忽地笑γ-生育酚甲基转移酶基因LaTMT的克隆与表达分析
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摘要
采用RACE技术从忽地笑〔Lycoris aurea(L’Hér.)Herb.〕叶片中克隆获得γ-生育酚甲基转移酶(γ-TMT)基因,命名为La TMT。序列分析结果显示:该基因c DNA全长1 458 bp,其中开放阅读框(ORF)长1 017 bp,编码338个氨基酸残基。La TMT基因编码蛋白质的理论相对分子质量37 560,理论等电点p I 8.70,为亲水性蛋白,无跨膜结构但具有信号肽结构;并具有S-腺苷甲硫氨酸(SAM)甲基转移酶保守结构域,包含3个SAM结合位点;该蛋白的二级结构中包含44.08%的α-螺旋、32.84%的无规则卷曲、12.72%的延伸链和10.36%的β-转角。序列比对和系统进化树分析结果显示:La TMT蛋白属于S-腺苷甲硫氨酸-依赖性γ-生育酚甲基转移酶家族,与其他植物γ-TMT蛋白的一致性为64%~75%;在NJ系统树上,La TMT蛋白与单子叶植物γ-TMT蛋白聚为同一大类,并与油棕(Elaeis guineensis Jacq.)Eg TMT和美洲油棕〔Elaeis oleifera(Kunth)Cortés〕Eo TMT聚为同一类,亲缘关系最近。基因表达分析结果显示:La TMT基因可在大肠杆菌中成功表达,且表达量随异丙基硫代半乳糖苷(IPTG)诱导时间的延长而增加;在忽地笑的根、叶片、花苞、子房、雄蕊、花瓣和鳞茎中La TMT基因均可表达,其中在叶片中的相对表达量最高,在子房、雄蕊和鳞茎中的相对表达量相对较低,具有明显的组织特异性。研究结果表明:忽地笑La TMT基因在进化过程中具有很高的保守性;该基因主要定位于叶绿体中,并与忽地笑对非生物逆境胁迫的抗性相关。
Using RACE method,γ-tocopherol methyltransferase( γ-TMT) gene was cloned from leaf of Lycoris aurea( L'Hér.) Herb.,which was named La TMT. The result of sequence analysis shows that fulllength c DNA of La TMT gene is 1 458 bp,in which,length of open reading frame( ORF) is 1 017 bp,and 338 amino acid residues are encoded. Theoretical relative molecular mass of the protein encoded by La TMT gene is 37 560,theoretical isoelectric point is p I 8. 70,which is a hydrophilic protein without transmembrane structure but with signal peptide structure. And this protein has S-adenosylmethionine( SAM) methyltransferase conserved domain and includes three SAM binding sites. Its tertiary structure includes 44. 08% of α-helix,32. 84% of random coil,12. 72% of extended strand and 10. 36% of β-turn. The analysis results of sequence alignment and phylogenetic tree show that La TMT protein belongs to S-adenosylmethionine-dependent γ-tocopherol methyltransferase family,and its identity with γ-TMT protein from other plants is 64%- 75%. In NJ phylogenetic tree,La TMT protein and γ-TMT protein from monocotyledon are clustered into the same large category, and this protein with Eg TMT of Elaeis guineensis Jacq. and Eo TMT of Elaeis oleifera( Kunth) Cortés is clustered into the same category,their relationship is close. The analysis result of genetic expression shows that La TMT gene can express successfully in Escherichia coli,and its expression increases with prolonging of inducing time of isopropyl thiogalactoside( IPTG). Also,La TMT gene can express in root,leaf,bud,ovary,stamen,petal and bulb of L. aurea,in which,relative expression is the highest in leaf,and that is relative low in ovary,stamen and bulb,with obvious tissue specificity. It is suggested that La TMT gene from L. aurea has high conservation in evolutionary process,the gene is mainly located in chloroplast and is related to resistance of abiotic adversity stress.
Using RACE method,γ-tocopherol methyltransferase( γ-TMT) gene was cloned from leaf of Lycoris aurea( L'Hér.) Herb.,which was named La TMT. The result of sequence analysis shows that fulllength c DNA of La TMT gene is 1 458 bp,in which,length of open reading frame( ORF) is 1 017 bp,and 338 amino acid residues are encoded. Theoretical relative molecular mass of the protein encoded by La TMT gene is 37 560,theoretical isoelectric point is p I 8. 70,which is a hydrophilic protein without transmembrane structure but with signal peptide structure. And this protein has S-adenosylmethionine( SAM) methyltransferase conserved domain and includes three SAM binding sites. Its tertiary structure includes 44. 08% of α-helix,32. 84% of random coil,12. 72% of extended strand and 10. 36% of β-turn. The analysis results of sequence alignment and phylogenetic tree show that La TMT protein belongs to S-adenosylmethionine-dependent γ-tocopherol methyltransferase family,and its identity with γ-TMT protein from other plants is 64%- 75%. In NJ phylogenetic tree,La TMT protein and γ-TMT protein from monocotyledon are clustered into the same large category, and this protein with Eg TMT of Elaeis guineensis Jacq. and Eo TMT of Elaeis oleifera( Kunth) Cortés is clustered into the same category,their relationship is close. The analysis result of genetic expression shows that La TMT gene can express successfully in Escherichia coli,and its expression increases with prolonging of inducing time of isopropyl thiogalactoside( IPTG). Also,La TMT gene can express in root,leaf,bud,ovary,stamen,petal and bulb of L. aurea,in which,relative expression is the highest in leaf,and that is relative low in ovary,stamen and bulb,with obvious tissue specificity. It is suggested that La TMT gene from L. aurea has high conservation in evolutionary process,the gene is mainly located in chloroplast and is related to resistance of abiotic adversity stress.
引文
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[13]HAVAUX M,EYMERY F,PORFIROVA S,et al.Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana[J].The Plant Cell,2005,17:3451-3469.
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[15]SHINTANI D,DELLAPENNA D.Elevating the vitamin E content of plants through metabolic engineering[J].Science,1998,282:2098-2100.
[16]RIMBACH G,MINIHANE A M,MAJEWICZ J,et al.Regulation of cell signalling by vitamin E[J].The Proceedings of the Nutrition Society,2002,61:415-425.
[17]JIN S,DANIELL H.Expression ofγ-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,2014,12:1274-1285.
[18]YUSUF M A,KUMAR D,RAJWANSHI R,et al.Overexpression ofγ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress:physiological and chlorophyll a fluorescence measurements[J].Biochimica et Biophysica Acta,2010,1797:1428-1438.
[2]NIKI E.Evidence for beneficial effects of vitamin E[J].The Korean Journal of Internal Medicine,2015,30:571-579.
[3]TRABER M G,SIES H.Vitamin E in humans:demand and delivery[J].Annual Review of Nutrition,1996,16:321-347.
[4]FRYER M J.The antioxidant effects of thylakoid vitamin E(α-tocopherol)[J].Plant,Cell and Environment,1992,15:381-392.
[5]NETSCHER T.Synthesis of vitamin E[J].Vitamins and Hormones,2007,76:155-202.
[6]DELLAPENNA D.A decade of progress in understanding vitamin E synthesis in plants[J].Journal of Plant Physiology,2005,162:729-737.
[7]LI Y,ZHOU Y,WANG Z,et al.Engineering tocopherol biosynthetic pathway in Arabidopsis leaves and its effect on antioxidant metabolism[J].Plant Science,2010,178:312-320.
[8]YUSUF M A,SARINL N B.Antioxidant value addition in human diets:genetic transformation of Brassica juncea withγ-TMT gene for increasedα-tocopherol content[J].Transgenic Research,2007,16:109-113.
[9]HARVEY A L.The pharmacology of galanthamine and its analogues[J].Pharmacology and Therapeutics,1995,68:113-128.
[10]WANG R,XU S,JIANG Y,et al.De novo sequence assembly and characterization of Lycoris aurea transcriptome using GS FLX titanium platform of 454 pyrosequencing[J].PLo S One,2013,8:e60449.
[11]别庆玲,徐晟,傅江燕,等.一个新的忽地笑O-甲基转移酶基因的克隆与原核表达[J].植物生理学报,2014,50(5):651-659.
[12]石鹏,曹红星,李东霞,等.油棕等植物γ-生育酚甲基转移酶的生物信息学分析[J].热带作物学报,2015,36(2):308-315.
[13]HAVAUX M,EYMERY F,PORFIROVA S,et al.Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana[J].The Plant Cell,2005,17:3451-3469.
[14]LI Z,KEASLING J D,NIYOGI K K.Overlapping photoprotective function of vitamin E and carotenoids in Chlamydomonas[J].Plant Physiol,2012,158:313-323.
[15]SHINTANI D,DELLAPENNA D.Elevating the vitamin E content of plants through metabolic engineering[J].Science,1998,282:2098-2100.
[16]RIMBACH G,MINIHANE A M,MAJEWICZ J,et al.Regulation of cell signalling by vitamin E[J].The Proceedings of the Nutrition Society,2002,61:415-425.
[17]JIN S,DANIELL H.Expression ofγ-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,2014,12:1274-1285.
[18]YUSUF M A,KUMAR D,RAJWANSHI R,et al.Overexpression ofγ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress:physiological and chlorophyll a fluorescence measurements[J].Biochimica et Biophysica Acta,2010,1797:1428-1438.