红掌AP2类转录因子AaSNB编码基因的克隆及表达分析
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摘要
红掌是重要的高档盆栽花卉。挖掘红掌观赏器官的关键控制基因,对于利用生物技术改良红掌品质意义重大。本研究采用RACE技术,克隆了一个AP2类转录因子的核苷酸序列,该序列全长2 201 bp,编码区长度为1 461 bp,预测编码486个氨基酸残基。通过NCBI数据库比对和生物信息学分析,发现该基因由两个较为保守的AP2结构域组成,该结构域前端连接有一段核定位信号,在预测氨基酸的氮端具有转录激活功能信号,认定该基因与水稻苞片控制基因SNB为同源类似物,将其命名为AaSNB。采用荧光定量RT-PCR方法初步分析了该基因在红掌不同幼嫩器官的表达情况,发现该基因在肉穗花序中表达量最高,苞片中表达量次之,在根系中也有极微量表达。综合分析认为该转录因子对于红掌苞片等的发育起到重要作用,讨论了利用该基因创制多苞片红掌新种质的可能性。本研究对发掘红掌苞片发育的关键控制基因,并利用其改良红掌品质提供了更多选择,对于丰富AP2转录因子家族也具有积极意义。
Anthurium is an importa nt high-grade potted flower. It is of great significance for improving the quality of Anthurium by biotechnology to dig out the key control genes of the ornamental organs of Anthurium. In this study, the nucleotide sequence of a AP2 type transcription factor was cloned using RACE technology. The full length of the sequence was 2 201 bp and the coding region was 1 641 bp long, which was predicted to encode 486 amino acid residues. Through NCBI database and bioinformatics analysis, it was found that the gene was composed of two relatively conservative AP2 domains, and the front end of this domain was connected with a nuclear location signal. Besides, a transcription activation signal was found at the nitrogen end of this predicted amino acid sequence. Thus, the gene was identified as a homologous analogue to the bract control gene SNB in rice and named AaSNB. The gene expression patterns in different young organs were analyzed by fluorescence quantitative RT-PCR method. The result indicated that the expression level in inflorescence was the highest,followed by the bract, and the content in root was very little. Overall, data analysis showed that this transcription factor played important roles in the development of bract. The possibility of using this gene to create supernumerarybract Anthurium was discussed. This study could provide more options for exploring key control genes of Anthurium to improve its ornamental quality, and could positively enrich the family members of AP2 transcription factor as well.
Anthurium is an importa nt high-grade potted flower. It is of great significance for improving the quality of Anthurium by biotechnology to dig out the key control genes of the ornamental organs of Anthurium. In this study, the nucleotide sequence of a AP2 type transcription factor was cloned using RACE technology. The full length of the sequence was 2 201 bp and the coding region was 1 641 bp long, which was predicted to encode 486 amino acid residues. Through NCBI database and bioinformatics analysis, it was found that the gene was composed of two relatively conservative AP2 domains, and the front end of this domain was connected with a nuclear location signal. Besides, a transcription activation signal was found at the nitrogen end of this predicted amino acid sequence. Thus, the gene was identified as a homologous analogue to the bract control gene SNB in rice and named AaSNB. The gene expression patterns in different young organs were analyzed by fluorescence quantitative RT-PCR method. The result indicated that the expression level in inflorescence was the highest,followed by the bract, and the content in root was very little. Overall, data analysis showed that this transcription factor played important roles in the development of bract. The possibility of using this gene to create supernumerarybract Anthurium was discussed. This study could provide more options for exploring key control genes of Anthurium to improve its ornamental quality, and could positively enrich the family members of AP2 transcription factor as well.
引文
Aukerman M.J.,and Sakai H.,200 3,Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes,Plant Cell,15(11):2730-2741
Bowman J.L.,Alvarez J.,Weigel D.,Meyerowitz E.M.,and Smyth D.R.,1993,Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes,Development,119(3):721-743
Chuck G.,Meeley R.,Irish E.,Sakai H.,and Hake S.,2007,The maize tasselseed4 microrna controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1,Nat.Genet.,39(12):1517-1521
Chuck G.,Meeley R.B.,and Hake S.,1998,The control of maize spikelet meristem fate by the apetala2-like gene indeterminate spikelet1,Genes Dev.,12(8):1145-1154
Coen E.S.,and Meyerowitz E.M.,1991,The war of the whorls:genetic interactions controlling flower development,Nature,353(6339):31-37
Debernardi J.M.,Lin H.,Chuck G.,Faris J.D.,and Dubcovsky J.,2017,MicroRNA172 plays a crucial role in wheat spike morphogenesis and grain threshability,Development,144(11):1966-1975
Denay G.,Chahtane H.,Tichtinsky G.,and Parcy F.,2017,Aflower is born:an update on Arabidopsis floral meristem formation,Curr.Opin.Plant Biol.,35:15-22
Dietz K.J.,Vogel M.O.,and Viehhauser A.,2010,AP2/EREBPtranscription factors are part of gene regulatory networks and integrate metabolic,hormonal and environmental signals in stress acclimation and retrograde signalling,Protoplasma,245(1-4):3-14
Dreni L.,and Kater M.M.,2014,MADS reloaded:evolution of the AGAMOUS subfamily genes,New Phytol.,201(3):717-732
Dreni L.,and Zhang D.,2016,Flower development:the evolutionary history and functions of the AGL6 subfamily MADS-box genes,J.Exp.Bot.,67(6):1625-1638
Drews G.N.,Bowman J.L.,and Meyerowitz E.M.,1991,Negative regulation of the Arabidopsis homeotic gene AGAMOUSby the APETALA2 product,Cell,65(6):991-1002
Gu C.,Guo Z.H.,Hao P.P.,Wang G.M.,Jin Z.M.,and Zhang S.L.,2017,Multiple regulatory roles of AP2/ERF transcription factor in angiosperm,Bot.Stud.,58(1):6
Jofuku K.D.,den Boer B.G.,Van Montagu M.,and Okamuro J.K.,1994,Control of Arabidopsis flower and seed development by the homeotic gene APETALA2,Plant Cell,6(9):1211-1225
Kagaya Y.,Ohmiya K.,and Hattori T.,1999,Rav1,a novel DNA-binding protein,binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants,Nucleic Acids Res.,27(2):470-478
Lee D.Y.,and An G.,2012,Two AP2 family genes,SUPERNU-MERARY BRACT(SNB)and OsINDETERMINATE SPIK-ELET 1(OsIDS1),synergistically control inflorescence architecture and floral meristem establishment in rice,Plant J.,69(3):445-461
Lee D.Y.,Lee J.,Moon S.,Park S.Y.,and An G.,2007,The rice heterochronic gene supernumerary bract regulates the transition from spikelet meristem to floral meristem,Plant J.,49(1):64-78
Li C.,Qiu J.,Yang G.,Huang S.,and Yin J.,2016,Isolation and characterization of a R2R3-MYB transcription factor gene related to anthocyanin biosynthesis in the spathes of Anthurium andraeanum(Hort.),Plant Cell Rep.,35(10):2151-2165
Li Z.,Wang J.,Zhang X.,and Xu L.,2015,Comparative transcriptome analysis of Anthurium"Albama"and its anthocyanin-Loss mutant,PLo S One,10(3):e0119027
Liang W.H.,Shang F.,Lin Q.T.,Lou C.,and Zhang J.,2014,Tillering and panicle branching genes in rice,Gene,537(1):1-5
Ma G.Y.,Liu H.C.,Zou Q.C.,,Zhu K.Y.,,Zhou J.H.,Zhan J.,and Tian D.Q.,2014,Cloning and expression analysis of Bclass gene AaPI from Anthurium andraeanum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(5):950-955(马广莹,刘慧春,邹清成,朱开元,周江华,詹菁,田丹青,2014,红掌花发育B类基因AaPI的克隆及表达分析,分子植物育种,12(5):950-955)
Ma G.Y.,Shi X.H.,Zhu K.Y.,Jin L.,Zou Q.C.,Liu H.C.,Zhang J.Q.,and Tian D.Q.,2017a,Gene cloning,expression and ectopic transformation analysis of AaMYB1 from Anthurium,Henongxue Bao(Journal of Nuclear Agricultural Sciences),31(9):1708-1716(马广莹,史小华,朱开元,金亮,邹清成,刘慧春,张加强,田丹青,2017a,红掌AaMYB1基因的克隆,表达及异源转化研究,核农学报,31(9):1708-1716)
Ma G.Y.,Zhu K.Y.,Shi X.H.,Zou Q.C.,Liu H.C.,Zhan J.,and Tian D.Q.,2017b,Cloning,sequence and expression analysis of two SOC1 genes from Anthurium,Zhejiang Daxue Xuebao(Journal of Zhejiang University),43(3):289-297(马广莹,朱开元,史小华,邹清成,刘慧春,詹菁,田丹青,2017b,红掌2个SOC1基因的克隆,序列与表达分析,浙江大学学报,43(3):289-297)
Melzer R.,and Theiben G.,2009,Reconstitution of'floral quartets'in vitro involving class B and class E floral homeotic proteins,Nucleic Acids Res.,37(8):2723-2736
Meyerowitz E.M.,Bowman J.L.,Brockman L.L.,Drews G.N.,Jack T.,Sieburth L.E.,and Weigel D.,1991,A genetic and molecular model for development in Arabidopsis thaliana,Dev.Suppl.,1(1):157-167
Ng M.,and Yanofsky M.F.,2000,Three ways to learn the ABCs,Curr.Opin.Plant Biol.,3(1):47-52
Ohme-Takagi M.,and Shinshi H.,1995,Ethylene-inducible DNAbinding proteins that interact with an ethylene-responsive element,Plant Cell,7(2):173-182
Ren D.Y.,Li Y.F.,Zhao F.M.,Sang X.C.,Shi J.Q.,Wang N.,Guo S.,Ling Y.H.,Zhang C.W.,Yang Z.L.,and He G.H.,2013,Multi-floret spikelet1,which encodes an AP2/ERFprotein,determines spikelet meristem fate and sterile lemma identity in rice,Plant Physiol.,162(2):872-884
Suzuki J.Y.,Amore T.D.,Calla B.,Palmer N.A.,Scully E.D.,Sattler S.E.,Sarath G.,Lichty J.S.,Myers R.Y.,Keith L.M.,Matsumoto T.K.,and Geib S.M.,2017,Organ-specific transcriptome profiling of metabolic and pigment biosynthesis pathways in the floral ornamental progenitor species Anthurium amnicola Dressler,Sci.Rep.,7(1):1596
Würschum T.,Gross-Hardt R.,and Laux T.,2006,APETALA2regulates the stem cell niche in the Arabidopsis shoot meristem,Plant Cell,18(2):295-307
Yang F.X.,Zhu G.F.,Wang Z.,Liu H.L.,and Huang D.,2015a,A putative mir172-targeted ceapetala2-like gene is involved in floral patterning regulation of the orchid Cymbidium ensifolium,Genet.Mol.Res.,14(4):12049-12061
Yang Y.X.,Chen X.X.,Xu B.,Li.X.,Ma Y.H.,and Wang G.D.,2015b,Phenotype and transcriptome analysis reveals chloroplast development and pigment biosynthesis together influenced the leaf color formation in mutants of Anthurium andraeanum'Sonate',Front Plant Sci.,6:139
Zhang H.,Zhu Y.Y.,Shao X.,Wang Y.,Li Y.Q.,and Guo W.D.,2015,Isolation and characterization of PpcCBF CBF/DREBtranscription factor cDNA of Chinese cherry(Prunus pseudocerasus),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):149-154(张弘,朱友银,邵姁,王月,李永强,郭卫东,2015,中国樱桃CBF/DREB转录因子PpcCBF的克隆与表达分析,基因组学与应用生物学,34(1):149-154)
Zhang J.Y.,Wang Q.J.,and Guo Z.R.,2012,Progresses on plant AP2/ERF transcription factors,Yichuan(Hereditas),34(7):835-847(张计育,王庆菊,郭忠仁,2012,植物AP2/ERF类转录因子研究进展,遗传,34(7):835-847)
Zhou Y.,Lu D.F.,Li C.Y.,Luo J.H.,Zhu B.F.,Zhu J.J.,Shangguan Y.Y.,Wang Z.X.,Sang T.,Zhou B.,and Han B.,2012,Genetic control of seed shattering in rice by the APETALA2transcription factor SHATTERING ABORTION1,Plant Cell,24(3):1034-1048
Bowman J.L.,Alvarez J.,Weigel D.,Meyerowitz E.M.,and Smyth D.R.,1993,Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes,Development,119(3):721-743
Chuck G.,Meeley R.,Irish E.,Sakai H.,and Hake S.,2007,The maize tasselseed4 microrna controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1,Nat.Genet.,39(12):1517-1521
Chuck G.,Meeley R.B.,and Hake S.,1998,The control of maize spikelet meristem fate by the apetala2-like gene indeterminate spikelet1,Genes Dev.,12(8):1145-1154
Coen E.S.,and Meyerowitz E.M.,1991,The war of the whorls:genetic interactions controlling flower development,Nature,353(6339):31-37
Debernardi J.M.,Lin H.,Chuck G.,Faris J.D.,and Dubcovsky J.,2017,MicroRNA172 plays a crucial role in wheat spike morphogenesis and grain threshability,Development,144(11):1966-1975
Denay G.,Chahtane H.,Tichtinsky G.,and Parcy F.,2017,Aflower is born:an update on Arabidopsis floral meristem formation,Curr.Opin.Plant Biol.,35:15-22
Dietz K.J.,Vogel M.O.,and Viehhauser A.,2010,AP2/EREBPtranscription factors are part of gene regulatory networks and integrate metabolic,hormonal and environmental signals in stress acclimation and retrograde signalling,Protoplasma,245(1-4):3-14
Dreni L.,and Kater M.M.,2014,MADS reloaded:evolution of the AGAMOUS subfamily genes,New Phytol.,201(3):717-732
Dreni L.,and Zhang D.,2016,Flower development:the evolutionary history and functions of the AGL6 subfamily MADS-box genes,J.Exp.Bot.,67(6):1625-1638
Drews G.N.,Bowman J.L.,and Meyerowitz E.M.,1991,Negative regulation of the Arabidopsis homeotic gene AGAMOUSby the APETALA2 product,Cell,65(6):991-1002
Gu C.,Guo Z.H.,Hao P.P.,Wang G.M.,Jin Z.M.,and Zhang S.L.,2017,Multiple regulatory roles of AP2/ERF transcription factor in angiosperm,Bot.Stud.,58(1):6
Jofuku K.D.,den Boer B.G.,Van Montagu M.,and Okamuro J.K.,1994,Control of Arabidopsis flower and seed development by the homeotic gene APETALA2,Plant Cell,6(9):1211-1225
Kagaya Y.,Ohmiya K.,and Hattori T.,1999,Rav1,a novel DNA-binding protein,binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants,Nucleic Acids Res.,27(2):470-478
Lee D.Y.,and An G.,2012,Two AP2 family genes,SUPERNU-MERARY BRACT(SNB)and OsINDETERMINATE SPIK-ELET 1(OsIDS1),synergistically control inflorescence architecture and floral meristem establishment in rice,Plant J.,69(3):445-461
Lee D.Y.,Lee J.,Moon S.,Park S.Y.,and An G.,2007,The rice heterochronic gene supernumerary bract regulates the transition from spikelet meristem to floral meristem,Plant J.,49(1):64-78
Li C.,Qiu J.,Yang G.,Huang S.,and Yin J.,2016,Isolation and characterization of a R2R3-MYB transcription factor gene related to anthocyanin biosynthesis in the spathes of Anthurium andraeanum(Hort.),Plant Cell Rep.,35(10):2151-2165
Li Z.,Wang J.,Zhang X.,and Xu L.,2015,Comparative transcriptome analysis of Anthurium"Albama"and its anthocyanin-Loss mutant,PLo S One,10(3):e0119027
Liang W.H.,Shang F.,Lin Q.T.,Lou C.,and Zhang J.,2014,Tillering and panicle branching genes in rice,Gene,537(1):1-5
Ma G.Y.,Liu H.C.,Zou Q.C.,,Zhu K.Y.,,Zhou J.H.,Zhan J.,and Tian D.Q.,2014,Cloning and expression analysis of Bclass gene AaPI from Anthurium andraeanum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(5):950-955(马广莹,刘慧春,邹清成,朱开元,周江华,詹菁,田丹青,2014,红掌花发育B类基因AaPI的克隆及表达分析,分子植物育种,12(5):950-955)
Ma G.Y.,Shi X.H.,Zhu K.Y.,Jin L.,Zou Q.C.,Liu H.C.,Zhang J.Q.,and Tian D.Q.,2017a,Gene cloning,expression and ectopic transformation analysis of AaMYB1 from Anthurium,Henongxue Bao(Journal of Nuclear Agricultural Sciences),31(9):1708-1716(马广莹,史小华,朱开元,金亮,邹清成,刘慧春,张加强,田丹青,2017a,红掌AaMYB1基因的克隆,表达及异源转化研究,核农学报,31(9):1708-1716)
Ma G.Y.,Zhu K.Y.,Shi X.H.,Zou Q.C.,Liu H.C.,Zhan J.,and Tian D.Q.,2017b,Cloning,sequence and expression analysis of two SOC1 genes from Anthurium,Zhejiang Daxue Xuebao(Journal of Zhejiang University),43(3):289-297(马广莹,朱开元,史小华,邹清成,刘慧春,詹菁,田丹青,2017b,红掌2个SOC1基因的克隆,序列与表达分析,浙江大学学报,43(3):289-297)
Melzer R.,and Theiben G.,2009,Reconstitution of'floral quartets'in vitro involving class B and class E floral homeotic proteins,Nucleic Acids Res.,37(8):2723-2736
Meyerowitz E.M.,Bowman J.L.,Brockman L.L.,Drews G.N.,Jack T.,Sieburth L.E.,and Weigel D.,1991,A genetic and molecular model for development in Arabidopsis thaliana,Dev.Suppl.,1(1):157-167
Ng M.,and Yanofsky M.F.,2000,Three ways to learn the ABCs,Curr.Opin.Plant Biol.,3(1):47-52
Ohme-Takagi M.,and Shinshi H.,1995,Ethylene-inducible DNAbinding proteins that interact with an ethylene-responsive element,Plant Cell,7(2):173-182
Ren D.Y.,Li Y.F.,Zhao F.M.,Sang X.C.,Shi J.Q.,Wang N.,Guo S.,Ling Y.H.,Zhang C.W.,Yang Z.L.,and He G.H.,2013,Multi-floret spikelet1,which encodes an AP2/ERFprotein,determines spikelet meristem fate and sterile lemma identity in rice,Plant Physiol.,162(2):872-884
Suzuki J.Y.,Amore T.D.,Calla B.,Palmer N.A.,Scully E.D.,Sattler S.E.,Sarath G.,Lichty J.S.,Myers R.Y.,Keith L.M.,Matsumoto T.K.,and Geib S.M.,2017,Organ-specific transcriptome profiling of metabolic and pigment biosynthesis pathways in the floral ornamental progenitor species Anthurium amnicola Dressler,Sci.Rep.,7(1):1596
Würschum T.,Gross-Hardt R.,and Laux T.,2006,APETALA2regulates the stem cell niche in the Arabidopsis shoot meristem,Plant Cell,18(2):295-307
Yang F.X.,Zhu G.F.,Wang Z.,Liu H.L.,and Huang D.,2015a,A putative mir172-targeted ceapetala2-like gene is involved in floral patterning regulation of the orchid Cymbidium ensifolium,Genet.Mol.Res.,14(4):12049-12061
Yang Y.X.,Chen X.X.,Xu B.,Li.X.,Ma Y.H.,and Wang G.D.,2015b,Phenotype and transcriptome analysis reveals chloroplast development and pigment biosynthesis together influenced the leaf color formation in mutants of Anthurium andraeanum'Sonate',Front Plant Sci.,6:139
Zhang H.,Zhu Y.Y.,Shao X.,Wang Y.,Li Y.Q.,and Guo W.D.,2015,Isolation and characterization of PpcCBF CBF/DREBtranscription factor cDNA of Chinese cherry(Prunus pseudocerasus),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):149-154(张弘,朱友银,邵姁,王月,李永强,郭卫东,2015,中国樱桃CBF/DREB转录因子PpcCBF的克隆与表达分析,基因组学与应用生物学,34(1):149-154)
Zhang J.Y.,Wang Q.J.,and Guo Z.R.,2012,Progresses on plant AP2/ERF transcription factors,Yichuan(Hereditas),34(7):835-847(张计育,王庆菊,郭忠仁,2012,植物AP2/ERF类转录因子研究进展,遗传,34(7):835-847)
Zhou Y.,Lu D.F.,Li C.Y.,Luo J.H.,Zhu B.F.,Zhu J.J.,Shangguan Y.Y.,Wang Z.X.,Sang T.,Zhou B.,and Han B.,2012,Genetic control of seed shattering in rice by the APETALA2transcription factor SHATTERING ABORTION1,Plant Cell,24(3):1034-1048