平邑甜茶生长素响应因子MhARF2基因克隆与表达分析
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摘要
采用RT-PCR技术克隆得到平邑甜茶(Malus hupehensis)生长素响应因子基因MhARF2,该基因开放阅读框(ORF)2 532 bp,编码843个氨基酸残基。基因结构分析表明,MhARF2包含13个内含子和14个外显子;进化分析表明,MhARF2属于ARF2成员,与苹果(Malus domestica)、白梨(Pyrus bretschneideri)、甜樱桃(Prunus avium)ARF2具有较高的同源性;MhARF2含有ARF家族特有的B3 DNA结合域、CTD结构域和Auxin-resp结构域;MhARF2定位于细胞核;MhARF2启动子区域含有光响应元件、生长素应答元件和MeJA响应元件、ABA响应元件等多个顺式作用元件。qRT-PCR结果表明,MhARF2在平邑甜茶叶、根、茎、花和果实中均表达,其中叶片中表达水平最高,根次之。30 mg·L~(-1) IAA处理下,MhARF2在根中的转录水平受到诱导,6 h时最高;在100mmol·L~(-1)NaCl和200 mmol·L~(-1)甘露醇处理下,根系中MhARF2转录水平均随胁迫时间的延长先上升后降低,这说明MhARF2可能参与调控平邑甜茶生长素信号转导以及渗透胁迫响应的过程。
The auxin response factor gene MhARF2 was cloned by RT-PCR technology from Malus hupehensis.The open reading frame(ORF)of MhARF2 was 2 532 bp and encoding a putative protein with 843 amino acids.The gene structure analysis showed that MhARF2 was consisted of 13 introns and 14 exons.Phylogenetic analysis showed that MhARF2 was a member of ARF2,and had high homology with ARF2 sequence of Malus domestica,Pyrus bretschneideri and Prunus avium.MhARF2 contained the B3 DNA-binding domain,CTD domain,and Auxin-resp domain,which were specific to the ARF family.And MhARF2 was localized in the nucleus.MhARF2 promoter region contained multiple cisacting elements,such as light response elements,auxin response elements,MeJA response elements,and ABA response elements.MhARF2 was expressed in leaves,roots,stems,flowers and fruits,and the expression level was the highest in leaves,and followed by roots.Under 30 mg·L~(-1) IAA treatment,the expression level of MhARF2 in the root of M.hupehensis was induced,and the highest level was at 6 h.However,the expression levels of MhARF2 under 100 mmol·L~(-1) NaCl and 200mmol·L~(-1)mannitol treatment were changed with time extension,which were both firstly raised and then dropped.These results indicated that MhARF2 might be involved in the regulation of auxin signal transduction and osmotic stress response in M.hupehensis.
The auxin response factor gene MhARF2 was cloned by RT-PCR technology from Malus hupehensis.The open reading frame(ORF)of MhARF2 was 2 532 bp and encoding a putative protein with 843 amino acids.The gene structure analysis showed that MhARF2 was consisted of 13 introns and 14 exons.Phylogenetic analysis showed that MhARF2 was a member of ARF2,and had high homology with ARF2 sequence of Malus domestica,Pyrus bretschneideri and Prunus avium.MhARF2 contained the B3 DNA-binding domain,CTD domain,and Auxin-resp domain,which were specific to the ARF family.And MhARF2 was localized in the nucleus.MhARF2 promoter region contained multiple cisacting elements,such as light response elements,auxin response elements,MeJA response elements,and ABA response elements.MhARF2 was expressed in leaves,roots,stems,flowers and fruits,and the expression level was the highest in leaves,and followed by roots.Under 30 mg·L~(-1) IAA treatment,the expression level of MhARF2 in the root of M.hupehensis was induced,and the highest level was at 6 h.However,the expression levels of MhARF2 under 100 mmol·L~(-1) NaCl and 200mmol·L~(-1)mannitol treatment were changed with time extension,which were both firstly raised and then dropped.These results indicated that MhARF2 might be involved in the regulation of auxin signal transduction and osmotic stress response in M.hupehensis.
引文
An JP,Song LQ,Zhao LL,et al(2018).Cloning and functional characterization of an auxin response factor gene MdARF5 in apple.Sci Agr Sin,51(7):1345-1352(in Chinese with English abstract)[安建平,宋来庆,赵玲玲等(2018).苹果生长素响应因子MdARF5的克隆与功能鉴定.中国农业科学,51(7):1345-1352]
Bouzroud S,Gouiaa S,Hu N,et al(2018).Auxin Response Factors(ARFs)are potential mediators of auxin action in tomato response to biotic and abiotic stress(Solanum lycopersicum).PLoS One,13(2):e0193517
Chandler JW(2016).Auxin response factors.Plant Cell Environ,39(5):1014-1028
Ellis CM,Nagpal P,Young JC,et al(2005).Auxin response factor1 and auxin response factor 2 regulate senescence and floral organ abscission in Arabidopsis thaliana.Development,132(20):4563-4574
Feng YY,Hou P,Li YN,et al(2012).Bioinformatic analysis and subcellular localization of Solanum lycopersicum ARF2.Prog Biochem Biophys,39(1):51-58(in Chinese with English abstract)[冯媛媛,侯佩,李颖楠等(2012).番茄ARF2蛋白的生物信息学分析与亚细胞定位.生物化学与生物物理进展,39(1):51-58]
Guan C,Wu B,Yu T,et al(2017)Spatial auxin signaling controls leaf flattening in Arabidopsis.Current Biol,27(19):2940-2950
Hu B,Jin J,Guo AY,et al(2015).GSDS 2.0:an upgraded gene feature visualization server.Bioinformatics,31(8):1296-1297
Hughes R,Spielman M,Schruff MC,et al(2008).Yield assessment of integument-led seed growth following targeted repair of auxin response factor 2.Plant Biotechnol J,6(8):758-769
Li HF,Ran K,He P,et al(2015).Genome-wide identification and expression analysis of auxin response factor(ARF)gene family in apple.Plant Physiol J,51(7):1045-1054(in Chinese with English abstract)[李慧峰,冉昆,何平等(2015).苹果生长素响应因子(ARF)基因家族全基因组鉴定及表达分析.植物生理学报,51(7):1045-1054]
Li SB,Xie ZZ,Hu CG,et al(2016)A review of auxin response factors(ARFs)in plants.Front Plant Sci,7:47
Li YL,Gao ZH,Song J,et al(2017).Auxin response factor(ARF)and its functions in plant growth and development.Plant Physiol J,53(10):1842-1858(in Chinese with English abstract)[李艳林,高志红,宋娟等(2017).植物生长素响应因子ARF与生长发育.植物生理学报,53(10):1842-1858]
Lim PO,Lee IC,Kim J,et al(2010).Auxin response factor 2(ARF2)plays a major role in regulating auxin-mediated leaf longevity.J Exp Bot,61(5):1419-1430
Liu GC,Ding ZJ(2018).Auxin regulates plant growth and development by mediating various environmental cues.Chinese Bull Bot,53(1):17-26(in Chinese with English abstract)[刘广超,丁兆军(2018).生长素介导环境信号调控植物的生长发育.植物学报,53(1):17-26]
Liu Z,Miao L,Huo R,et al(2018).ARF2-ARF4 and ARF5are essential for female and male gametophyte development in Arabidopsis.Plant Cell Physiol,59(1):179-189
Ljung K(2013).Auxin metabolism and homeostasis during plant development.Development,140(5):943-950
Luo J,Zhou JJ,Zhang JZ(2018).Aux/IAA gene family in plants:molecular structure,regulation,and function.Int JMol Sci,19(1):259-276
Meng LS,Wang ZB,Yao SQ,et al(2015).The ARF2-ANT-COR15A gene cascade regulates ABA-signaling-mediated resistance of large seeds to drought in Arabidopsis.JCell Sci,128(21):3922-3932
Okushima Y,Mitina I,Quach HL,et al(2005).AUXIN RE-SPONSE FACTOR 2(ARF2):a pleiotropic developmental regulator.Plant J,43(1):29-46
Promchuea S,Zhu Y,Chen Z,et al(2017).ARF2 coordinates with PLETHORAs and PINs to orchestrate ABA-mediated root meristem activity in Arabidopsis.J Integr Plant Biol,59(1):30-43
Ran K,Yang HQ,Sun XL,et al(2016).A rapid and simple protocol for isolation of functional RNA from different tissues of multiple fruit trees.Acta Hortic,1110:163-170
Ren Z,Liu R,Gu W,et al(2017).The Solanum lycopersicum auxin response factor SlARF2 participates in regulating lateral root formation and flower organ senescence.Plant Sci,256:103-111
Roosjen M,Paque S,Weijers D(2017).Auxin response factors:output control in auxin biology.J Exp Bot,69(4):179-188
Schefe JH,Lehmann KE,Buschmann IR,et al(2006).Quantitative real-time RT-PCR data analysis:current concepts and the novel‘gene expression's CT difference’formula.J Mol Med,84:901-910
Schruff MC,Spielman M,Tiwari S,et al(2006).The AUXINRESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling,cell division,and the size of seeds and other organs.Development,133(2):251-261
Sun RW,Liu YJ,Wang X,et al(2018).Bioinformatics identification of auxin response factor genes and expression profiles under drought stress in wheat.J Plant Genet Resour,19(1):122-134(in Chinese with English abstract)[孙仁玮,刘永杰,王翔等(2018).小麦ARF基因家族生物信息学分析及在干旱胁迫下的表达特性研究.植物遗传资源学报,19(1):122-134]
Vert G,Walcher CL,Chory J,et al(2008).Integration of auxin and brassinosteroid pathways by auxin response factor2.Proc Natl Acad Sci USA,105(28):9829-9834
Wang D,Pei K,Fu Y,et al(2007).Genome-wide analysis of the auxin response factors(ARF)gene family in rice(Oryza sativa).Gene,394:13-24
Wang L,Hua D,He J,et al(2011).Auxin Response Factor2(ARF2)and its regulated homeodomain gene HB33 mediate abscisic acid response in Arabidopsis.PLoS Genet,7(7):e1002172
Wang YC,Xu HF,Wang N,et al(2017).Molecular cloning and expression analysis of an auxin signaling related gene MdARF3 in red flesh apple.Acta Hortic Sin,44(4):633-643(in Chinese with English abstract)[王意程,许海峰,王楠等(2017).红肉苹果愈伤组织生长素信号相关基因MdARF3的克隆与表达分析.园艺学报,44(4):633-643]
Yang HQ,Fan WG(2012).Advances in research of apple root system architecture and it’s regulation.Acta Hortic Sin,39(9):1673-1678(in Chinese with English abstract)[杨洪强,范伟国.苹果根系构型及其调控研究进展.园艺学报,39(9):1673-1678]
Zhao S,Zhang ML,Ma TL,et al(2016).Phosphorylation of ARF2 relieves its repression of transcription of the K+transporter gene HAK5 in response to low potassium stress.Plant Cell,28(12):3005-3019
Bouzroud S,Gouiaa S,Hu N,et al(2018).Auxin Response Factors(ARFs)are potential mediators of auxin action in tomato response to biotic and abiotic stress(Solanum lycopersicum).PLoS One,13(2):e0193517
Chandler JW(2016).Auxin response factors.Plant Cell Environ,39(5):1014-1028
Ellis CM,Nagpal P,Young JC,et al(2005).Auxin response factor1 and auxin response factor 2 regulate senescence and floral organ abscission in Arabidopsis thaliana.Development,132(20):4563-4574
Feng YY,Hou P,Li YN,et al(2012).Bioinformatic analysis and subcellular localization of Solanum lycopersicum ARF2.Prog Biochem Biophys,39(1):51-58(in Chinese with English abstract)[冯媛媛,侯佩,李颖楠等(2012).番茄ARF2蛋白的生物信息学分析与亚细胞定位.生物化学与生物物理进展,39(1):51-58]
Guan C,Wu B,Yu T,et al(2017)Spatial auxin signaling controls leaf flattening in Arabidopsis.Current Biol,27(19):2940-2950
Hu B,Jin J,Guo AY,et al(2015).GSDS 2.0:an upgraded gene feature visualization server.Bioinformatics,31(8):1296-1297
Hughes R,Spielman M,Schruff MC,et al(2008).Yield assessment of integument-led seed growth following targeted repair of auxin response factor 2.Plant Biotechnol J,6(8):758-769
Li HF,Ran K,He P,et al(2015).Genome-wide identification and expression analysis of auxin response factor(ARF)gene family in apple.Plant Physiol J,51(7):1045-1054(in Chinese with English abstract)[李慧峰,冉昆,何平等(2015).苹果生长素响应因子(ARF)基因家族全基因组鉴定及表达分析.植物生理学报,51(7):1045-1054]
Li SB,Xie ZZ,Hu CG,et al(2016)A review of auxin response factors(ARFs)in plants.Front Plant Sci,7:47
Li YL,Gao ZH,Song J,et al(2017).Auxin response factor(ARF)and its functions in plant growth and development.Plant Physiol J,53(10):1842-1858(in Chinese with English abstract)[李艳林,高志红,宋娟等(2017).植物生长素响应因子ARF与生长发育.植物生理学报,53(10):1842-1858]
Lim PO,Lee IC,Kim J,et al(2010).Auxin response factor 2(ARF2)plays a major role in regulating auxin-mediated leaf longevity.J Exp Bot,61(5):1419-1430
Liu GC,Ding ZJ(2018).Auxin regulates plant growth and development by mediating various environmental cues.Chinese Bull Bot,53(1):17-26(in Chinese with English abstract)[刘广超,丁兆军(2018).生长素介导环境信号调控植物的生长发育.植物学报,53(1):17-26]
Liu Z,Miao L,Huo R,et al(2018).ARF2-ARF4 and ARF5are essential for female and male gametophyte development in Arabidopsis.Plant Cell Physiol,59(1):179-189
Ljung K(2013).Auxin metabolism and homeostasis during plant development.Development,140(5):943-950
Luo J,Zhou JJ,Zhang JZ(2018).Aux/IAA gene family in plants:molecular structure,regulation,and function.Int JMol Sci,19(1):259-276
Meng LS,Wang ZB,Yao SQ,et al(2015).The ARF2-ANT-COR15A gene cascade regulates ABA-signaling-mediated resistance of large seeds to drought in Arabidopsis.JCell Sci,128(21):3922-3932
Okushima Y,Mitina I,Quach HL,et al(2005).AUXIN RE-SPONSE FACTOR 2(ARF2):a pleiotropic developmental regulator.Plant J,43(1):29-46
Promchuea S,Zhu Y,Chen Z,et al(2017).ARF2 coordinates with PLETHORAs and PINs to orchestrate ABA-mediated root meristem activity in Arabidopsis.J Integr Plant Biol,59(1):30-43
Ran K,Yang HQ,Sun XL,et al(2016).A rapid and simple protocol for isolation of functional RNA from different tissues of multiple fruit trees.Acta Hortic,1110:163-170
Ren Z,Liu R,Gu W,et al(2017).The Solanum lycopersicum auxin response factor SlARF2 participates in regulating lateral root formation and flower organ senescence.Plant Sci,256:103-111
Roosjen M,Paque S,Weijers D(2017).Auxin response factors:output control in auxin biology.J Exp Bot,69(4):179-188
Schefe JH,Lehmann KE,Buschmann IR,et al(2006).Quantitative real-time RT-PCR data analysis:current concepts and the novel‘gene expression's CT difference’formula.J Mol Med,84:901-910
Schruff MC,Spielman M,Tiwari S,et al(2006).The AUXINRESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling,cell division,and the size of seeds and other organs.Development,133(2):251-261
Sun RW,Liu YJ,Wang X,et al(2018).Bioinformatics identification of auxin response factor genes and expression profiles under drought stress in wheat.J Plant Genet Resour,19(1):122-134(in Chinese with English abstract)[孙仁玮,刘永杰,王翔等(2018).小麦ARF基因家族生物信息学分析及在干旱胁迫下的表达特性研究.植物遗传资源学报,19(1):122-134]
Vert G,Walcher CL,Chory J,et al(2008).Integration of auxin and brassinosteroid pathways by auxin response factor2.Proc Natl Acad Sci USA,105(28):9829-9834
Wang D,Pei K,Fu Y,et al(2007).Genome-wide analysis of the auxin response factors(ARF)gene family in rice(Oryza sativa).Gene,394:13-24
Wang L,Hua D,He J,et al(2011).Auxin Response Factor2(ARF2)and its regulated homeodomain gene HB33 mediate abscisic acid response in Arabidopsis.PLoS Genet,7(7):e1002172
Wang YC,Xu HF,Wang N,et al(2017).Molecular cloning and expression analysis of an auxin signaling related gene MdARF3 in red flesh apple.Acta Hortic Sin,44(4):633-643(in Chinese with English abstract)[王意程,许海峰,王楠等(2017).红肉苹果愈伤组织生长素信号相关基因MdARF3的克隆与表达分析.园艺学报,44(4):633-643]
Yang HQ,Fan WG(2012).Advances in research of apple root system architecture and it’s regulation.Acta Hortic Sin,39(9):1673-1678(in Chinese with English abstract)[杨洪强,范伟国.苹果根系构型及其调控研究进展.园艺学报,39(9):1673-1678]
Zhao S,Zhang ML,Ma TL,et al(2016).Phosphorylation of ARF2 relieves its repression of transcription of the K+transporter gene HAK5 in response to low potassium stress.Plant Cell,28(12):3005-3019