苹果N-乙酰基-5-羟色胺-O-甲基转移酶基因的克隆及原核表达
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摘要
以‘富士’和‘嘎啦’苹果(Malus pumila)叶片总RNA为模板,利用反转录PCR(RT-PCR)技术,克隆N-乙酰基-5-羟色胺-O-甲基转移酶(ASMT)基因,通过DNAMAN、MEGA 5.1、Prot Param等生物信息学软件对基因cDNA序列、系统进化关系、理化性质等进行分析,将克隆的苹果ASMT基因与表达载体pET32a连接,构建原核表达载体并转入大肠杆菌(Escherichia coli)表达菌株Rosetta(DE3)中,优化原核表达条件,用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测融合蛋白的可溶性,用蛋白免疫印迹法(western blot)和酶活性测定验证目的蛋白的表达。结果表明,获得了苹果ASMT的全长cDNA序列,命名为MpASMT1(Gen Bank登记号MF135479)。该基因开放阅读框(ORF)为1 077 bp,编码358个氨基酸。MpASMT1编码蛋白的理论分子量约为39.7 kDa,等电点为5.42,是非分泌型、疏水的稳定蛋白;其263位组氨酸(His)为催化位点,225位谷氨酸为底物S-腺苷甲硫氨酸结合位点。多序列比对及系统发生树分析表明,MpASMT1编码的氨基酸序列与珠美海棠(M.zumi)MzASMT1(KJ123721)同源性最高,相似性达99.2%,其次为葡萄(Vitis vinifera)ASMT(LOC100248671),氨基酸序列相似性为66.4%。原核表达结果显示,经0.25~2.0 mmol·L~(-1)异丙基-β-d-硫代半乳糖苷(IPTG)诱导的重组质粒可特异性表达分子量约58 kDa的融合蛋白,且低温条件(15和25°C)能够提高该蛋白的可溶性表达;western blot分析表明融合表达蛋白能与His单克隆抗体特异性结合,纯化蛋白的酶活性为7.8 pmol·mg~(-1)(蛋白),进一步证实MpASMT1编码蛋白成功表达。
N-acetylserotonin O-methyltransferase gene(MpASMT1) was amplified by reverse transcription PCR from leaves of Malus pumila Mill. ‘Fuji' and ‘Gala'. Full-length c DNA sequence, phylogenetic relationship and physicochemical properties of this gene were analyzed by a variety of bioinformatics softwares such as DNAMAN, MEGA 5.1 and Prot Param. Then, a recombinant plasmid pET-MpASMT1 was constructed using pET32 a as a fused expression vector. Optimal conditions for recombinant protein expression were determined in Escherichia coli Rosetta(DE3) including inducing time, isopropyl β-d-thiogalactopyranoside(IPTG) concentration and inducing temperature. Recombinant protein expression and solubility of MpASMT1 were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Western blot and enzyme activity assay were used to confirm the successful expression of MpASMT1 in E. coli. The results show that fulllength cDNA sequence of MpASMT1 from M. pumila was obtained and its Gen Bank accession number is MF135479. Its open reading frame(ORF) of 1 077 bp encodes 358 amino acid residues with a predicted molecular weight of 39.7 kDa and an isoelectric point value of 5.42. MpASMT1 is a non-secretory and hydrophobic stable protein. The 263 aa histidine is a catalytic site and 225 aa glutamate is a substrate binding site of S-adenylyl methionine. Multiple sequence alignment and phylogenetic analysis showed that the encoded protein had the closest genetic relationship with M. zumi MzASMT1(Gen Bank: KJ123721) and Vitis vinifera ASMT(GenBank: LOC100248671), and the similarities of amino acid sequences were 99.2% and 66.4%, respectively. Prokaryotic expression results showed that efficient expression of recombinant MpASMT1 protein about 58 kDa was realized when induced by 0.25–2.0 mmol·L~(-1) IPTG in E. coli. However, lower temperature(15 and 25°C) could increase the solubility of recombinant protein. Western blot analysis showed that the recombinant protein could be specifically recognized by His monoclonal antibody, and the enzyme activity of purified MpASMT1 protein was 7.8 pmol·mg~(-1)(protein), which further confirms that MpASMT1 protein was successfully expressed.
N-acetylserotonin O-methyltransferase gene(MpASMT1) was amplified by reverse transcription PCR from leaves of Malus pumila Mill. ‘Fuji' and ‘Gala'. Full-length c DNA sequence, phylogenetic relationship and physicochemical properties of this gene were analyzed by a variety of bioinformatics softwares such as DNAMAN, MEGA 5.1 and Prot Param. Then, a recombinant plasmid pET-MpASMT1 was constructed using pET32 a as a fused expression vector. Optimal conditions for recombinant protein expression were determined in Escherichia coli Rosetta(DE3) including inducing time, isopropyl β-d-thiogalactopyranoside(IPTG) concentration and inducing temperature. Recombinant protein expression and solubility of MpASMT1 were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Western blot and enzyme activity assay were used to confirm the successful expression of MpASMT1 in E. coli. The results show that fulllength cDNA sequence of MpASMT1 from M. pumila was obtained and its Gen Bank accession number is MF135479. Its open reading frame(ORF) of 1 077 bp encodes 358 amino acid residues with a predicted molecular weight of 39.7 kDa and an isoelectric point value of 5.42. MpASMT1 is a non-secretory and hydrophobic stable protein. The 263 aa histidine is a catalytic site and 225 aa glutamate is a substrate binding site of S-adenylyl methionine. Multiple sequence alignment and phylogenetic analysis showed that the encoded protein had the closest genetic relationship with M. zumi MzASMT1(Gen Bank: KJ123721) and Vitis vinifera ASMT(GenBank: LOC100248671), and the similarities of amino acid sequences were 99.2% and 66.4%, respectively. Prokaryotic expression results showed that efficient expression of recombinant MpASMT1 protein about 58 kDa was realized when induced by 0.25–2.0 mmol·L~(-1) IPTG in E. coli. However, lower temperature(15 and 25°C) could increase the solubility of recombinant protein. Western blot analysis showed that the recombinant protein could be specifically recognized by His monoclonal antibody, and the enzyme activity of purified MpASMT1 protein was 7.8 pmol·mg~(-1)(protein), which further confirms that MpASMT1 protein was successfully expressed.
引文
Aghdam MS,Fard JR(2017).Melatonin treatment attenuates postharvest decay and maintains nutritional quality of strawberry fruits(Fragaria×anannasa cv.Selva)by enhancing GABA shunt activity.Food Chem,221:1650-1657
Arnao MB,Hernández-Ruiz J(2014).Melatonin:plant growth regulator and/or biostimulator during stress?Trends Plant Sci,19(12):789-797
Arnao MB,Hernández-Ruiz J(2015).Functions of melatonin in plants:a review.J Pineal Res,59(2):133-150
Ben-Abdallah M,Bondet V,Fauchereau F,et al(2011).Production of soluble,active acetyl serotonin methyl transferase in Leishmania tarentolae.Protein Expres Purif,75(1):114-118
Byeon Y,Back K(2014).Melatonin synthesis in rice seedlings in vivo is enhanced at high temperatures and under dark conditions due to increased serotonin N-acetyltransferase and N-acetylserotonin methyltransferase activities.J Pineal Res,56(2):189-195
Byeon Y,Back K(2016).Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.Appl Microbiol Biot,100(15):6683-6691
Byeon Y,Lee HJ,Lee HY,et al(2016).Cloning and functional characterization of the Arabidopsis N-acetylserotonin O-methyltransferase responsible for melatonin synthesis.J Pineal Res,60(1):65-73
Carillo-Vico A,Lardone PJ,álvarez-Sánchez N,et al(2013).Melatonin:buffering the immune system.Int J Mol Sci,14(4):8638-8683
Dubbels R,Reiter RJ,Klenke E,et al(1995).Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry.JPineal Res,18(1):28-31
Fu NX,Zhai F,Jiang M,et al(2013).c DNA cloning sequence analysis and prokaryotic expression of farnesyl pyrophosphate synthase from Epicauta gorhami Marseul(Coleoptera:Meloidae).Sci Agr Sin,47(2):273-283(in Chinese with English abstract)[付楠霞,翟枫,姜鸣等(2013).锯角豆芫菁法尼基焦磷酸合酶的c DNA克隆、序列分析及原核表达.中国农业科学,47(2):273-283]
Hardeland R,Madrid JA,Tan DX,et al(2012).Melatonin,the circadian multioscillator system and health:the need for detailed analyses of peripheral melatonin signaling.JPineal Res,52(2):139-166
Hernández-Ruiz J,Cano A,Arnao MB(2004).Melatonin:a growth-stimulating compound present in lupin tissues.Planta,220(1):140-144
Kang K,Kong K,Park S,et al(2011).Molecular cloning of a plant N-acetylserotonin methyltransferase and its expression characteristics in rice.J Pineal Res,50(3):304-309
Kanjanaphachoat P,Wei BY,Lo SF,et al(2012).Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.Plant Mol Biol,78(6):525-543
Kim M,Seo H,Park C,et al(2016).Examination of the auxin hypothesis of phytomelatonin action in classical auxin assay systems in maize.J Plant Physiol,l190:67-71
Lee HY,Byeon Y,Back K(2014).Melatonin as a signal molecule triggering defense responses against pathogen attack in Arabidopsis and tobacco.J Pineal Res,57(3):262-268
Park S,Byeon Y,Back K(2013a).Functional analyses of three ASMT gene family members in rice plants.J Pineal Res,55(4):409-415
Park S,Byeon Y,Kim YS,et al(2013b).Kinetic analysis of purified recombinant rice N-acetylserotonin methyltransferase and peak melatonin production in etiolated rice shoots.J Pineal Res,54(2):139-144
Park S,Kang K,Lee K,et al(2009).Induction of serotonin biosynthesis is uncoupled from the coordinated induction of tryptophan biosynthesis in pepper fruits(Capsicum annuum)upon pathogen infection.Planta,230(6):1197-1206
Reiter RJ,Tan DX,Rosales-Corral S,et al(2013).The universal nature,unequal distribution and antioxidant functions of melatonin and its derivatives.Mini Rev Med Chem,13(3):373-384
Reiter RJ,Tan DX,Zhou Z,et al(2015).Phytomelatonin:assisting plants to survive and thrive.Molecules,20(4):7396-7437
Schein CH,Noteborn MHM(1988).Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature.Nat Biotechnol,6(3):291-294
Tan DX,Hardeland R,Manchester LC,et al(2012).Functional roles of melatonin in plants,and perspectives in nutritional and agricultural science.J Exp Bot,63(2):577-597
Wang B,Li BH,Dong XL,et al(2015).Effects of temperature,wetness duration and moisture on the conidial germination,infection and disease incubation period of Glomerella cingulata.Plant Dis,99(2):249-256
Wang CX,Zhang ZF,Li BH,et al(2012).First report of Glomerella leaf spot of apple caused by Glomerella cingulata in China.Plant Dis,96(6):912
Wang R,Yang XL,Xu H,et al(2016).Research progress of melatonin biosynthesis and metabolism in higher plants.Plant Physiol J,52(5):615-627(in Chinese with English abstract)[王蕊,杨小龙,须晖等(2016).高等植物褪黑素的合成和代谢研究进展.植物生理学报,52(5):615-627]
Wang WX,Zhang RM,Sun Y,et al(2016).Effect of exogenous melatonin on the antioxidant system of cucumber seedling under nitrate stress.Acta Hortic Sin,43(4):695-703(in Chinese with English abstract)[王伟香,张锐敏,孙艳等(2016).外源褪黑素对硝酸盐胁迫条件下黄瓜幼苗抗氧化系统的影响.园艺学报,43(4):695-703]
Ye T,Hao YH,Yu L,et al(2017).A simple,rapid method for determination of melatonin in plant tissues by UPLCcoupled with high resolution orbitrap mass spectrometry.Front Plant Sci,8:64
Yin L,Wang P,Li M,et al(2013).Exogenous melatonin improves Malus resistance to Marssonina apple blotch.JPineal Res,54(4):426-434
Zeng XL,Zheng RR,Luo J,et al(2016).Cloning and characterization of cinnamate 4-hydroxylase(C4H)genes from Osmanthus fragrans.Acta Hortic Sin,43(3):525-537(in Chinese with English abstract)[曾祥玲,郑日如,罗靖等(2016).桂花C4H基因的克隆与表达特性分析.园艺学报,43(3):525-537]
Zhang L,Wang CX,Li BH,et al(2015).Sexual reproduction,biological characteristics and pathogenicity of ascospores of Glomerella cingulata.Mycosystema,34(6):1101-1110(in Chinese with English abstract)[张路,王彩霞,李保华等(2015).围小丛壳Glomerella cingulata子囊孢子交配繁殖、生物学特性及致病性.菌物学报,34(6):1101-1110]
Zhang N,Sun Q,Zhang H,et al(2015).Roles of melatonin in abiotic stress resistance in plants.J Exp Bot,66(3):647-656
Zuo B,Zheng X,He P,et al(2014).Overexpression of Mz ASMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis thaliana plants.J Pineal Res,57(4):408-417
Arnao MB,Hernández-Ruiz J(2014).Melatonin:plant growth regulator and/or biostimulator during stress?Trends Plant Sci,19(12):789-797
Arnao MB,Hernández-Ruiz J(2015).Functions of melatonin in plants:a review.J Pineal Res,59(2):133-150
Ben-Abdallah M,Bondet V,Fauchereau F,et al(2011).Production of soluble,active acetyl serotonin methyl transferase in Leishmania tarentolae.Protein Expres Purif,75(1):114-118
Byeon Y,Back K(2014).Melatonin synthesis in rice seedlings in vivo is enhanced at high temperatures and under dark conditions due to increased serotonin N-acetyltransferase and N-acetylserotonin methyltransferase activities.J Pineal Res,56(2):189-195
Byeon Y,Back K(2016).Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.Appl Microbiol Biot,100(15):6683-6691
Byeon Y,Lee HJ,Lee HY,et al(2016).Cloning and functional characterization of the Arabidopsis N-acetylserotonin O-methyltransferase responsible for melatonin synthesis.J Pineal Res,60(1):65-73
Carillo-Vico A,Lardone PJ,álvarez-Sánchez N,et al(2013).Melatonin:buffering the immune system.Int J Mol Sci,14(4):8638-8683
Dubbels R,Reiter RJ,Klenke E,et al(1995).Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry.JPineal Res,18(1):28-31
Fu NX,Zhai F,Jiang M,et al(2013).c DNA cloning sequence analysis and prokaryotic expression of farnesyl pyrophosphate synthase from Epicauta gorhami Marseul(Coleoptera:Meloidae).Sci Agr Sin,47(2):273-283(in Chinese with English abstract)[付楠霞,翟枫,姜鸣等(2013).锯角豆芫菁法尼基焦磷酸合酶的c DNA克隆、序列分析及原核表达.中国农业科学,47(2):273-283]
Hardeland R,Madrid JA,Tan DX,et al(2012).Melatonin,the circadian multioscillator system and health:the need for detailed analyses of peripheral melatonin signaling.JPineal Res,52(2):139-166
Hernández-Ruiz J,Cano A,Arnao MB(2004).Melatonin:a growth-stimulating compound present in lupin tissues.Planta,220(1):140-144
Kang K,Kong K,Park S,et al(2011).Molecular cloning of a plant N-acetylserotonin methyltransferase and its expression characteristics in rice.J Pineal Res,50(3):304-309
Kanjanaphachoat P,Wei BY,Lo SF,et al(2012).Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.Plant Mol Biol,78(6):525-543
Kim M,Seo H,Park C,et al(2016).Examination of the auxin hypothesis of phytomelatonin action in classical auxin assay systems in maize.J Plant Physiol,l190:67-71
Lee HY,Byeon Y,Back K(2014).Melatonin as a signal molecule triggering defense responses against pathogen attack in Arabidopsis and tobacco.J Pineal Res,57(3):262-268
Park S,Byeon Y,Back K(2013a).Functional analyses of three ASMT gene family members in rice plants.J Pineal Res,55(4):409-415
Park S,Byeon Y,Kim YS,et al(2013b).Kinetic analysis of purified recombinant rice N-acetylserotonin methyltransferase and peak melatonin production in etiolated rice shoots.J Pineal Res,54(2):139-144
Park S,Kang K,Lee K,et al(2009).Induction of serotonin biosynthesis is uncoupled from the coordinated induction of tryptophan biosynthesis in pepper fruits(Capsicum annuum)upon pathogen infection.Planta,230(6):1197-1206
Reiter RJ,Tan DX,Rosales-Corral S,et al(2013).The universal nature,unequal distribution and antioxidant functions of melatonin and its derivatives.Mini Rev Med Chem,13(3):373-384
Reiter RJ,Tan DX,Zhou Z,et al(2015).Phytomelatonin:assisting plants to survive and thrive.Molecules,20(4):7396-7437
Schein CH,Noteborn MHM(1988).Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature.Nat Biotechnol,6(3):291-294
Tan DX,Hardeland R,Manchester LC,et al(2012).Functional roles of melatonin in plants,and perspectives in nutritional and agricultural science.J Exp Bot,63(2):577-597
Wang B,Li BH,Dong XL,et al(2015).Effects of temperature,wetness duration and moisture on the conidial germination,infection and disease incubation period of Glomerella cingulata.Plant Dis,99(2):249-256
Wang CX,Zhang ZF,Li BH,et al(2012).First report of Glomerella leaf spot of apple caused by Glomerella cingulata in China.Plant Dis,96(6):912
Wang R,Yang XL,Xu H,et al(2016).Research progress of melatonin biosynthesis and metabolism in higher plants.Plant Physiol J,52(5):615-627(in Chinese with English abstract)[王蕊,杨小龙,须晖等(2016).高等植物褪黑素的合成和代谢研究进展.植物生理学报,52(5):615-627]
Wang WX,Zhang RM,Sun Y,et al(2016).Effect of exogenous melatonin on the antioxidant system of cucumber seedling under nitrate stress.Acta Hortic Sin,43(4):695-703(in Chinese with English abstract)[王伟香,张锐敏,孙艳等(2016).外源褪黑素对硝酸盐胁迫条件下黄瓜幼苗抗氧化系统的影响.园艺学报,43(4):695-703]
Ye T,Hao YH,Yu L,et al(2017).A simple,rapid method for determination of melatonin in plant tissues by UPLCcoupled with high resolution orbitrap mass spectrometry.Front Plant Sci,8:64
Yin L,Wang P,Li M,et al(2013).Exogenous melatonin improves Malus resistance to Marssonina apple blotch.JPineal Res,54(4):426-434
Zeng XL,Zheng RR,Luo J,et al(2016).Cloning and characterization of cinnamate 4-hydroxylase(C4H)genes from Osmanthus fragrans.Acta Hortic Sin,43(3):525-537(in Chinese with English abstract)[曾祥玲,郑日如,罗靖等(2016).桂花C4H基因的克隆与表达特性分析.园艺学报,43(3):525-537]
Zhang L,Wang CX,Li BH,et al(2015).Sexual reproduction,biological characteristics and pathogenicity of ascospores of Glomerella cingulata.Mycosystema,34(6):1101-1110(in Chinese with English abstract)[张路,王彩霞,李保华等(2015).围小丛壳Glomerella cingulata子囊孢子交配繁殖、生物学特性及致病性.菌物学报,34(6):1101-1110]
Zhang N,Sun Q,Zhang H,et al(2015).Roles of melatonin in abiotic stress resistance in plants.J Exp Bot,66(3):647-656
Zuo B,Zheng X,He P,et al(2014).Overexpression of Mz ASMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis thaliana plants.J Pineal Res,57(4):408-417