割手密鸟氨酸转氨酶(Ssδ-OAT-2)基因的克隆与表达分析
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摘要
鸟氨酸转氨酶是合成脯氨酸的关键酶之一,在植物适应逆境胁迫中起重要作用。本研究以割手密为研究材料,克隆得到了鸟氨酸转氨酶基因c DNA序列全长,将该基因命名为Ssδ-OAT-2(Gen Bank登陆号:KX714116)。该序列全长1 373 bp,包含一个1 365 bp的开放读码框(ORF),编码454个氨基酸,相对分子质量49.54 k D。同源比对分析表明,Ssδ-OAT-2基因同其近缘属甘蔗的同源性最高(99%),其次是高粱(98%),与其它禾本科植物的同源性约为92%。通过构建与GFP融合表达载体,转洋葱表皮细胞进行瞬时表达,结果表明,该蛋白质主要定位于细胞质和细胞膜上。Ssδ-OAT-2基因的获得为甘蔗抗逆基因工程提供候选基因资源。
Ornithine-δ-aminotransferase is considered to be one of the key enzymes for proline biosynthesis, so that it plays an important role in adaptation to environmental stress in plants. Full-length c DNAs of ornithine-δ-aminotransferase, named as Ssδ-OAT-2, were isolated from Saccharum spontaneum using homologous clone strategy. The sequence of this gene was then deposited in Gen Bank database with the accession number KX714116. Sequence analysis showed that the full length of the Ssδ-OAT-2 gene was 1 373 bp, the open reading frame was 1 365 bp, coding 454 amino acids, and the molecular weight of coded protein was 49.54 k D. The amino acid sequence blast results showed that δ-OAT gene from Saccharum spontaneum shared the highest homology(99%) with relative genera plant Saccharum officinarum, then it had 98% homology with Sorghum bicolor, and had about 92% homology with other grass family. Through the construction of expression vector with GFP fusion,onion epidermal cells did transient expression. The results showed that the protein was mainly located in cytoplasm and cell membrane. The cloning of Ssδ-OAT-2 gene might provide candidate gene resources for resistance gene engineering in sugarcane.
Ornithine-δ-aminotransferase is considered to be one of the key enzymes for proline biosynthesis, so that it plays an important role in adaptation to environmental stress in plants. Full-length c DNAs of ornithine-δ-aminotransferase, named as Ssδ-OAT-2, were isolated from Saccharum spontaneum using homologous clone strategy. The sequence of this gene was then deposited in Gen Bank database with the accession number KX714116. Sequence analysis showed that the full length of the Ssδ-OAT-2 gene was 1 373 bp, the open reading frame was 1 365 bp, coding 454 amino acids, and the molecular weight of coded protein was 49.54 k D. The amino acid sequence blast results showed that δ-OAT gene from Saccharum spontaneum shared the highest homology(99%) with relative genera plant Saccharum officinarum, then it had 98% homology with Sorghum bicolor, and had about 92% homology with other grass family. Through the construction of expression vector with GFP fusion,onion epidermal cells did transient expression. The results showed that the protein was mainly located in cytoplasm and cell membrane. The cloning of Ssδ-OAT-2 gene might provide candidate gene resources for resistance gene engineering in sugarcane.
引文
Bernstein R.L.,and Henke R.R.,1982,Rapid proline uptake in cultured tobacco cells and inhibition by a proline analog,FEBS Lett.,141(2):283-286
Chen L.P.,and He D.Y.,2010,Research advance on drought and salt resistant genes in transgenic plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(3):542-549(陈丽萍,何道一,2010,植物抗旱耐盐基因的研究进展,基因组学与应用生物学,29(3):542-549)
Delauney A.J.,Hu C.A.,Kishor P.B.,and Verma D.P.,1993,Cloning of ornithineδ-aminotransferase c DNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis,J.Biol.Chem.,268(25):18673-18678
Hmida-Sayari A.,Gargouri-Bouzid R.,Bidani A.,Jaoua L.,SavouréA.,and Jaoua S.,2005,Overexpression of△1-pyrroline-5-carboxylate synthetase increases proline production and confers salt tolerance in transgenic potato plants,Plant Science,169(4):746-752
Hong Z.L.,Lakkineni K.,Zhang Z.M.,and Verma D.P.,2000,Removal of feedback inhibition of△1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress,Plant Physiol.,122(4):1129-1136
Jiao R.,Liu G.S.,Liu H.B.,Wang S.L.,Hou N.,Wang Q.Z.,Jin Y.R.,Liu C.K.,Feng X.G.,and Hu X.M.,2011,Cloning and expression analysis of orn-δ-aminotransferase gene Ntδ-OAT in Nicotiana tabacum,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),12(4):588-593(焦蓉,刘贯山,刘好宝,王树林,侯娜,王全贞,靳义荣,刘朝科,冯祥国,胡晓明,2011,普通烟草鸟氨酸转氨酶基因Ntδ-OAT的克隆与表达分析,植物遗传资源学报,12(4):588-593)
Li W.,2008,Cloning the key genes EaδOAT and Ea PSCS involved in proline metabolism o f Erianthus arundinaceum and their function analysis,Thesis for M.S.,Agronomy Institute,Fujian Agriculture and Forestry University,Supervisor Zhang M.Q.,pp.8-10(李伟,2008,斑茅脯氨酸代谢关键基因EaδOAT和Ea P5CS克隆及功能分析,硕士学位论文福建农林大学农学院,导师:张木清,pp.8-10)
Robert J.G.,Elizabeth P.,and Georgina M.I.,1997,Green-fluo rescent protein fusions for efficient characterization of nuclear targeting,Plant J.,11(3):573-586
Roosens N.H.,Thu T.T.,Iskandar H.M.,and Jacobs M.,1998,Isolation of the omithine-δ-aminotransferase c DNA and effec of salt stress on its expression in Arabidopsis thaliana,Plan Physiol.,117(1):263-271
Slama I.,Messedi D.,Ghnaya T.,Savoure A.,and Abdelly C.2006,Effects of water deficit on growth and proline metabolism in Sesuvium portulacastrum,Environmental and Experimental Botany,56(3):231-238
Str觃nsk觃J.,Kopecny D.,Tylichov觃M.,Snégaroff J.,and譒ebela M.,2008,Ornithine delta-aminotran-sferase:an enzyme implicated in salt tolerance in higher plants,Plant Signaling&Behavior,3(11):929-935
Verbruggen N.,and Hermans C.,2008,Proline accumulation in plants:a review,Amino Acids,35(4):753-759
Wang W.D.,Shu Z.F.,Du Y.L.,Li X.H.,and Wang Y.H.,2014Cloning and expression analysis of ornithine-δ-aminotransferase gene Csδ-OAT in Camellia sinensis,Yuanyi Xuebao(Acta Horticulturae Sinica),41(12):2465-2473(王伟东,疏再发,杜昱林,黎星辉,王玉花,2014,茶树鸟氨酸转氨酶基因Csδ-OAT的克隆与表达分析,园艺学报,41(12)2465-2473)
Wang P.,Yu F.,Huang Y.J.,Chen S.Y.,and Deng Z.H.,2015,Physical localization of 5S r DNA on the chromosomes of different ploidy Saccharum spontaneum,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1761-1768(王平,余凡,黄永吉,陈胜烨,邓祖湖,2015,5Sr DNA在不同倍性割手密染色体上的物理定位,基因组学与应用生物学,34(8):1761-1768)
Wu L.Q.,Fan Z.M.,Guo L.,Li Y.Q.,Zhang W.J.,Qu L.J.,and Chen Z.L.,2003,Over-expression of an Arabidopsisδ-OATgene enhances salt and drought tolerance in transgenic rice,Chinese Science Bulletin,48(23):2594-2600
Yu G.H.,Liu Z.H.,Zeng F.H.,Li L.,and Wu Z.H.,2002,The relationship between the proline accumulation and the activities of te key enzymes concerned its synthetase,Zhanjiang Shifan Xueyuan Xuebao(Journal of Zhanjiang Normal College),23(6):57-60(余光辉,刘正辉,曾富华,李玲,吴志华,2002,脯氨酸累积与其合成关键酶活性的关系,湛江师范学院学报,23(6):57-60)
Zhao F.G.,and Liu Y.L.,2000,Study on determination of ADCand TGase activities,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),36(5):442-445(赵福庚,刘友良,2000,精氨酸脱羧酶和谷酰胺转移酶活性的测定方法,植物生理学通讯,36(5):442-445)
Zhao R.X.,Zhu H.S.,Cheng Y.H.,Dong K.H.,2008,Research progress on proline and its biosynthesis enzymes in plant,Caoye Kexue(Pratacultural Science),25(2):90-97(赵瑞雪,朱慧森,程钰宏,董宽虎,2008,植物脯氨酸及其合成酶系研究进展,草业科学,25(2):90-97)
Chen L.P.,and He D.Y.,2010,Research advance on drought and salt resistant genes in transgenic plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(3):542-549(陈丽萍,何道一,2010,植物抗旱耐盐基因的研究进展,基因组学与应用生物学,29(3):542-549)
Delauney A.J.,Hu C.A.,Kishor P.B.,and Verma D.P.,1993,Cloning of ornithineδ-aminotransferase c DNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis,J.Biol.Chem.,268(25):18673-18678
Hmida-Sayari A.,Gargouri-Bouzid R.,Bidani A.,Jaoua L.,SavouréA.,and Jaoua S.,2005,Overexpression of△1-pyrroline-5-carboxylate synthetase increases proline production and confers salt tolerance in transgenic potato plants,Plant Science,169(4):746-752
Hong Z.L.,Lakkineni K.,Zhang Z.M.,and Verma D.P.,2000,Removal of feedback inhibition of△1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress,Plant Physiol.,122(4):1129-1136
Jiao R.,Liu G.S.,Liu H.B.,Wang S.L.,Hou N.,Wang Q.Z.,Jin Y.R.,Liu C.K.,Feng X.G.,and Hu X.M.,2011,Cloning and expression analysis of orn-δ-aminotransferase gene Ntδ-OAT in Nicotiana tabacum,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),12(4):588-593(焦蓉,刘贯山,刘好宝,王树林,侯娜,王全贞,靳义荣,刘朝科,冯祥国,胡晓明,2011,普通烟草鸟氨酸转氨酶基因Ntδ-OAT的克隆与表达分析,植物遗传资源学报,12(4):588-593)
Li W.,2008,Cloning the key genes EaδOAT and Ea PSCS involved in proline metabolism o f Erianthus arundinaceum and their function analysis,Thesis for M.S.,Agronomy Institute,Fujian Agriculture and Forestry University,Supervisor Zhang M.Q.,pp.8-10(李伟,2008,斑茅脯氨酸代谢关键基因EaδOAT和Ea P5CS克隆及功能分析,硕士学位论文福建农林大学农学院,导师:张木清,pp.8-10)
Robert J.G.,Elizabeth P.,and Georgina M.I.,1997,Green-fluo rescent protein fusions for efficient characterization of nuclear targeting,Plant J.,11(3):573-586
Roosens N.H.,Thu T.T.,Iskandar H.M.,and Jacobs M.,1998,Isolation of the omithine-δ-aminotransferase c DNA and effec of salt stress on its expression in Arabidopsis thaliana,Plan Physiol.,117(1):263-271
Slama I.,Messedi D.,Ghnaya T.,Savoure A.,and Abdelly C.2006,Effects of water deficit on growth and proline metabolism in Sesuvium portulacastrum,Environmental and Experimental Botany,56(3):231-238
Str觃nsk觃J.,Kopecny D.,Tylichov觃M.,Snégaroff J.,and譒ebela M.,2008,Ornithine delta-aminotran-sferase:an enzyme implicated in salt tolerance in higher plants,Plant Signaling&Behavior,3(11):929-935
Verbruggen N.,and Hermans C.,2008,Proline accumulation in plants:a review,Amino Acids,35(4):753-759
Wang W.D.,Shu Z.F.,Du Y.L.,Li X.H.,and Wang Y.H.,2014Cloning and expression analysis of ornithine-δ-aminotransferase gene Csδ-OAT in Camellia sinensis,Yuanyi Xuebao(Acta Horticulturae Sinica),41(12):2465-2473(王伟东,疏再发,杜昱林,黎星辉,王玉花,2014,茶树鸟氨酸转氨酶基因Csδ-OAT的克隆与表达分析,园艺学报,41(12)2465-2473)
Wang P.,Yu F.,Huang Y.J.,Chen S.Y.,and Deng Z.H.,2015,Physical localization of 5S r DNA on the chromosomes of different ploidy Saccharum spontaneum,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1761-1768(王平,余凡,黄永吉,陈胜烨,邓祖湖,2015,5Sr DNA在不同倍性割手密染色体上的物理定位,基因组学与应用生物学,34(8):1761-1768)
Wu L.Q.,Fan Z.M.,Guo L.,Li Y.Q.,Zhang W.J.,Qu L.J.,and Chen Z.L.,2003,Over-expression of an Arabidopsisδ-OATgene enhances salt and drought tolerance in transgenic rice,Chinese Science Bulletin,48(23):2594-2600
Yu G.H.,Liu Z.H.,Zeng F.H.,Li L.,and Wu Z.H.,2002,The relationship between the proline accumulation and the activities of te key enzymes concerned its synthetase,Zhanjiang Shifan Xueyuan Xuebao(Journal of Zhanjiang Normal College),23(6):57-60(余光辉,刘正辉,曾富华,李玲,吴志华,2002,脯氨酸累积与其合成关键酶活性的关系,湛江师范学院学报,23(6):57-60)
Zhao F.G.,and Liu Y.L.,2000,Study on determination of ADCand TGase activities,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),36(5):442-445(赵福庚,刘友良,2000,精氨酸脱羧酶和谷酰胺转移酶活性的测定方法,植物生理学通讯,36(5):442-445)
Zhao R.X.,Zhu H.S.,Cheng Y.H.,Dong K.H.,2008,Research progress on proline and its biosynthesis enzymes in plant,Caoye Kexue(Pratacultural Science),25(2):90-97(赵瑞雪,朱慧森,程钰宏,董宽虎,2008,植物脯氨酸及其合成酶系研究进展,草业科学,25(2):90-97)