MiR172介导的AP2-like转录因子表达对大岩桐花期调控的研究（英文）
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摘要
目的:本文通过研究microRNA172a(miR172a)对成花途径中关键靶基因APETALA2类(AP2-like)的调控作用及其机理分析,探索通过操纵miRNA表达改变观赏植物花期的基因工程分子育种新策略。创新点:本研究基于植物miR172序列和功能的高度保守性,通过转基因的方法操纵大岩桐miR172的表达,进而影响AP2-like基因的表达,并起到调控花期的作用。方法:本研究借用拟南芥miR172a的已知序列构建组成型过表达载体35S:miR172a和抑制miR172表达的35S:MIM172载体。利用农杆菌介导法成功获得了35S:miR172a过表达株系以及抑制miR172作用的35S:MIM172株系,并利用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆得到了大岩桐AP2-likecDNA全序列,并通过实时荧光定量聚合酶链式反应技术(qPCR)检测转基因株系中AP2-like的表达变化。结论:短日照条件下,35S:miR172a过表达株系花期比野生型提前(47.00±2.16)天;35S:MIM172株系花期延迟(7.00±4.28)天。在35S:miR172a过表达株系中miR172的表达水平明显上升,其靶基因SsAP2-like的表达量明显下降;35S:MIM172株系中miR172的积累水平受到抑制,而SsAP2-like的表达量明显上升,表明miR172介导调控SsAP2的表达对大岩桐成花转变具有促进作用。通过改变miR172的表达调控关键靶基因进而改变花期的方法可以作为调节观赏植物开花时间的有效策略。
We investigated the microRNA172(miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA(cDNA) of gloxinia(Sinningia speciosa) APETALA2-like(SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up-or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172 a, whereas the expression pattern of miR172 a was negatively correlated with that of miR156 a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
We investigated the microRNA172(miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA(cDNA) of gloxinia(Sinningia speciosa) APETALA2-like(SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up-or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172 a, whereas the expression pattern of miR172 a was negatively correlated with that of miR156 a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
引文
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Aukerman MJ,Sakai H,2003.Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes.Plant Cell,15(11):2730-2741.https://doi.org/10.1105/tpc.016238
Chen XM,2005.MicroRNA biogenesis and function in plants.FEBS Lett,579(26):5923-5931.https://doi.org/10.1016/j.febslet.2005.07.071
Ferreira ESG,Silva EM,da Silva Azevedo M,2014.MicroRNA156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development.Plant J,78(4):604-618https://doi.org/10.1111/tpj.12493
Franco-Zorrilla JM,Valli A,Todesco M,et al.,2007.Target mimicry provides a new mechanism for regulation of microRNA activity.Nat Genet,39(8):1033-1037.https://doi.org/10.1038/ng2079
Hong YG,Jackson S,2015.Floral induction and flower formation-the role and potential applications of miRNAs.Plant Biotechnol J,13(3):282-292.https://doi.org/10.1111/pbi.12340
Huijser P,Schmid M,2011.The control of developmental phase transitions in plants.Development,138(19):4117-4129.https://doi.org/10.1242/dev.063511
Kramer JA,2001.Omiga?:a PC-based sequence analysis tool.Mol Biotechnol,19(1):97-106.https://doi.org/10.1385/MB:19:1:097
Lee YS,Lee DY,Cho LH,et al.,2014.Rice mi R172 induces flowering by suppressing Os IDS1 and SNB,two AP2genes that negatively regulate expression of Ehd1 and florigens.Rice,7(1):31.https://doi.org/10.1186/s12284-014-0031-4
Li XY,Bian HW,Song DF,et al.,2013.Flowering time control in ornamental gloxinia(Sinningia speciosa)by manipulation of miR159 expression.Ann Bot,111(5):791-799.https://doi.org/10.1093/aob/mct034
Mathieu J,Yant LJ,Mürdter F,et al.,2009.Repression of flowering by the miR172 target SMZ.PLo S Biol,7(7):e1000148.https://doi.org/10.1371/journal.pbio.1000148
Mlotshwa S,Yang ZY,Kim YJ,et al.,2006.Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana.Plant Mol Biol,61(4-5):781-793.https://doi.org/10.1007/s11103-006-0049-0
Morea EGO,da Silva EM,e Silva GFF,et al.,2016.Functional and evolutionary analyses of the miR156 and miR529families in land plants.BMC Plant Biol,16:40.https://doi.org/10.1186/s12870-016-0716-5
Teotia S,Tang GL,2015.To bloom or not to bloom:role of microRNAs in plant flowering.Mol Plant,8(3):359-377.https://doi.org/10.1016/j.molp.2014.12.018
Terzi LC,Simpson GG,2008.Regulation of flowering time by RNA processing.In:Reddy ASN,Golovkin M(Eds.),Nuclear Pre-mRNA Processing in Plants.Springer,Berlin,Heidelberg,p.201-218.https://doi.org/10.1007/978-3-540-76776-3_11
Todesco M,Rubio-Somoza I,Paz-Ares J,et al.,2010.A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.PLoS Genet,6(7):e1001031.https://doi.org/10.1371/journal.pgen.1001031
Varkonyi-Gasic E,Hellens RP,2010.q RT-PCR of small RNAs.In:Kovalchuk I,Zemp FJ(Eds.),Plant Epigenetics:Methods and Protocols.Humana Press,Totowa,p.109-122.https://doi.org/10.1007/978-1-60761-646-7_10
Wang YN,Wang LX,Zou YM,et al.,2014.Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation.Plant Cell,26(12):4782-4801.https://doi.org/10.1105/tpc.114.131607
Wu G,Park MY,Conway SR,et al.,2009.The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis.Cell,138(4):750-759.https://doi.org/10.1016/j.cell.2009.06.031
Xu ML,Hu TQ,Zhao JF,et al.,2016.Developmental functions of miR156-regulated SQUAMOSA PROMOTERBINDING PROTEIN-LIKE(SPL)genes in Arabidopsis thaliana.PLoS Genet,12(8):e1006263.https://doi.org/10.1371/journal.pgen.1006263
Yamaguchi A,Abe M,2012.Regulation of reproductive development by non-coding RNA in Arabidopsis:to flower or not to flower.J Plant Res,125(6):693-704.https://doi.org/10.1007/s10265-012-0513-7
Yang L,Conway SR,Poethig RS,2011.Vegetative phase change is mediated by a leaf-derived signal that represses the transcription of miR156.Development,138(2):245-249.https://doi.org/10.1242/dev.058578
Yant L,Mathieu J,Dinh TT,et al.,2010.Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2.Plant Cell,22(7):2156-2170.https://doi.org/10.1105/tpc.110.075606
Yao JL,Tomes S,Xu J,et al.,2016.How micro RNA172affects fruit growth in different species is dependent on fruit type.Plant Signal Behav,11(4):e1156833.https://doi.org/10.1080/15592324.2016.1156833
Zhang MZ,Ye D,Wang LL,et al.,2008.Overexpression of the cucumber LEAFY homolog CFL and hormone treatments alter flower development in gloxinia(Sinningia speciosa).Plant Mol Biol,67(4):419-427.https://doi.org/10.1007/s11103-008-9330-8
Zhu QH,Helliwell CA,2011.Regulation of flowering time and floral patterning by miR172.J Exp Bot,62(2):487-495.https://doi.org/10.1093/jxb/erq295
Zhu QH,Upadhyaya NM,Gubler F,et al.,2009.Overexpression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice(Oryza sativa).BMC Plant Biol,9:149.https://doi.org/10.1186/1471-2229-9-149
Aukerman MJ,Sakai H,2003.Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes.Plant Cell,15(11):2730-2741.https://doi.org/10.1105/tpc.016238
Chen XM,2005.MicroRNA biogenesis and function in plants.FEBS Lett,579(26):5923-5931.https://doi.org/10.1016/j.febslet.2005.07.071
Ferreira ESG,Silva EM,da Silva Azevedo M,2014.MicroRNA156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development.Plant J,78(4):604-618https://doi.org/10.1111/tpj.12493
Franco-Zorrilla JM,Valli A,Todesco M,et al.,2007.Target mimicry provides a new mechanism for regulation of microRNA activity.Nat Genet,39(8):1033-1037.https://doi.org/10.1038/ng2079
Hong YG,Jackson S,2015.Floral induction and flower formation-the role and potential applications of miRNAs.Plant Biotechnol J,13(3):282-292.https://doi.org/10.1111/pbi.12340
Huijser P,Schmid M,2011.The control of developmental phase transitions in plants.Development,138(19):4117-4129.https://doi.org/10.1242/dev.063511
Kramer JA,2001.Omiga?:a PC-based sequence analysis tool.Mol Biotechnol,19(1):97-106.https://doi.org/10.1385/MB:19:1:097
Lee YS,Lee DY,Cho LH,et al.,2014.Rice mi R172 induces flowering by suppressing Os IDS1 and SNB,two AP2genes that negatively regulate expression of Ehd1 and florigens.Rice,7(1):31.https://doi.org/10.1186/s12284-014-0031-4
Li XY,Bian HW,Song DF,et al.,2013.Flowering time control in ornamental gloxinia(Sinningia speciosa)by manipulation of miR159 expression.Ann Bot,111(5):791-799.https://doi.org/10.1093/aob/mct034
Mathieu J,Yant LJ,Mürdter F,et al.,2009.Repression of flowering by the miR172 target SMZ.PLo S Biol,7(7):e1000148.https://doi.org/10.1371/journal.pbio.1000148
Mlotshwa S,Yang ZY,Kim YJ,et al.,2006.Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana.Plant Mol Biol,61(4-5):781-793.https://doi.org/10.1007/s11103-006-0049-0
Morea EGO,da Silva EM,e Silva GFF,et al.,2016.Functional and evolutionary analyses of the miR156 and miR529families in land plants.BMC Plant Biol,16:40.https://doi.org/10.1186/s12870-016-0716-5
Teotia S,Tang GL,2015.To bloom or not to bloom:role of microRNAs in plant flowering.Mol Plant,8(3):359-377.https://doi.org/10.1016/j.molp.2014.12.018
Terzi LC,Simpson GG,2008.Regulation of flowering time by RNA processing.In:Reddy ASN,Golovkin M(Eds.),Nuclear Pre-mRNA Processing in Plants.Springer,Berlin,Heidelberg,p.201-218.https://doi.org/10.1007/978-3-540-76776-3_11
Todesco M,Rubio-Somoza I,Paz-Ares J,et al.,2010.A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.PLoS Genet,6(7):e1001031.https://doi.org/10.1371/journal.pgen.1001031
Varkonyi-Gasic E,Hellens RP,2010.q RT-PCR of small RNAs.In:Kovalchuk I,Zemp FJ(Eds.),Plant Epigenetics:Methods and Protocols.Humana Press,Totowa,p.109-122.https://doi.org/10.1007/978-1-60761-646-7_10
Wang YN,Wang LX,Zou YM,et al.,2014.Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation.Plant Cell,26(12):4782-4801.https://doi.org/10.1105/tpc.114.131607
Wu G,Park MY,Conway SR,et al.,2009.The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis.Cell,138(4):750-759.https://doi.org/10.1016/j.cell.2009.06.031
Xu ML,Hu TQ,Zhao JF,et al.,2016.Developmental functions of miR156-regulated SQUAMOSA PROMOTERBINDING PROTEIN-LIKE(SPL)genes in Arabidopsis thaliana.PLoS Genet,12(8):e1006263.https://doi.org/10.1371/journal.pgen.1006263
Yamaguchi A,Abe M,2012.Regulation of reproductive development by non-coding RNA in Arabidopsis:to flower or not to flower.J Plant Res,125(6):693-704.https://doi.org/10.1007/s10265-012-0513-7
Yang L,Conway SR,Poethig RS,2011.Vegetative phase change is mediated by a leaf-derived signal that represses the transcription of miR156.Development,138(2):245-249.https://doi.org/10.1242/dev.058578
Yant L,Mathieu J,Dinh TT,et al.,2010.Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2.Plant Cell,22(7):2156-2170.https://doi.org/10.1105/tpc.110.075606
Yao JL,Tomes S,Xu J,et al.,2016.How micro RNA172affects fruit growth in different species is dependent on fruit type.Plant Signal Behav,11(4):e1156833.https://doi.org/10.1080/15592324.2016.1156833
Zhang MZ,Ye D,Wang LL,et al.,2008.Overexpression of the cucumber LEAFY homolog CFL and hormone treatments alter flower development in gloxinia(Sinningia speciosa).Plant Mol Biol,67(4):419-427.https://doi.org/10.1007/s11103-008-9330-8
Zhu QH,Helliwell CA,2011.Regulation of flowering time and floral patterning by miR172.J Exp Bot,62(2):487-495.https://doi.org/10.1093/jxb/erq295
Zhu QH,Upadhyaya NM,Gubler F,et al.,2009.Overexpression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice(Oryza sativa).BMC Plant Biol,9:149.https://doi.org/10.1186/1471-2229-9-149