕千,等.小麦TaHTAS-5A基因的克隆、表达分析及亚细胞定位[J].麦类作物学报,2018,38(10):1 137-1 145.LIU Y,ZHAO Y,Lü Q,et al.Cloning,expression analysis and subcellular localization of TaHTAS-5A gene from wheat (Triticum aestivum)[J].Journal of Triticeae Crops,2018,38(10):1 137-1 145.
[15] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2[-Delta Delta C(T)] method[J].Methods,2001,25(4):402-408.
[16] ECKARDT N A.Vitamin E-defective mutants of Arabidopsis tell tales of convergent evolution[J].Plant Cell,2003,15(10):2 233-2 235.
[17] 郭娟,游文慧,彭秋菊,等.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[J/OL].分子植物育种,2019:1-13.[2019-03-10].http://kns.cnki.net/kcms/detail/46.1068.S.20180824.1008.018.html.
[18] DELLAPENNA D.A decade of progress in understanding vitamin E synthesis in plants[J].Journal of Plant Physiology,2005,162(7):729-737.
[19] CROWELL E F,MCGRATH J M,DOUCHES D S.Accumulation of vitamin E in potato (Solanum tuberosum) tubers[J].Transgenic Research,2008,17(2):205-217.
[20] 贾会丽,王学敏,高洪文,等.紫花苜蓿γ-生育酚甲基转移酶(γ-TMT)基因的克隆与逆境下的表达分析[J].草业学报,2012,21(6):198-206.JIA H L,WANG X M,GAO H W,et al.Cloning the gene of γ-tocopherol methyltransferase(γ-TMT) from alfalfa and expression analysis in adverse situations[J].Acta Prataculturae Sinica,2012,21(6):198-206.
[21] GHIMIRE B K,SEONG E S,CHAN O L,et al.Improved antioxidant activity in transgenic Perilla frutescens plants via overexpression of the γ-tocopherol methyltransferase (γ-tmt) gene[J].Protoplasma,2015,252(5):1 285-1 290.
[22] ALI-REZA A,MOHAMAD H,et al.Specific roles of alpha-and gamma-tocopherol in abiotic stress responses of transgenic tobacco[J].Plant Physiology,2007,143(4):1 720-1 738.
[23] YUSUF M A,KUMAR D,RAJWANSHI R,et al.Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress:physiological and chlorophyll a fluorescence measurements[J].BBA-Bioenergetics,2010,1 797(8):1 428-1 438.
[24] SHUANGXIA J,HENRY D.Expression of γ-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stresses by reducing reactive oxygen species[J].Plant Biotechnology Journal,2015,12(9):1 274-1 285.
[25] JIANG J,JIA H,FENG G,et al.Overexpression of Medicago sativa TMT elevates the α-tocopherol content in Arabidopsis seeds,alfalfa leaves,and delays dark-induced leaf senescence[J].Plant Science An International Journal of Experimental Plant Biology,2016,249:93-104.
[26] CHANG Y J,KIM Y H,KIM H S,et al.Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco[J].Plant Physiology & Biochemistry,2016,106:118-128.
[27] 姜霁珊.紫花苜蓿维生素E合成基因γ-TMT、HPT和HPPD分离、遗传转化及功能分析[D].北京:中国农业科学院,2016.
[28] ZHANG G Y,LIU R R,XU G,et al.Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase[J].Transgenic Research,2013,22(1):89-99.
[29] MUTHUKRISHNAN A,KONDETI S,JEEVARAJ T,et al.Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars[J].Applied Biochemistry & Biotechnology,2014,172(4):1 763-1 776.