芝麻COX Ⅶa基因克隆、表达与序列特征分析
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摘要
为获得芝麻细胞色素氧化酶Ⅶa亚基基因及其蛋白质序列特征,研究其组织表达特性以及可能的功能。本研究利用NCBI数据库中注释的芝麻COXⅦa基因序列,设计特异PCR引物,以RT-PCR技术进行克隆和表达分析,并利用生物信息学方法分析其序列特征,预测其蛋白质亚细胞定位、跨膜结构和三级结构等。结果表明,克隆的芝麻SiCOXⅦa1和SiCOXⅦa2基因的cDNA序列长度分别为481 bp和416 bp,二者编码的蛋白质序列长度为67个氨基酸残基,具有88%的相似性,属于同源蛋白,均定位于线粒体中,在蛋白序列中35～57 aa形成一个跨膜螺旋区;预测的SiCOXⅦa三级结构均由2段α-螺旋结构组成,但SiCOXⅦa2与SiCOXⅦa1序列的C端形成的高级结构存在较小的差异。表达分析表明,二者在芝麻花蕾、花冠、根、茎和叶中均有表达,且表达模式相似,均在生长旺盛的组织中高丰度表达。本研究为揭示芝麻COX蛋白复合物的结构和功能提供了理论指导。
To obtain cDNA and its protein sequence characteristic of the cytochrome c oxidase 7 a subunits(COX Ⅶa) in Sesamum indicum., and study their tissue expression characteristics and possible functions, the cDNA sequences of COX Ⅶa in Sesamum indicum were annotated in NCBI database. Specific primers were designed for RT-PCR cloning and expression analysis, and the sequence characteristics were analyzed by bioinformatics to predict protein subcellular localization, transmembrane structure and tertiary structure. The results showed that the cloned SiCOX Ⅶa1 and SiCOX Ⅶa2 were 481 bp and 416 bp in length, respectively. The length of the protein sequence encoded by the two genes was 67 amino acid residues with 88% similarity. They belonged to homologous proteins, both located in mitochondria and formed a transmembrane helix region in 35～57 aa of the protein sequence. The predicted tertiary structure of SiCOX Ⅶa1 was composed of two segments of a-helix structure, but there was little difference between the advanced structure formed at C-teminal of SiCOX Ⅶa2 and SiCOX Ⅶa1.The gene expression analysis indicated that SiCOX Ⅶ a1 and SiCOX Ⅶ a2 were expressed in the buds, corolla,roots, stems and leaves of Sesamum indicum, and their expression patterns were similar, and they were abundantly expressed in the growing tissues. This study could provide theoretical guidance for revealing the structure and function of Sesamum indicum COX protein complex.
To obtain cDNA and its protein sequence characteristic of the cytochrome c oxidase 7 a subunits(COX Ⅶa) in Sesamum indicum., and study their tissue expression characteristics and possible functions, the cDNA sequences of COX Ⅶa in Sesamum indicum were annotated in NCBI database. Specific primers were designed for RT-PCR cloning and expression analysis, and the sequence characteristics were analyzed by bioinformatics to predict protein subcellular localization, transmembrane structure and tertiary structure. The results showed that the cloned SiCOX Ⅶa1 and SiCOX Ⅶa2 were 481 bp and 416 bp in length, respectively. The length of the protein sequence encoded by the two genes was 67 amino acid residues with 88% similarity. They belonged to homologous proteins, both located in mitochondria and formed a transmembrane helix region in 35～57 aa of the protein sequence. The predicted tertiary structure of SiCOX Ⅶa1 was composed of two segments of a-helix structure, but there was little difference between the advanced structure formed at C-teminal of SiCOX Ⅶa2 and SiCOX Ⅶa1.The gene expression analysis indicated that SiCOX Ⅶ a1 and SiCOX Ⅶ a2 were expressed in the buds, corolla,roots, stems and leaves of Sesamum indicum, and their expression patterns were similar, and they were abundantly expressed in the growing tissues. This study could provide theoretical guidance for revealing the structure and function of Sesamum indicum COX protein complex.
引文
Aggeler R.,and Capaldi R.A.,1990,Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme.Cloning,sequencing,and characterization of the nuclear-encoded gene,J.Biol.Chem.,265(27):16389-16393
Anthony G.,Stroh A.,Lottspeich F.,and Kadenbach B.,1990,Different isozymes of cytochrome c oxidase are expressed in bovine smooth muscle and skeletal or heart muscle,FEBSLett.,277(1):97-100
Budak H.,Akpinar B.A.,Unver T.,and Turktas M.,2013,Proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanoLC-ESI-MS/MS,Plant Mol.Biol.,83(1):1-15
Circu M.L.,and Aw T.Y.,2010,Reactive oxygen species,cellular redox systems and apoptosis,Free Radic.Biol.Med.,48(6):749-762
Ducos E.,Touzet P.,and Boutry M.,2001,The male sterile G cytoplasm of wild beet displays modified mitochondrial respiratory complexes,Plant J.,26(2):171-180
Fan X.F.,Wang J.S.,Liu H.Z.,Zhou L.,Wang X.J.,and Zhao J.H.,2016,Cloning and sequences analysis of COXVIIa gene in wheat,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):271-279(范小芳,王俊生,刘红占,周琳,王晓娟,赵锦慧,2016,小麦COXVIIa基因克隆及其蛋白序列,分子植物育种,14(2):271-279)
Hüttemann M.,Frank V.,and Kadenbach B.,1999,The possible role of isoforms of cytochrome c oxidase subunit VIa in mammalian thermogenesis,Cell.Mol.Life.Sci.,55(11):1482-1490
Hüttemann M.,Hellingc S.,Sanderson T.H.,Sinkler C.,Samavati L.,Mahapatra G.,Varughese A.,Lu G.,Liu J.,Ramzan R.,Vogt S.,Grossman L.I.,Doan J.W.,Marcus K.,and Lee I.,2012,Regulation of mitochondrial respiration and apoptosis through cell signaling:cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation,Biochim.Biophys.Acta,1817(4):598-609
Li P.,Li X.H.,and Qiu D.L.,2007,Relationship between the gene edition of COX3 transcripts and restoration of"V"type male sterile line,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Science Edition)),(S):1007-1032(李鹏,李新华,邱登林,2007,COX3基因转录本的编辑与V型小麦育性恢复的关系,湖南农业大学学报(自然科学版),(S):1007-1032)
Lightowlers R.,Ewart G.,Aggeler R.,Zhang Y.Z.,Calavetta L.,and Capaldi R.A.,1990,Isolation and characterization of the cDNAs encoding two isoforms of subunit COX of bovine cytochrome c oxidase,J.Biol.Chem.,265(5):2677-2681
Ma J.T.,Han C.B.,Zhou Y.,Zhao J.Z.,Jing W.,and Zou H.W.,2012,Altered expression of mitochondrial cytochrome c oxidase I and NADH dehydrogenase 4 transcripts associated with gastric tumorigenesis and tumor dedifferentiation,Mol.Med.Rep.,5(6):1526-1530
Mansilla N.,Garcia L.,Gonzalez D.H.,and Welchen E.,2015,At COX10,a protein involved in haem o synthesis during cytochrome c oxidase biogenesis,is essential for plant embryogenesis and modulates the progression of senescence,J.Exp.Bot.,66(21):6761-6775
Radin I.,Mansilla N.,Rodel G.,and Steinebrunner I.,2015,The Arabidopsis COX11 homolog is essential for cytochrome c oxidase activity,Front.Plant Sci.,6(255):1091
Seelan R.S.,and Grossman L.I.,1991,Cytochrome c oxidase subunit VIIa isoforms.Characterization and expression of bovine cDNAs,J.Biol.Chem.,266(29):19752-19757
Srinivasan S.,and Avadhani N.G.,2012,Cytochrome c oxidase dysfunction in oxidative stress,Free Radic.Biol.Med.,53(6):1252-1263
Sun J.,Rao Y.M.,Le M.W.,Yan T.X.,Yan X.W.,and Zhou H.Y.,2010,Effects of drought stress on sesame growth and yield characteristics and comprehensive evaluation of drought tolerance,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),32(4):525-533(孙建,饶月亮,乐美旺,颜廷献,颜小文,周红英,2010,干旱胁迫对芝麻生长与产量性状的影响及其抗旱性综合评价,中国油料作物学报,32(4):525-533)
Taanman J.W.,and Capaldi R.A.,1993,Subunit VIa of yeast cytochrome c oxidase is not necessary for assembly of the enzyme complex but modulates the enzyme activity.Isolation and characterization of the nuclear-coded gene,J.Biol.Chem.,268(25):18754-18761
Vijayasarathy C.,Biunno I.,Lenka N.,Yang M.,Basu A.,Hall I.P.,and Avadhani N.G.,1998,Variations in the subunit content and catalytic activity of the cytochrome c oxidase complex from different tissues and different cardiac compartments,Biochim.Biophys.Acta,1371(1):71-82
Wang Y.H.,Gao T.M.,Sima H.Q.,Wei S.L.,and Li C.M.,2010,The advantages,problems and countermeasures in sesame production of China,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),39(12):133-135(王永宏,高桐梅,司马青焕,卫双玲,李春明,2010,我国芝麻生产优势、存在问题及对策研究,河南农业科学,39(12):133-135)
Zhang H.,Chen Z.Y.,Li Y.S.,and Zhu Y.G.,2007,The mitochondrial genomic mutation and function in the cytoplasmic male sterile line of maxie rice,Wuhan Zhiwuxue Yanjiu(Journal of Wuhan Botanical Research),25(3):313-315(张鸿,陈祖玉,李阳生,朱英国,2007,马协型水稻细胞质雄性不育系线粒体基因组的变异与功能的研究,武汉植物学研究,25(3):313-315)
Anthony G.,Stroh A.,Lottspeich F.,and Kadenbach B.,1990,Different isozymes of cytochrome c oxidase are expressed in bovine smooth muscle and skeletal or heart muscle,FEBSLett.,277(1):97-100
Budak H.,Akpinar B.A.,Unver T.,and Turktas M.,2013,Proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanoLC-ESI-MS/MS,Plant Mol.Biol.,83(1):1-15
Circu M.L.,and Aw T.Y.,2010,Reactive oxygen species,cellular redox systems and apoptosis,Free Radic.Biol.Med.,48(6):749-762
Ducos E.,Touzet P.,and Boutry M.,2001,The male sterile G cytoplasm of wild beet displays modified mitochondrial respiratory complexes,Plant J.,26(2):171-180
Fan X.F.,Wang J.S.,Liu H.Z.,Zhou L.,Wang X.J.,and Zhao J.H.,2016,Cloning and sequences analysis of COXVIIa gene in wheat,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):271-279(范小芳,王俊生,刘红占,周琳,王晓娟,赵锦慧,2016,小麦COXVIIa基因克隆及其蛋白序列,分子植物育种,14(2):271-279)
Hüttemann M.,Frank V.,and Kadenbach B.,1999,The possible role of isoforms of cytochrome c oxidase subunit VIa in mammalian thermogenesis,Cell.Mol.Life.Sci.,55(11):1482-1490
Hüttemann M.,Hellingc S.,Sanderson T.H.,Sinkler C.,Samavati L.,Mahapatra G.,Varughese A.,Lu G.,Liu J.,Ramzan R.,Vogt S.,Grossman L.I.,Doan J.W.,Marcus K.,and Lee I.,2012,Regulation of mitochondrial respiration and apoptosis through cell signaling:cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation,Biochim.Biophys.Acta,1817(4):598-609
Li P.,Li X.H.,and Qiu D.L.,2007,Relationship between the gene edition of COX3 transcripts and restoration of"V"type male sterile line,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Science Edition)),(S):1007-1032(李鹏,李新华,邱登林,2007,COX3基因转录本的编辑与V型小麦育性恢复的关系,湖南农业大学学报(自然科学版),(S):1007-1032)
Lightowlers R.,Ewart G.,Aggeler R.,Zhang Y.Z.,Calavetta L.,and Capaldi R.A.,1990,Isolation and characterization of the cDNAs encoding two isoforms of subunit COX of bovine cytochrome c oxidase,J.Biol.Chem.,265(5):2677-2681
Ma J.T.,Han C.B.,Zhou Y.,Zhao J.Z.,Jing W.,and Zou H.W.,2012,Altered expression of mitochondrial cytochrome c oxidase I and NADH dehydrogenase 4 transcripts associated with gastric tumorigenesis and tumor dedifferentiation,Mol.Med.Rep.,5(6):1526-1530
Mansilla N.,Garcia L.,Gonzalez D.H.,and Welchen E.,2015,At COX10,a protein involved in haem o synthesis during cytochrome c oxidase biogenesis,is essential for plant embryogenesis and modulates the progression of senescence,J.Exp.Bot.,66(21):6761-6775
Radin I.,Mansilla N.,Rodel G.,and Steinebrunner I.,2015,The Arabidopsis COX11 homolog is essential for cytochrome c oxidase activity,Front.Plant Sci.,6(255):1091
Seelan R.S.,and Grossman L.I.,1991,Cytochrome c oxidase subunit VIIa isoforms.Characterization and expression of bovine cDNAs,J.Biol.Chem.,266(29):19752-19757
Srinivasan S.,and Avadhani N.G.,2012,Cytochrome c oxidase dysfunction in oxidative stress,Free Radic.Biol.Med.,53(6):1252-1263
Sun J.,Rao Y.M.,Le M.W.,Yan T.X.,Yan X.W.,and Zhou H.Y.,2010,Effects of drought stress on sesame growth and yield characteristics and comprehensive evaluation of drought tolerance,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),32(4):525-533(孙建,饶月亮,乐美旺,颜廷献,颜小文,周红英,2010,干旱胁迫对芝麻生长与产量性状的影响及其抗旱性综合评价,中国油料作物学报,32(4):525-533)
Taanman J.W.,and Capaldi R.A.,1993,Subunit VIa of yeast cytochrome c oxidase is not necessary for assembly of the enzyme complex but modulates the enzyme activity.Isolation and characterization of the nuclear-coded gene,J.Biol.Chem.,268(25):18754-18761
Vijayasarathy C.,Biunno I.,Lenka N.,Yang M.,Basu A.,Hall I.P.,and Avadhani N.G.,1998,Variations in the subunit content and catalytic activity of the cytochrome c oxidase complex from different tissues and different cardiac compartments,Biochim.Biophys.Acta,1371(1):71-82
Wang Y.H.,Gao T.M.,Sima H.Q.,Wei S.L.,and Li C.M.,2010,The advantages,problems and countermeasures in sesame production of China,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),39(12):133-135(王永宏,高桐梅,司马青焕,卫双玲,李春明,2010,我国芝麻生产优势、存在问题及对策研究,河南农业科学,39(12):133-135)
Zhang H.,Chen Z.Y.,Li Y.S.,and Zhu Y.G.,2007,The mitochondrial genomic mutation and function in the cytoplasmic male sterile line of maxie rice,Wuhan Zhiwuxue Yanjiu(Journal of Wuhan Botanical Research),25(3):313-315(张鸿,陈祖玉,李阳生,朱英国,2007,马协型水稻细胞质雄性不育系线粒体基因组的变异与功能的研究,武汉植物学研究,25(3):313-315)
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