芍药分生组织决定基因APETALA2(AP2)的克隆及生物信息学分析
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摘要
花分生组织决定基因APETALA2(AP2)属于植物ABCDE模型基因,在花器官发育过程中起着重要的调控作用。为进一步了解芍药AP2基因的生物学功能,利用RACE扩增和测序技术克隆plAP2基因序列,利用生物信息学在线程序对其序列特征、蛋白结构及功能、亚细胞定位进行预测,并利用MEGA 5.0构建不同植物AP2分子进化树,最后利用qPCR检测其在内外花瓣中的差异表达情况。结果显示,克隆获得芍药AP2基因(plAP2)cDNA序列全长1 935 bp,其ORF全长为1 578 bp,编码525个氨基酸。蛋白结构与功能分析表明,plAP2蛋白为亲水性不稳定蛋白,无跨膜结构和信号肽,表明为非分泌蛋白;核定位信号位于氨基酸序列139-147(KKSRRGPRS);二级结构包括α-螺旋(24%)、β-折叠(19%)、β-转角(28%)和无规则卷曲(28%);plAP2蛋白存在8个糖基化位点和64个磷酸化位点,plAP2蛋白包含2个相同的保守结构域:AP2/(Ethylene-Responsive factors,ERF)(151-213aa和243-306aa)。亚细胞定位主要在细胞质(45.0%)中,少量分布于微体、线粒体基质间隙和溶酶体;进化树分析表明,芍药AP2基因与牡丹高度同源且亲缘关系最近;qPCR检测显示外瓣AP2表达量均极显著高于内瓣(P<0.01)。克隆出芍药AP2全长cDNA序列,系统地揭示了plAP2蛋白基本结构、功能位点区域、细胞定位以及组织表达情况,为今后深入研究plAP2基因功能提供基础素材和理论参考。
Flower-meristem-identity gene APETALA2( AP2) belonging to ABCDE model plays a role in regulating the development of plant floral organ. In order to understand the biological function of P. lactiflora AP2 gene,the plAP2 gene sequence of Paeonia lactiflora was cloned using RACE and sequencing technique technology,meanwhile the sequence signature,protein structure and function,subcellular localization were analyzed by bioinformatics methods. Molecular phylogenetic tree of AP2 from different plants were constructed by MEGA 5. 0 software. Finally,we detected the differential expression between inner-petal and outer-petal by qPCR. Results showed,the full-length cDNA sequence of plAP2 gene was 1 935 bp and consisted of a 1 578 bp open reading frame( ORF) encoding 525 amino acid proteins. Analysis of protein structure and function showed,plAP2 was a hydrophilic and unstable protein,meanwhile plAP2 was also non-secretory protein without signal peptide and transmembrane structure. Nuclear localization signal located between 139 th and 147 th amino acid( KKSRRGPRS). The secondary structure of plAP2 protein contained 24% α-helices,19% β-sheet,28% β-turn and 28% random coil. plAP2 protein had eight glycosylation sites and sixty-four phosphorylation sites. plAP2 protein had two same conservative domain: AP2/EthyleneResponsive factors( ERF),which was located in the region between 151 and 213,between 243 and 306,respec-tively. Prediction of subcellular localization showed that plAP2 located mostly in cytoplasm( 45. 0%),rarely in microbody,mitochondrial matrix and lysosome. Analysis of evolutionary tree showed that the AP2 of Paeonia lactiflora were highly homologous and close to Paeonia suffruticosa. qPCR showed the expression of AP2 in outer-petal was extremely significant than that in inner-petal( P < 0. 01). In this study,we cloned the complete cDNA sequence of AP2 gene in Paeonia lactiflora and systematically revealed the basic protein structure,functional sites,domains,cellular localization and expression level,which will provide basic materials and theoretical references for further studying plAP2 gene function.
Flower-meristem-identity gene APETALA2( AP2) belonging to ABCDE model plays a role in regulating the development of plant floral organ. In order to understand the biological function of P. lactiflora AP2 gene,the plAP2 gene sequence of Paeonia lactiflora was cloned using RACE and sequencing technique technology,meanwhile the sequence signature,protein structure and function,subcellular localization were analyzed by bioinformatics methods. Molecular phylogenetic tree of AP2 from different plants were constructed by MEGA 5. 0 software. Finally,we detected the differential expression between inner-petal and outer-petal by qPCR. Results showed,the full-length cDNA sequence of plAP2 gene was 1 935 bp and consisted of a 1 578 bp open reading frame( ORF) encoding 525 amino acid proteins. Analysis of protein structure and function showed,plAP2 was a hydrophilic and unstable protein,meanwhile plAP2 was also non-secretory protein without signal peptide and transmembrane structure. Nuclear localization signal located between 139 th and 147 th amino acid( KKSRRGPRS). The secondary structure of plAP2 protein contained 24% α-helices,19% β-sheet,28% β-turn and 28% random coil. plAP2 protein had eight glycosylation sites and sixty-four phosphorylation sites. plAP2 protein had two same conservative domain: AP2/EthyleneResponsive factors( ERF),which was located in the region between 151 and 213,between 243 and 306,respec-tively. Prediction of subcellular localization showed that plAP2 located mostly in cytoplasm( 45. 0%),rarely in microbody,mitochondrial matrix and lysosome. Analysis of evolutionary tree showed that the AP2 of Paeonia lactiflora were highly homologous and close to Paeonia suffruticosa. qPCR showed the expression of AP2 in outer-petal was extremely significant than that in inner-petal( P < 0. 01). In this study,we cloned the complete cDNA sequence of AP2 gene in Paeonia lactiflora and systematically revealed the basic protein structure,functional sites,domains,cellular localization and expression level,which will provide basic materials and theoretical references for further studying plAP2 gene function.
引文
[1]曾英,胡金勇,李志坚.植物MADS盒基因与花器官的进化发育[J].植物生理学通讯,2001,37(4):281-287.
[2]Coen E S,Meyerowitz E M.The war of the whorls:genetic interactions controlling flower development[J].Nature,1991,353(6339):31-37.
[3]Angenent G C,Franken J,Busscher M,et al.A novel class of MADS box genes is involved in ovule development in petunia[J].The Plant Cell,1995,7(10):1569-1582.
[4]Colombo L,Franken J,Koetje E,et al.The petunia MADS box gene FBP11 determines ovule identity[J].The Plant Cell,1995,7(11):1859-1868.
[5]Pelaz S,Ditta G S,Baumann E,et al.B and C floral organ identity functions require SEPALLATA MADS-box genes[J].Nature,2000,405(6783):200-203.
[6]Pelaz S,Gustafson-Brown C,Kohalmi S E,et al.APETALA1 and SEPALLATA3 interact to promote flower development[J].The Plant Journal:for Cell and Molecular Biology,2001,26(4):385-394.
[7]Maes T,Van De Steene N,Zethof J,et al.Petunia Ap2-like genes and their role in flower and seed development[J].The Plant Cell,2001,13(2):229-244.
[8]Jofuku K D,Den Boer B G,Van Montagu M,et al.Control of arabidopsis flower and seed development by the homeotic gene APETALA2[J].The Plant Cell,1994,6(9):1211-1225.
[9]任磊.牡丹花器官发育相关基因的克隆与表达[D].北京:中国林业科学研究院,2011.
[10]宿红艳,周盛梅,王磊,等.草莓APETALA2同源基因的克隆及表达分析[J].西北植物学报,2005,25(10):1937-1942.
[11]Zhuang J,Deng D X,Yao Q H,et al.Discovery,phylogeny and expression patterns of AP2-like genes in maize[J].Plant Growth Regulation,2010,62(1):51-58.
[12]Zhuang J,Yao Q H,Xiong A S,et al.Isolation,phylogeny and expression patterns of AP2-like genes in apple(Malus×domestica Borkh)[J].Plant Molecular Biology Reporter,2011,29(1):209-216.
[13]罗睿雄,赵志常,高爱平,等.芒果4CL基因的克隆及其表达分析[J].华北农学报,2016,31(1):57-62.
[14]雷海英,白凤麟,刘建霞,等.玉米Zmcen基因的克隆,表达与生物信息学分析[J].华北农学报,2016,31(3):18-24.
[15]侯艳霞,汤浩茹,林源秀,等.基于RACE方法的草莓Faapx-c基因克隆、表达及其生物信息学分析[J].北方园艺,2016(11):88-92.
[16]Zhao D,Jiang Y,Ning C,et al.Transcriptome sequencing of a chimaera reveals coordinated expression of anthocyanin biosynthetic genes mediating yellow formation in herbaceous peony(Paeonia lactiflora Pall.)[J].BMC Genomics,2014,15(1):689.
[17]Petersen T N,Brunak S,Von Heijne G,et al.Signal P4.0:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785-786.
[18]Nguyen Ba A N,Pogoutse A,Provart N,et al.NLStradamus:a simple Hidden Markov Model for nuclear localization signal prediction[J].BMC Bioinformatics,2009,10(1):202.
[19]La Cour T,Kiemer L,Mlgaard A,et al.Analysis and prediction of leucine-rich nuclear export signals[J].Protein Engineering,Design&Selection,2004,17(6):527-536.
[20]Mellquist J L,Kasturi L,Spitalnik S L,et al.The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency[J].Biochemistry,1998,37(19):6833-6837.
[21]Kim J H,Lee J,Oh B,et al.Prediction of phosphorylation sites using SVMs[J].Bioinformatics,2004,20(17):3179-3184.
[22]Blom N,Gammeltoft S,Brunak S.Sequence and structure-based prediction of eukaryotic protein phosphorylation sites[J].Journal of Molecular Biology,1999,294(5):1351-1362.
[23]Marchler-Bauer A,Derbyshire M K,Gonzales N R,et al.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,2015,43:222-226.
[24]Schultz J,Milpetz F,Bork P,et al.SMART,a simple modular architecture research tool:identification of signaling domains[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(11):5857-5864.
[25]Emanuelsson O,Nielsen H,Brunak S,et al.Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[J].Journal of Molecular Biology,2000,300(4):1005-1016.
[26]Wilkins M R,Gasteiger E,Bairoch A,et al.Protein identification and analysis tools in the Ex PASy server[J].Methods in Molecular Biology,1999,112(112):531-552.
[27]Weigel D.The APETALA2 domain is related to a novel type of DNA binding domain[J].The Plant Cell,1995,7(4):388-389.
[28]丰锦,陈信波.抗逆相关AP2/EREBP转录因子研究进展[J].生物技术通报,2011(7):1-6.
[29]赵利峰,柴团耀.AP2/EREBP转录因子在植物发育和胁迫应答中的作用[J].植物学通报,2008,25(1):89-101.
[30]韩志萍,安利佳,侯和胜.AP2/EREBP转录因子的结构与功能[J].中国农学通报,2006,22(2):33-38.
[31]Ohme-Takagi M,Shinshi H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].The Plant Cell,1995,7(2):173-182.
[32]Zhuang J,Anyia A,Vidmar J,et al.Discovery and expression assessment of the AP2-like genes in Hordeum vulgare[J].Acta Physiologiae Plantarum,2011,33(5):1639-1649.
[33]张剑,徐桂霞,薛皓月,等.植物进化发育生物学的形成与研究进展[J].植物学通报,2007,24(1):1-30.
[34]张计育,王庆菊,郭忠仁.植物AP2/ERF类转录因子研究进展[J].遗传,2012,4(7):835-884.
[35]Kyozuka J,Kobayashi T,Morita M,et al.Spatially and temporally regulated expression of rice MADS box genes with similarity to Arabidopsis class A,B and C genes[J].Plant&Cell Physiology,2000,41(6):710-718.
[2]Coen E S,Meyerowitz E M.The war of the whorls:genetic interactions controlling flower development[J].Nature,1991,353(6339):31-37.
[3]Angenent G C,Franken J,Busscher M,et al.A novel class of MADS box genes is involved in ovule development in petunia[J].The Plant Cell,1995,7(10):1569-1582.
[4]Colombo L,Franken J,Koetje E,et al.The petunia MADS box gene FBP11 determines ovule identity[J].The Plant Cell,1995,7(11):1859-1868.
[5]Pelaz S,Ditta G S,Baumann E,et al.B and C floral organ identity functions require SEPALLATA MADS-box genes[J].Nature,2000,405(6783):200-203.
[6]Pelaz S,Gustafson-Brown C,Kohalmi S E,et al.APETALA1 and SEPALLATA3 interact to promote flower development[J].The Plant Journal:for Cell and Molecular Biology,2001,26(4):385-394.
[7]Maes T,Van De Steene N,Zethof J,et al.Petunia Ap2-like genes and their role in flower and seed development[J].The Plant Cell,2001,13(2):229-244.
[8]Jofuku K D,Den Boer B G,Van Montagu M,et al.Control of arabidopsis flower and seed development by the homeotic gene APETALA2[J].The Plant Cell,1994,6(9):1211-1225.
[9]任磊.牡丹花器官发育相关基因的克隆与表达[D].北京:中国林业科学研究院,2011.
[10]宿红艳,周盛梅,王磊,等.草莓APETALA2同源基因的克隆及表达分析[J].西北植物学报,2005,25(10):1937-1942.
[11]Zhuang J,Deng D X,Yao Q H,et al.Discovery,phylogeny and expression patterns of AP2-like genes in maize[J].Plant Growth Regulation,2010,62(1):51-58.
[12]Zhuang J,Yao Q H,Xiong A S,et al.Isolation,phylogeny and expression patterns of AP2-like genes in apple(Malus×domestica Borkh)[J].Plant Molecular Biology Reporter,2011,29(1):209-216.
[13]罗睿雄,赵志常,高爱平,等.芒果4CL基因的克隆及其表达分析[J].华北农学报,2016,31(1):57-62.
[14]雷海英,白凤麟,刘建霞,等.玉米Zmcen基因的克隆,表达与生物信息学分析[J].华北农学报,2016,31(3):18-24.
[15]侯艳霞,汤浩茹,林源秀,等.基于RACE方法的草莓Faapx-c基因克隆、表达及其生物信息学分析[J].北方园艺,2016(11):88-92.
[16]Zhao D,Jiang Y,Ning C,et al.Transcriptome sequencing of a chimaera reveals coordinated expression of anthocyanin biosynthetic genes mediating yellow formation in herbaceous peony(Paeonia lactiflora Pall.)[J].BMC Genomics,2014,15(1):689.
[17]Petersen T N,Brunak S,Von Heijne G,et al.Signal P4.0:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785-786.
[18]Nguyen Ba A N,Pogoutse A,Provart N,et al.NLStradamus:a simple Hidden Markov Model for nuclear localization signal prediction[J].BMC Bioinformatics,2009,10(1):202.
[19]La Cour T,Kiemer L,Mlgaard A,et al.Analysis and prediction of leucine-rich nuclear export signals[J].Protein Engineering,Design&Selection,2004,17(6):527-536.
[20]Mellquist J L,Kasturi L,Spitalnik S L,et al.The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency[J].Biochemistry,1998,37(19):6833-6837.
[21]Kim J H,Lee J,Oh B,et al.Prediction of phosphorylation sites using SVMs[J].Bioinformatics,2004,20(17):3179-3184.
[22]Blom N,Gammeltoft S,Brunak S.Sequence and structure-based prediction of eukaryotic protein phosphorylation sites[J].Journal of Molecular Biology,1999,294(5):1351-1362.
[23]Marchler-Bauer A,Derbyshire M K,Gonzales N R,et al.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,2015,43:222-226.
[24]Schultz J,Milpetz F,Bork P,et al.SMART,a simple modular architecture research tool:identification of signaling domains[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(11):5857-5864.
[25]Emanuelsson O,Nielsen H,Brunak S,et al.Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[J].Journal of Molecular Biology,2000,300(4):1005-1016.
[26]Wilkins M R,Gasteiger E,Bairoch A,et al.Protein identification and analysis tools in the Ex PASy server[J].Methods in Molecular Biology,1999,112(112):531-552.
[27]Weigel D.The APETALA2 domain is related to a novel type of DNA binding domain[J].The Plant Cell,1995,7(4):388-389.
[28]丰锦,陈信波.抗逆相关AP2/EREBP转录因子研究进展[J].生物技术通报,2011(7):1-6.
[29]赵利峰,柴团耀.AP2/EREBP转录因子在植物发育和胁迫应答中的作用[J].植物学通报,2008,25(1):89-101.
[30]韩志萍,安利佳,侯和胜.AP2/EREBP转录因子的结构与功能[J].中国农学通报,2006,22(2):33-38.
[31]Ohme-Takagi M,Shinshi H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].The Plant Cell,1995,7(2):173-182.
[32]Zhuang J,Anyia A,Vidmar J,et al.Discovery and expression assessment of the AP2-like genes in Hordeum vulgare[J].Acta Physiologiae Plantarum,2011,33(5):1639-1649.
[33]张剑,徐桂霞,薛皓月,等.植物进化发育生物学的形成与研究进展[J].植物学通报,2007,24(1):1-30.
[34]张计育,王庆菊,郭忠仁.植物AP2/ERF类转录因子研究进展[J].遗传,2012,4(7):835-884.
[35]Kyozuka J,Kobayashi T,Morita M,et al.Spatially and temporally regulated expression of rice MADS box genes with similarity to Arabidopsis class A,B and C genes[J].Plant&Cell Physiology,2000,41(6):710-718.