日本蛇根草OjGT2基因的克隆与序列分析
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摘要
糖基转移酶是广泛存在于内质网和高尔基体内的一大类酶,参与体内重要生物活性物质如糖蛋白和糖脂中糖链的形成。本实验以日本蛇根草为研究材料,根据转录组测得的OjGT2基因序列设计引物,通过分子生物学方法克隆获得日本蛇根草OjGT2基因的cDNA序列,并完成了该基因及其编码蛋白质的生物信息学分析。分析显示,OjGT2基因的c DNA长度为1 041 bp,共编码346个AA,预测蛋白相对分子质量为52.37 kD,等电点为5.07,归属于糖基转移酶GTB超家族,含有多个糖基转移酶功能保守域,与其它植物糖基转移酶具有很高的同源性。研究结果将为该基因功能的解析及日本蛇根草糖基化代谢产物的研究提供参考。
Glycosyltransferase is a kind o f enzyme that exists widely in the eddopplasmic reticulum and golgi,which participates in the formation of sugar chains in important bioactive substances such as glycoproteins and glycolipids. In this study, Ophiorrhiza japonica was used as the research material, and primer was designed according to the results of OjGT2 transcriptome sequencing result. The complete cDNA sequence of OjGT2 was successfully cloned by molecular methods. The bioinformatics analysis of OjGT2 gene and its coding protein was completed. The analysis showed that full-length c DNA of OjGT2 gene was 1 041 bp, encoding 346 amino acids.The predicted molecular weight of the protein was 52.37 kD and its isoelectric point was 5.07. It belonged to the GTB superfamily of glycosyltransferases. It contained many conserved functional domains of glycosyltransferases and had high homology with other plant glycosyltransferases. The results would provide a reference for the functional analysis of the gene and the study of glycosylated metabolites of Ophiorrhiza japonica.
Glycosyltransferase is a kind o f enzyme that exists widely in the eddopplasmic reticulum and golgi,which participates in the formation of sugar chains in important bioactive substances such as glycoproteins and glycolipids. In this study, Ophiorrhiza japonica was used as the research material, and primer was designed according to the results of OjGT2 transcriptome sequencing result. The complete cDNA sequence of OjGT2 was successfully cloned by molecular methods. The bioinformatics analysis of OjGT2 gene and its coding protein was completed. The analysis showed that full-length c DNA of OjGT2 gene was 1 041 bp, encoding 346 amino acids.The predicted molecular weight of the protein was 52.37 kD and its isoelectric point was 5.07. It belonged to the GTB superfamily of glycosyltransferases. It contained many conserved functional domains of glycosyltransferases and had high homology with other plant glycosyltransferases. The results would provide a reference for the functional analysis of the gene and the study of glycosylated metabolites of Ophiorrhiza japonica.
引文
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Bowles D.,Isayenkova J.,Lim E.k.,and Poppenberger B.,2005,Glycosyltransferases:managers of small molecules,Curr.Opin.Plant Biol.,8(3):254-263
Campbell J.A.,Davies G.J.,Bulone V.,and Henrissat B.,1997,A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities,Biochem.J.,326(Pt 3):929-939
Gourmelen A.,Blondelet-Rouault M.H.,and Pernodet J.L.,1998,Characterization of a glycosyl transferase inactivating macrolides,encoded by gimA from Streptomyces ambofaciens,Antimicrob Agents Chemother.,42(10):2612-2619
He X.M.,and Liu H.W.,2002,Formation of unusual sugars:mechanistic studies and biosynthetic applications,Annu.Rev.Biochem.,71:701-754
Hoffmeister D.,Ichinose K.,and Bechthold A.,2001,Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity,Chem.Biol.,8(6):557-567
Jones P.,and Vogt T.,2001,Glycosyltransferases in secondary plant metabolism:tranquilizer sand stimulant controllers,Planta,213(2):164-174
Lim E.K.,and Bowles D.J.,2004,Plant glycosyltransferases involved in cellular homeostasis,EMBO J.,23(15):2915-2922
Oberthür M.,Leimkuhler C.,Kruger R.G.,Lu W.,Walsh C.T.,and Kahne D.,2005,A systematic investigation of the synthetic utility of glycopeptide glycosyltransferases,J.Am.Chem.Soc.,127(30):10747-10752
Salas A.P.,Zhu L.,Sánchez C.,Brana A.F.,Rohr J.,Méndez C.,and Salas J.A.,2005,Deciphering the late steps in the biosynthesis of the anti-tumour indolocarbazole staurosporine:sugar donor substrate flexibility of the StaG glycosyltransferase,Mol.Microbiol.,58(1):17-27
Salas J.A.,and Méndez C.,2005,Biosynthesis pathways for deoxysugars in antibiotic-producing actinomycetes:isolation,characterization and generation of nov el glycosylated derivatives,J.Mol.Microbiol.Biotechnol.,9(2):77-85
Sánchez C.,Zhu L.,Brana A.F.,Salas A.P.,Rohr J.,Méndez C.,and Salas J.A.,2005,Combinatorial biosynthesis of antitumor indolocarbazole compounds,Proc.Natl.Acad.Sci.USA,102(2):461-266
Shen H.,Lin J.,Li Y.K.,Pan R.R.,Peng G.,and Sun W.,2018,Prokaryotic expression vector construction and recombinantprotein purification of CHI gene from Ophiorrhiza japonica,Guizhou Shifan Daxue Xuebao(Journal of Guizhou Normal University(Natural Sciences)),36(4):36-39(申欢,林建,李欲轲,潘蓉蓉,彭贵,孙威,2018,日本蛇根草CHI基因原核表达载体的构建及重组蛋白的纯化,贵州师范大学学报(自然科学版),36(4):36-39)
Walsh C.T.,Losey H.C.,and Freel Meyers C.L.,2003,Antibiotic glycosyltransferases,Biochem.Soc.Trans.,31(Pt 3):487-492
Wang J.,and Hou B.K.,2008,Glycosylation and glycosyltransferases of small molecular compounds of plant,ZhiwuShenglixue Tongxun(Plant Physiology Communications),44(5):997-1003(王军,侯丙凯,2008,植物小分子化合物的糖基化与糖基转移酶,植物生理通讯,44(5):997-1003)
Wohlert S.E.,Blanco G.,LombóF.,Fernández E.,Brana A.,Reich S.,Udvarnoki G.,Mendez C.,Decker H.,Frevert J.,Salas J.,and Rohr J.,1998,Novel hybrid tetracenomycins through combinatorial biosynthesis using a glycosyltransferase encoded by the elm genes in cosmid 16F4 and which shows a broad sugar substrate specificity,J.Am.Chem.Soc.,120(41):10596-10601
Bowles D.,Isayenkova J.,Lim E.k.,and Poppenberger B.,2005,Glycosyltransferases:managers of small molecules,Curr.Opin.Plant Biol.,8(3):254-263
Campbell J.A.,Davies G.J.,Bulone V.,and Henrissat B.,1997,A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities,Biochem.J.,326(Pt 3):929-939
Gourmelen A.,Blondelet-Rouault M.H.,and Pernodet J.L.,1998,Characterization of a glycosyl transferase inactivating macrolides,encoded by gimA from Streptomyces ambofaciens,Antimicrob Agents Chemother.,42(10):2612-2619
He X.M.,and Liu H.W.,2002,Formation of unusual sugars:mechanistic studies and biosynthetic applications,Annu.Rev.Biochem.,71:701-754
Hoffmeister D.,Ichinose K.,and Bechthold A.,2001,Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity,Chem.Biol.,8(6):557-567
Jones P.,and Vogt T.,2001,Glycosyltransferases in secondary plant metabolism:tranquilizer sand stimulant controllers,Planta,213(2):164-174
Lim E.K.,and Bowles D.J.,2004,Plant glycosyltransferases involved in cellular homeostasis,EMBO J.,23(15):2915-2922
Oberthür M.,Leimkuhler C.,Kruger R.G.,Lu W.,Walsh C.T.,and Kahne D.,2005,A systematic investigation of the synthetic utility of glycopeptide glycosyltransferases,J.Am.Chem.Soc.,127(30):10747-10752
Salas A.P.,Zhu L.,Sánchez C.,Brana A.F.,Rohr J.,Méndez C.,and Salas J.A.,2005,Deciphering the late steps in the biosynthesis of the anti-tumour indolocarbazole staurosporine:sugar donor substrate flexibility of the StaG glycosyltransferase,Mol.Microbiol.,58(1):17-27
Salas J.A.,and Méndez C.,2005,Biosynthesis pathways for deoxysugars in antibiotic-producing actinomycetes:isolation,characterization and generation of nov el glycosylated derivatives,J.Mol.Microbiol.Biotechnol.,9(2):77-85
Sánchez C.,Zhu L.,Brana A.F.,Salas A.P.,Rohr J.,Méndez C.,and Salas J.A.,2005,Combinatorial biosynthesis of antitumor indolocarbazole compounds,Proc.Natl.Acad.Sci.USA,102(2):461-266
Shen H.,Lin J.,Li Y.K.,Pan R.R.,Peng G.,and Sun W.,2018,Prokaryotic expression vector construction and recombinantprotein purification of CHI gene from Ophiorrhiza japonica,Guizhou Shifan Daxue Xuebao(Journal of Guizhou Normal University(Natural Sciences)),36(4):36-39(申欢,林建,李欲轲,潘蓉蓉,彭贵,孙威,2018,日本蛇根草CHI基因原核表达载体的构建及重组蛋白的纯化,贵州师范大学学报(自然科学版),36(4):36-39)
Walsh C.T.,Losey H.C.,and Freel Meyers C.L.,2003,Antibiotic glycosyltransferases,Biochem.Soc.Trans.,31(Pt 3):487-492
Wang J.,and Hou B.K.,2008,Glycosylation and glycosyltransferases of small molecular compounds of plant,ZhiwuShenglixue Tongxun(Plant Physiology Communications),44(5):997-1003(王军,侯丙凯,2008,植物小分子化合物的糖基化与糖基转移酶,植物生理通讯,44(5):997-1003)
Wohlert S.E.,Blanco G.,LombóF.,Fernández E.,Brana A.,Reich S.,Udvarnoki G.,Mendez C.,Decker H.,Frevert J.,Salas J.,and Rohr J.,1998,Novel hybrid tetracenomycins through combinatorial biosynthesis using a glycosyltransferase encoded by the elm genes in cosmid 16F4 and which shows a broad sugar substrate specificity,J.Am.Chem.Soc.,120(41):10596-10601