忽地笑α-葡萄糖苷酶编码基因的分子克隆与原核表达
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摘要
利用RACE技术从忽地笑(Lycoris aurea (L'Hér.) Herb.)花瓣中克隆获得一个α-葡萄糖苷酶编码基因Lau Agl。该基因cDNA全长2 810 bp,开放阅读框为2 613 bp,编码含有870个氨基酸残基的LauAgl蛋白质。LauAgl蛋白质氨基酸序列具有α-葡萄糖苷酶特征性的保守结构域和天冬氨酸残基组成的活性中心,其N端含有一个由21个氨基酸组成的信号肽序列,还具有一些蛋白质糖基化的潜在位点(N-X-S/T共有序列)。LauAgl蛋白质与石刁柏、椰子、油棕等其他植物的α-葡萄糖苷酶的氨基酸序列一致性达到63%以上。利用pET表达系统,通过截掉其N-端信号肽序列,可以实现LauAgl蛋白质在大肠杆菌中的诱导表达。本研究为Lau Agl蛋白质的功能鉴定及其在忽地笑种子萌发与生长发育中的作用研究提供理论基础。
In this stud y, a α-glucosidase encoding gene LauAgl was cloned from the petals of Lycoris aurea(L'Hér.) Herb. by using RACE technology. The full-length cDNA of LauAgl was 2 810 bp with a 2 613 bp open reading frame, encoding a LauAgl protein containing 870 amino acids residues. LauAgl protein amino acid sequence contained the active center composed of conservative domain and aspartic acid residue characterized byɑ-glucosidase, the N-terminal of which contained a signal peptide sequence consisting of 21 amino acids, and also had some potential sites(N-X-S/T common sequence) for possible glycosylation. The amino acid sequence of LauAgl protein shared more than 63% consistent with that of α-glucosidases from Asparagus officinalis L., Cocos nucifera L., Elaeis guineensis Jacq. and other plants. Based on the pET expression system, LauAgl protein was successfully induced to express in Escherichia coli by truncating N-terminal signal peptide sequence. This study would provide a theoretical basis for the functional identification of LauAgl protein and its role in germination,growth and development of Lycoris aurea seeds.
In this stud y, a α-glucosidase encoding gene LauAgl was cloned from the petals of Lycoris aurea(L'Hér.) Herb. by using RACE technology. The full-length cDNA of LauAgl was 2 810 bp with a 2 613 bp open reading frame, encoding a LauAgl protein containing 870 amino acids residues. LauAgl protein amino acid sequence contained the active center composed of conservative domain and aspartic acid residue characterized byɑ-glucosidase, the N-terminal of which contained a signal peptide sequence consisting of 21 amino acids, and also had some potential sites(N-X-S/T common sequence) for possible glycosylation. The amino acid sequence of LauAgl protein shared more than 63% consistent with that of α-glucosidases from Asparagus officinalis L., Cocos nucifera L., Elaeis guineensis Jacq. and other plants. Based on the pET expression system, LauAgl protein was successfully induced to express in Escherichia coli by truncating N-terminal signal peptide sequence. This study would provide a theoretical basis for the functional identification of LauAgl protein and its role in germination,growth and development of Lycoris aurea seeds.
引文
Chiba S.,Kanaya K.I.,Hiromi K.,and Shimomura T.,1979,Substrate specificity and subsite affinities of buckwheatα-glucosidase,Agric.Biol.Chem.,43(2):237-242
Gillmor C.S.,Poindexter P.,Lorieau J.,Palcic M.M.,and Somerville C.,2002,α-Glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis,J.Cell Biol.,156(6):1003-1013
Guo Y.,Pigni N.B.,Zheng Y.,de Andrade J.P.,Torras-Claveria L.,Borges Wde S.,Viladomat F.,Codina C.,and Bastida J.,2014,Analysis of bioactive Amaryllidaceae alkaloid profiles in Lycoris species by GC-MS,Nat.Prod.Commun.,9(8):1081-1086
Hall T.A.,1999,Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT,Nucleic Acids Symp.Ser.,41:95-98
Im H.,and Henson C.A.,1995,Characterization of high p Iα-glucosidase from germinated barley seeds:substrate specificity,subsite affinities and active-site residues,Carbohydr.Res.,277(1):145-159
Konishi Y.,Okamoto A.,Takahashi J.,Aitani M.,and Nakatani N.,1994,Effects of Bay m 1099,anα-glucosidase inhibitor,on starch metabolism in germinating wheat seeds,Biosci.Biotechnol.Biochem.,58(1):135-139
Ma R.,Xu S.,Zhao Y.C.,Xia B.,and Wang R.,2016,Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea,Front.Plant Sci.,7:536
Malá譒.,DvorákováH.,Hrabal R.,and KrálováB.,1999,Towards regioselective synthesis of oligosaccharides by use ofα-glucosidases with different substrate specificity,Carbohydr.Res.,322(3-4):209-218
Matsui H.,Chiba S.,and Shimomura T.,1978,Substrate specificity of anα-glucosidase in sugar beet seed,Agric.Biol.Chem.,42(10):1855-1860
Novikova I.Y.,and Tulaganov A.A.,2002,Physicochemical methods for the analysis of galanthamine(Review),Pharm.Chem.J.,36(11):623-627
Okuyama M.,Saburi W.,Mori H.,and Kimura A.,2016,α-Glucosidases andα-1,4-glucan lyases:structures,functions,and physiological actions,Cell.Mol.Life Sci.,73(14):2727-2751
Omasits U.,Ahrens C.H.,Muller S.,and Wollscheid B.,2014,Protter:interactive protein feature visualization and integration with experimental proteomic data,Bioinformatics,30(6):884-886
Qian B.B.,Li Y.K.,Li J.,Cheng L.,Xia B.,and Wang R.,2018,Molecular cloning and expression analysis of CYP98Afrom Lycoris aurea,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(11):3533-3541(钱彬彬,李宜奎,李洁,程丽,夏冰,汪仁,2018,忽地笑CYP98A的分子克隆与表达分析,分子植物育种,16(11):3533-3541)
Stanley D.,Rejzek M.,Naested H.,Smedley M.,Otero S.,Fahy B.,Thorpe F.,Nash R.J.,Harwood W.,Svensson B.,Denyer K.,Field R.A.,and Smith A.M.,2011,The role ofα-glucosidase in germinating barley grains,Plant Physiol.,155(2):932-943
Sugimoto M.,Furui S.,Sasaki K.,and Suzuki Y.,2003,Transglucosylation activities of multiple forms ofα-glucosidase from spinach.Biosci,Biotechnol.Biochem.,67(5):1160-1163
Sun Z.T.,and Henson C.A.,1991,A quantitative assessment of the importance of barley seed alpha-amylase,beta-amylase,debranching enzyme,and alpha-glucosidase in starch degradation,Arch.Biochem.Biophys.,284(2):298-305
Tagami T.,Yamashita K.,Okuyama M.,Mori H.,Yao M.,and Kimura A.,2013,Molecular basis for the recognition of long-chain substrates by plant alpha-glucosidases,J.Biol.Chem.,288(26):19296-19303
Wang R.,Xu S.,Jiang Y.M.,Jiang J.,Li X.D.,Liang L.J.,He J.,Peng F.,and Xia B.,2013,De novo sequence assembly and characterization of Lycoris aurea transcriptome using GSFLX titanium platform of 454 pyrosequencing,PLo S One,8(4):e60449
Gillmor C.S.,Poindexter P.,Lorieau J.,Palcic M.M.,and Somerville C.,2002,α-Glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis,J.Cell Biol.,156(6):1003-1013
Guo Y.,Pigni N.B.,Zheng Y.,de Andrade J.P.,Torras-Claveria L.,Borges Wde S.,Viladomat F.,Codina C.,and Bastida J.,2014,Analysis of bioactive Amaryllidaceae alkaloid profiles in Lycoris species by GC-MS,Nat.Prod.Commun.,9(8):1081-1086
Hall T.A.,1999,Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT,Nucleic Acids Symp.Ser.,41:95-98
Im H.,and Henson C.A.,1995,Characterization of high p Iα-glucosidase from germinated barley seeds:substrate specificity,subsite affinities and active-site residues,Carbohydr.Res.,277(1):145-159
Konishi Y.,Okamoto A.,Takahashi J.,Aitani M.,and Nakatani N.,1994,Effects of Bay m 1099,anα-glucosidase inhibitor,on starch metabolism in germinating wheat seeds,Biosci.Biotechnol.Biochem.,58(1):135-139
Ma R.,Xu S.,Zhao Y.C.,Xia B.,and Wang R.,2016,Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea,Front.Plant Sci.,7:536
Malá譒.,DvorákováH.,Hrabal R.,and KrálováB.,1999,Towards regioselective synthesis of oligosaccharides by use ofα-glucosidases with different substrate specificity,Carbohydr.Res.,322(3-4):209-218
Matsui H.,Chiba S.,and Shimomura T.,1978,Substrate specificity of anα-glucosidase in sugar beet seed,Agric.Biol.Chem.,42(10):1855-1860
Novikova I.Y.,and Tulaganov A.A.,2002,Physicochemical methods for the analysis of galanthamine(Review),Pharm.Chem.J.,36(11):623-627
Okuyama M.,Saburi W.,Mori H.,and Kimura A.,2016,α-Glucosidases andα-1,4-glucan lyases:structures,functions,and physiological actions,Cell.Mol.Life Sci.,73(14):2727-2751
Omasits U.,Ahrens C.H.,Muller S.,and Wollscheid B.,2014,Protter:interactive protein feature visualization and integration with experimental proteomic data,Bioinformatics,30(6):884-886
Qian B.B.,Li Y.K.,Li J.,Cheng L.,Xia B.,and Wang R.,2018,Molecular cloning and expression analysis of CYP98Afrom Lycoris aurea,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(11):3533-3541(钱彬彬,李宜奎,李洁,程丽,夏冰,汪仁,2018,忽地笑CYP98A的分子克隆与表达分析,分子植物育种,16(11):3533-3541)
Stanley D.,Rejzek M.,Naested H.,Smedley M.,Otero S.,Fahy B.,Thorpe F.,Nash R.J.,Harwood W.,Svensson B.,Denyer K.,Field R.A.,and Smith A.M.,2011,The role ofα-glucosidase in germinating barley grains,Plant Physiol.,155(2):932-943
Sugimoto M.,Furui S.,Sasaki K.,and Suzuki Y.,2003,Transglucosylation activities of multiple forms ofα-glucosidase from spinach.Biosci,Biotechnol.Biochem.,67(5):1160-1163
Sun Z.T.,and Henson C.A.,1991,A quantitative assessment of the importance of barley seed alpha-amylase,beta-amylase,debranching enzyme,and alpha-glucosidase in starch degradation,Arch.Biochem.Biophys.,284(2):298-305
Tagami T.,Yamashita K.,Okuyama M.,Mori H.,Yao M.,and Kimura A.,2013,Molecular basis for the recognition of long-chain substrates by plant alpha-glucosidases,J.Biol.Chem.,288(26):19296-19303
Wang R.,Xu S.,Jiang Y.M.,Jiang J.,Li X.D.,Liang L.J.,He J.,Peng F.,and Xia B.,2013,De novo sequence assembly and characterization of Lycoris aurea transcriptome using GSFLX titanium platform of 454 pyrosequencing,PLo S One,8(4):e60449