黄瓜microRNA171的克隆与功能分析
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摘要
为了解microRNA171(miR171)对黄瓜的调控作用,从华北型黄瓜‘北京截头’中克隆了Csa-miR171家族的4个miRNA前体序列Csa-miR171a、Csa-miR171b、Csa-miR171c、Csa-miR171d,对前体序列进行比对与进化分析,预测成熟序列的靶基因并进一步开展了拟南芥遗传转化功能研究。结果表明,4个前体共剪切出两种成熟序列Csa-miR171a和Csa-miR171b,系统进化显示,Csa-miR171a、Csa-miR171b、Csa-miR171c、Csa-miR171d分别与甜瓜Cme-miR171e、Cme-miR171c、Cme-miR171a、Cme-miR171b同源性最近,与拟南芥、水稻、番茄等较远;Csa-miR171a、Csa-miR171b成熟序列主要靶向GRAS转录因子蛋白家族基因,拟南芥中过表达Csa-miR171a前体改变了叶片形态、颜色、抽薹时间、根、果实、花器官形态等多个表型性状。
To investigate the function of microRNA171(miR171)in cucumber,four precursors Csa-miR171a,Csa-miR171b,Csa-miR171c,Csa-miR171d of Csa-miR171 family genes were cloned in the northern Chinese cucumber‘Beijingjietou'and followed by sequence alignment and phylogenetic analysis.Target genes of the mature sequence of Csa-miR171 were predicted and functional analysis was performed in Arabidopsis by genetic transformation. The results showed that the two mature sequences,Csa-miR171a and Csa-miR171b were cleaved from the four Csa-miR171 precursors. Phylogenetic analysis showed that Csa-miR171a,Csa-miR171 b,Csa-miR171c,Csa-miR171d were closer to Cme-miR171e,Cme-miR171c,Cme-miR171a, Cme-miR171b in melon than those in Arabidopsis, rice and tomato, etc. GRAS transcription factor family was predicted to be the main target gene of Csa-miR171. Overexpression of Csa-miR171a in Arabidopsis significantly affected the phenotypes including leaf morphology and leaf color,blooming time,root,fruit and floral morphology.
To investigate the function of microRNA171(miR171)in cucumber,four precursors Csa-miR171a,Csa-miR171b,Csa-miR171c,Csa-miR171d of Csa-miR171 family genes were cloned in the northern Chinese cucumber‘Beijingjietou'and followed by sequence alignment and phylogenetic analysis.Target genes of the mature sequence of Csa-miR171 were predicted and functional analysis was performed in Arabidopsis by genetic transformation. The results showed that the two mature sequences,Csa-miR171a and Csa-miR171b were cleaved from the four Csa-miR171 precursors. Phylogenetic analysis showed that Csa-miR171a,Csa-miR171 b,Csa-miR171c,Csa-miR171d were closer to Cme-miR171e,Cme-miR171c,Cme-miR171a, Cme-miR171b in melon than those in Arabidopsis, rice and tomato, etc. GRAS transcription factor family was predicted to be the main target gene of Csa-miR171. Overexpression of Csa-miR171a in Arabidopsis significantly affected the phenotypes including leaf morphology and leaf color,blooming time,root,fruit and floral morphology.
引文
Achard P,Harberd N P.2003.Ethylene regulates Arabidopsis development via the modulation of DELLA protein growth repressor function.Plant Cell,15(12):2816-2825.
Arteagavázquez M,Caballeropérez J,Viellecalzada J P.2006.A family of microRNAs present in plants and animals.Plant Cell,18(12):3355.
Axtell M J,Bartel D P.2005.Antiquity of microRNAs and their targets in land plants.Plant Cell,17(6):1658-1673.
Bartel D P.2004.MicroRNAs.Genomics,biogenesis,mechanism,and function.Cell,116(2):281-297.
Bolle C,Koncz C,Chua N H.2000.PAT1.A new member of the GRAS family is involved in phytochrome a signal transduction.Genes&Development,14(10):1269-1278.
Cerovic Z G,Masdoumier G,Ghozlen N B,Ghozlen N B.2012.A new optical leaf-clip meter for simultaneous non-destructive assessment of leaf chlorophyll and epidermal flavonoids.Physiologia Plantarum,146(3):251.
Curaba J,Talbot M,Li Z,Helliwell C.2013.Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley.BMC Plant Biology,13(1):1-10.
Di Laurenzio L,Wysocka-Diller J,Malamy J E,Pysh L,Helariutta Y,Freshour G,Hahn M G,Feldmann K A,Benfey P N.1996.The SCARECROWgene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.Cell,86(3):423.
Djami-Tchatchou A T,Dubery I A.2015.Lipopolysaccharide perception leads to dynamic alterations in the microtranscriptome of Arabidopsis thaliana cells and leaf tissues.Bmc Plant Biology,15(1):1-19.
Doyle J J,Doyle J L.1987.A rapid DNA isolation procedure for small quantities of leaf tissue.Phytochem Bull,19:11-15.
Fan T,Li X,Yang W,Xia K,Ouyang J,Zhang M.2015.Rice osa-miR171c mediates phase change from vegetative to reproductive development and shoot apical meristem maintenance by repressing four OsHAM transcription factors.PLoS ONE,10(5):e0125833.
Felsenstein J.1985.Confidence limits on phylogenies:an approach using the bootstrap.Evolution,39(4):783-791.
Fu X,Gao X,Liu X.2015.Integration of ethylene and gibberellin signaling//Wen C K.Ethylene in plants.Netherlands:Dordrecht:Springer.
Fu X,Harberd N P.2003.Auxin promotes Arabidopsis root growth by modulating gibberellin response.Nature,421(6924):740-743.
Gong Shu-min,Ding Yan-fei,Zhu Cheng.2015.The role of miRNAs in plant seed development.Genetics,37(6):554-560.(in Chinese)龚淑敏,丁艳菲,朱诚.2015.miRNA在植物种子发育过程中的作用.遗传,37(6):554-560.
Goulas Y,Cerovic Z G,Cartelat A,Moya I.2004.Dualex:a new instrument for field measurements of epidermal ultraviolet absorbance by chlorophyll fluorescence.Appl Opt,43(23):4488-4496.
Hendelman A,Kravchik M,Stav R,Frank W,Arazi T.2016.Tomato HAIRY MERISTEM genes are involved in meristem maintenance and compound leaf morphogenesis.Journal of Experimental Botany,67(21):6187-6200.
Huang W,Peng S,Xian Z,Yang L.2016.Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis.Plant Biotechnology Journal,15(4):472.
Jonesrhoades M W,Bartel D P.2004.Computational identification of plant microRNAs and their targets,including a stress-induced miRNA.Molecular Cell,14(6):787-799.
Kumar S,Stecher G,Tamura K.2016.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets.Molecular Biology&Evolution,33(7):1870.
Lauressergues D,Delaux P M,Formey D,Lelandais-Briere C,Fort S,Cottaz S,Becard G,Niebel A,Roux C,Combier J P.2012.The microRNAmiR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2.Plant Journal,72(3):512-522.
Llave C,Kasschau K D,Rector M A,Carrington J C.2002a.Endogenous and silencing-associated small RNAs in plants.Plant Cell,14(7):1605-1619.
Llave C,Xie Z,Kasschau K D,Carrington J C.2002b.Cleavage of scarecrow-like m RNA targets directed by a class of Arabidopsis mi RNA.Science,297(5589):2053-2056.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
Ma Z,Axtell M J.2010.Arabidopsis lyrata small RNAs:transient MIRNA and small interfering RNA loci within the Arabidopsis genus.Plant Cell,22(4):1090-1103.
Ma Z,Hu X,Cai W,Huang W,Zhou X,Luo Q,Yang H,Wang J,Huang J.2014.Arabidopsis miR171-targeted scarecrow-like proteins bind to GT cis-elements and mediate gibberellin-regulated chlorophyll biosynthesis under light conditions.PLoS Genetics,10(8):e1004519
Millar A A,Waterhouse P M.2005.Plant and animal microRNAs:similarities and differences.Functional&Integrative Genomics,5(3):129-135.
Murray H G,Thompson W F.1980.Rapid isolation of higher weight DNA.Nucleic Acides Research,8:4321.
Rhoades M W,Reinhart B J,Lim L P,Burge C B,Bartel B,Bartel D P.2002.Prediction of plant microRNA targets.Cell,110(4):513-520.
Sabatini S,Heidstra R,Wildwater M,Scheres B.2003.SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem.Genes&Development,17(3):354-358.
Saitou N,Nei M.1987.The neighbor-joining method:a new method for reconstructing phylogenetic trees.Molecular Biology and Evolution,4(4):406-425.
Schumacher K,Schmitt T,Rossberg M,Schmitz G,Theres K.1999.The Lateral suppressor(Ls)gene of tomato encodes a new member of the VHIIDprotein family.Proceedings of the National Academy of Sciences of the United States of America,96(1):290-295.
Stuurman J,J?ggi F,Kuhlemeier C.2002.Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells.Genes&Development,16(17):2213-2218.
Sun T P.2010.Gibberellin-GID1-DELLA:a pivotal regulatory module for plant growth and development.Plant Physiology,154(2):567.
Tang G.2010.Plant microRNAs:an insight into their gene structures and evolution.Seminars in Cell&Developmental Biology,21:782-789.
Tian C,Wan P,Sun S,Li J,Chen M.2004.Genome-side analysis of the GRAS gene family in rice and arabidopsis.Plant Molecular Biology,54(4):519-532.
Wang L,Mai Y X,Zhang Y C,Qian L,Yang H Q.2010.MicroRNA171c-targeted SCL6-II,SCL6-III,and SCL6-IV genes regulate shoot branching in Arabidopsis.Molecular Plant,3(5):794-806.
Wang Yan-fang,Zhao Yan-hong,Zhang Ping,Zhou Rui-lian.2015a.Analysis of the evolution and function of soybean miR-171 gene family.Plant Genetic Resources,16(5):1089-1092.(in Chinese)王艳芳,赵彦宏,张萍,周瑞莲.2015a.大豆miR-171基因家族的进化与功能分析.植物遗传资源学报,16(5):1089-1092.
Wang Yan-fang,Zhou Rui-lian,Zhao Yan-hong.2015b.Evolutionary analysis of miR-171 gene family and prediction of target genes.Life Science Research,19(6):479-483.(in Chinese)王艳芳,周瑞莲,赵彦宏.2015b.miR-171基因家族进化分析及靶基因预测.生命科学研究,19(6):479-483.
Wei Qiang,Liang Yong-hong,Li Guang-lin.2013.Evolution of plant miRNAs.Hereditas,35(3):315-323.(in Chinese)魏强,梁永宏,李广林.2013.植物miRNA的进化.遗传,35(3):315-323.
Yang M,Yang Q,Fu T,Zhou Y.2011.Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance.Plant Cell Reports,30(3):373-388.
Yue Chuan,Zeng Jian-ming,Cao Hong-li,Wang Xin-chao,Zhang Zhi-fang.2012.Metabolism of gibberellin in higher plants and its signal transduction pathway.Journal of Plant Physiology,48(2):118-128.(in Chinese)岳川,曾建明,曹红利,王新超,章志芳.2012.高等植物赤霉素代谢及其信号转导通路.植物生理学报,48(2):118-128.
Zamore P D,Haley B.2005.Ribo-gnome:The big world of small rRNAs.Science,309(5740):1519-1524.
Zhang B,Pan X,Cannon C H,Cobb G P,Anderson T A.2006.Conservation and divergence of plant microRNA genes.Plant Journal,46(2):243-259.
Zhu X,Leng X,Sun X,Mu Q,Wang B,Li X,Wang C,Fang J.2015.Discovery of conservation and diversification of genes by phylogenetic analysis based on global genomes.The Plant Genome,8(2):1-11.
Arteagavázquez M,Caballeropérez J,Viellecalzada J P.2006.A family of microRNAs present in plants and animals.Plant Cell,18(12):3355.
Axtell M J,Bartel D P.2005.Antiquity of microRNAs and their targets in land plants.Plant Cell,17(6):1658-1673.
Bartel D P.2004.MicroRNAs.Genomics,biogenesis,mechanism,and function.Cell,116(2):281-297.
Bolle C,Koncz C,Chua N H.2000.PAT1.A new member of the GRAS family is involved in phytochrome a signal transduction.Genes&Development,14(10):1269-1278.
Cerovic Z G,Masdoumier G,Ghozlen N B,Ghozlen N B.2012.A new optical leaf-clip meter for simultaneous non-destructive assessment of leaf chlorophyll and epidermal flavonoids.Physiologia Plantarum,146(3):251.
Curaba J,Talbot M,Li Z,Helliwell C.2013.Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley.BMC Plant Biology,13(1):1-10.
Di Laurenzio L,Wysocka-Diller J,Malamy J E,Pysh L,Helariutta Y,Freshour G,Hahn M G,Feldmann K A,Benfey P N.1996.The SCARECROWgene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.Cell,86(3):423.
Djami-Tchatchou A T,Dubery I A.2015.Lipopolysaccharide perception leads to dynamic alterations in the microtranscriptome of Arabidopsis thaliana cells and leaf tissues.Bmc Plant Biology,15(1):1-19.
Doyle J J,Doyle J L.1987.A rapid DNA isolation procedure for small quantities of leaf tissue.Phytochem Bull,19:11-15.
Fan T,Li X,Yang W,Xia K,Ouyang J,Zhang M.2015.Rice osa-miR171c mediates phase change from vegetative to reproductive development and shoot apical meristem maintenance by repressing four OsHAM transcription factors.PLoS ONE,10(5):e0125833.
Felsenstein J.1985.Confidence limits on phylogenies:an approach using the bootstrap.Evolution,39(4):783-791.
Fu X,Gao X,Liu X.2015.Integration of ethylene and gibberellin signaling//Wen C K.Ethylene in plants.Netherlands:Dordrecht:Springer.
Fu X,Harberd N P.2003.Auxin promotes Arabidopsis root growth by modulating gibberellin response.Nature,421(6924):740-743.
Gong Shu-min,Ding Yan-fei,Zhu Cheng.2015.The role of miRNAs in plant seed development.Genetics,37(6):554-560.(in Chinese)龚淑敏,丁艳菲,朱诚.2015.miRNA在植物种子发育过程中的作用.遗传,37(6):554-560.
Goulas Y,Cerovic Z G,Cartelat A,Moya I.2004.Dualex:a new instrument for field measurements of epidermal ultraviolet absorbance by chlorophyll fluorescence.Appl Opt,43(23):4488-4496.
Hendelman A,Kravchik M,Stav R,Frank W,Arazi T.2016.Tomato HAIRY MERISTEM genes are involved in meristem maintenance and compound leaf morphogenesis.Journal of Experimental Botany,67(21):6187-6200.
Huang W,Peng S,Xian Z,Yang L.2016.Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis.Plant Biotechnology Journal,15(4):472.
Jonesrhoades M W,Bartel D P.2004.Computational identification of plant microRNAs and their targets,including a stress-induced miRNA.Molecular Cell,14(6):787-799.
Kumar S,Stecher G,Tamura K.2016.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets.Molecular Biology&Evolution,33(7):1870.
Lauressergues D,Delaux P M,Formey D,Lelandais-Briere C,Fort S,Cottaz S,Becard G,Niebel A,Roux C,Combier J P.2012.The microRNAmiR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2.Plant Journal,72(3):512-522.
Llave C,Kasschau K D,Rector M A,Carrington J C.2002a.Endogenous and silencing-associated small RNAs in plants.Plant Cell,14(7):1605-1619.
Llave C,Xie Z,Kasschau K D,Carrington J C.2002b.Cleavage of scarecrow-like m RNA targets directed by a class of Arabidopsis mi RNA.Science,297(5589):2053-2056.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
Ma Z,Axtell M J.2010.Arabidopsis lyrata small RNAs:transient MIRNA and small interfering RNA loci within the Arabidopsis genus.Plant Cell,22(4):1090-1103.
Ma Z,Hu X,Cai W,Huang W,Zhou X,Luo Q,Yang H,Wang J,Huang J.2014.Arabidopsis miR171-targeted scarecrow-like proteins bind to GT cis-elements and mediate gibberellin-regulated chlorophyll biosynthesis under light conditions.PLoS Genetics,10(8):e1004519
Millar A A,Waterhouse P M.2005.Plant and animal microRNAs:similarities and differences.Functional&Integrative Genomics,5(3):129-135.
Murray H G,Thompson W F.1980.Rapid isolation of higher weight DNA.Nucleic Acides Research,8:4321.
Rhoades M W,Reinhart B J,Lim L P,Burge C B,Bartel B,Bartel D P.2002.Prediction of plant microRNA targets.Cell,110(4):513-520.
Sabatini S,Heidstra R,Wildwater M,Scheres B.2003.SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem.Genes&Development,17(3):354-358.
Saitou N,Nei M.1987.The neighbor-joining method:a new method for reconstructing phylogenetic trees.Molecular Biology and Evolution,4(4):406-425.
Schumacher K,Schmitt T,Rossberg M,Schmitz G,Theres K.1999.The Lateral suppressor(Ls)gene of tomato encodes a new member of the VHIIDprotein family.Proceedings of the National Academy of Sciences of the United States of America,96(1):290-295.
Stuurman J,J?ggi F,Kuhlemeier C.2002.Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells.Genes&Development,16(17):2213-2218.
Sun T P.2010.Gibberellin-GID1-DELLA:a pivotal regulatory module for plant growth and development.Plant Physiology,154(2):567.
Tang G.2010.Plant microRNAs:an insight into their gene structures and evolution.Seminars in Cell&Developmental Biology,21:782-789.
Tian C,Wan P,Sun S,Li J,Chen M.2004.Genome-side analysis of the GRAS gene family in rice and arabidopsis.Plant Molecular Biology,54(4):519-532.
Wang L,Mai Y X,Zhang Y C,Qian L,Yang H Q.2010.MicroRNA171c-targeted SCL6-II,SCL6-III,and SCL6-IV genes regulate shoot branching in Arabidopsis.Molecular Plant,3(5):794-806.
Wang Yan-fang,Zhao Yan-hong,Zhang Ping,Zhou Rui-lian.2015a.Analysis of the evolution and function of soybean miR-171 gene family.Plant Genetic Resources,16(5):1089-1092.(in Chinese)王艳芳,赵彦宏,张萍,周瑞莲.2015a.大豆miR-171基因家族的进化与功能分析.植物遗传资源学报,16(5):1089-1092.
Wang Yan-fang,Zhou Rui-lian,Zhao Yan-hong.2015b.Evolutionary analysis of miR-171 gene family and prediction of target genes.Life Science Research,19(6):479-483.(in Chinese)王艳芳,周瑞莲,赵彦宏.2015b.miR-171基因家族进化分析及靶基因预测.生命科学研究,19(6):479-483.
Wei Qiang,Liang Yong-hong,Li Guang-lin.2013.Evolution of plant miRNAs.Hereditas,35(3):315-323.(in Chinese)魏强,梁永宏,李广林.2013.植物miRNA的进化.遗传,35(3):315-323.
Yang M,Yang Q,Fu T,Zhou Y.2011.Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance.Plant Cell Reports,30(3):373-388.
Yue Chuan,Zeng Jian-ming,Cao Hong-li,Wang Xin-chao,Zhang Zhi-fang.2012.Metabolism of gibberellin in higher plants and its signal transduction pathway.Journal of Plant Physiology,48(2):118-128.(in Chinese)岳川,曾建明,曹红利,王新超,章志芳.2012.高等植物赤霉素代谢及其信号转导通路.植物生理学报,48(2):118-128.
Zamore P D,Haley B.2005.Ribo-gnome:The big world of small rRNAs.Science,309(5740):1519-1524.
Zhang B,Pan X,Cannon C H,Cobb G P,Anderson T A.2006.Conservation and divergence of plant microRNA genes.Plant Journal,46(2):243-259.
Zhu X,Leng X,Sun X,Mu Q,Wang B,Li X,Wang C,Fang J.2015.Discovery of conservation and diversification of genes by phylogenetic analysis based on global genomes.The Plant Genome,8(2):1-11.
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