水稻转录因子基因OsSHR2的表达特征及其在营养生长中的调控作用
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摘要
【目的】水稻OsSHR2(LOC_Os03g31880)基因为拟南芥At SHR的同源基因,与OsSHR1、OsSCR1和OsSCR2同属于水稻GRAS转录因子家族。已有研究报道,转录因子基因SHR和SCR共同调控植物根系、叶片的发育,并参与各项生命活动。本研究旨在阐明OsSHR2在水稻中的时空表达特征及其在营养生长中的调控作用。【方法】通过生物信息学分析、表达模式分析、萌发动力学分析和水培实验验证该基因的功能。【结果】生物信息学分析发现OsSHR2、OsSHR1、OsSCR1和OsSCR2与拟南芥和其他物种的SHR亚家族和SCR亚家族成员具有很高的序列一致性;表达模式和p OsSHR2::GUS材料染色分析发现,OsSHR2在整个生长发育过程中的根系、叶片、维管组织和生殖器官中表达强烈,并集中在根尖的中柱、侧根原基和叶片及茎维管组织的中心表达,在野生型的地上部和根系中,OsSHR2受缺磷影响下调表达;对获得的OsSHR2的CRISPR-Cas9突变体osshr2进行种子萌发实验和水培实验,发现与野生型相比,osshr2的萌发时间延后,萌发率降低,在正常供磷和缺磷处理下,osshr2的地上部和根系长度显著小于野生型。【结论】OsSHR2在地上部和根系的发育、维管组织形成以及营养与生殖生长中具有重要作用,这为今后OsSHR2在分子育种等领域的应用奠定理论基础。
【Objective】OsSHR2(LOC_Os03 g31880) is a homologous gene of At SHR of Arabidopsis thaliana, which falls into GRAS transcription factor family together with OsSHR1, OsSCR1 and OsSCR2 in rice. It has been reported that the transcription factor genes SHR and SCR regulate the development of roots and leaves, and participate in various life activities. We analyzed the temporal and spatial expression patterns and the way OsSHR2 regulates vegetative growth in rice.【Method】The function of OsSHR2 was verified by bioinformatics analysis, expression pattern analysis, germination kinetic analysis and hydroponic experiments.【Result】 Biomechanical analysis showed that OsSHR2, OsSHR1, OsSCR1 and OsSCR2 had high homology with SHR subfamily and SCR subfamily in Arabidopsis thaliana and other species. The expression pattern analysis by q RT-PCR and p OsSHR2::GUS staining showed that OsSHR2 was strongly expressed in the roots, leaves, vascular tissues and reproductive organs during the whole vegetative and reproductive growth stages, and especially in the stele of the root tip, lateral root primordium and the central of leaf and stem vascular tissue. Additionally, the relative expression of OsSHR2 was down-regulated in Pi-deficiency in shoots and roots of wild type. The seed germination and hydroponic experiment analysis of CRISPR-Cas9 mutant osshr2 showed that seed germination of osshr2 was delayed and with lower germination rate than that of WT, in addition the length of shoot and root of osshr2 were significantly shorter than WT under Pi-sufficient and Pi-deficient conditions.【Conclusion】OsSHR2 plays an important role in the development of shoots and roots, the formation of vascular tissue and various activity in vegetative and reproductive growth, which lays an important theoretical basis for the application of OsSHR2 in molecular breeding.
【Objective】OsSHR2(LOC_Os03 g31880) is a homologous gene of At SHR of Arabidopsis thaliana, which falls into GRAS transcription factor family together with OsSHR1, OsSCR1 and OsSCR2 in rice. It has been reported that the transcription factor genes SHR and SCR regulate the development of roots and leaves, and participate in various life activities. We analyzed the temporal and spatial expression patterns and the way OsSHR2 regulates vegetative growth in rice.【Method】The function of OsSHR2 was verified by bioinformatics analysis, expression pattern analysis, germination kinetic analysis and hydroponic experiments.【Result】 Biomechanical analysis showed that OsSHR2, OsSHR1, OsSCR1 and OsSCR2 had high homology with SHR subfamily and SCR subfamily in Arabidopsis thaliana and other species. The expression pattern analysis by q RT-PCR and p OsSHR2::GUS staining showed that OsSHR2 was strongly expressed in the roots, leaves, vascular tissues and reproductive organs during the whole vegetative and reproductive growth stages, and especially in the stele of the root tip, lateral root primordium and the central of leaf and stem vascular tissue. Additionally, the relative expression of OsSHR2 was down-regulated in Pi-deficiency in shoots and roots of wild type. The seed germination and hydroponic experiment analysis of CRISPR-Cas9 mutant osshr2 showed that seed germination of osshr2 was delayed and with lower germination rate than that of WT, in addition the length of shoot and root of osshr2 were significantly shorter than WT under Pi-sufficient and Pi-deficient conditions.【Conclusion】OsSHR2 plays an important role in the development of shoots and roots, the formation of vascular tissue and various activity in vegetative and reproductive growth, which lays an important theoretical basis for the application of OsSHR2 in molecular breeding.
引文
[1]朱义旺,林雅容,陈亮.我国水稻分子育种研究进展.厦门大学学报,2016,55(5):661-671.Zhu Y W,Lin Y R,Chen L.Research progress of rice molecular breeding in China.J Xiamen Univ,2016,55(5):661-671.(in Chinese with English abstract)
[2]Riechmann J L,Heard J,Martin G,Reuber L,Jiang C Z,Keddie J,Adam L,Pineda O,Ratcliffe O J,Samaha R R,Creelman R,Pilgrim M,Broun P,Zhang J Z,Ghandehari D,Sherman B K,Yu G L.Arabidopsis transcription factors:Genome-wide comparative analysis among eukaryotes.Science,2000,290(5499):2105-2110.
[3]Pysh L D,Wysocka-Diller J W,Christine C,David B,Benfey P N.The GRAS gene family in Arabidopsis:Sequence characterization and basic expression analysis of the SCARECROW-LIKE genes.Plant J Cell&Mol Biol,1999,18(1):111.
[4]Cui H,Levesque M P,Vernoux T,Jung J W,Paquette A J,Gallagher K L,Wang J Y,Blilou I,Scheres B,Benfey P N.An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of endodermis in plants.Science,2007,316(5823):421-425.
[5]Dolan L.SCARECROWs at the Border.Science,2007,316(5823):377-378.
[6]Wu S,Lee C M,Hayashi T,Pricea S,Divolb F,Henryb S,Pauluzzib G,Perinb C,Gallaghera K L.A plausible mechanism,based upon short-root movement,for regulating the number of cortex cell layers in roots.Proc Natl Acad Sci USA,2014,111(45):16184-16189.
[7]Benfey P N,Scheres B.Root development.Curr Biol,2000,10(22):813-815.
[8]Benfey P N,Linstead P J,Roberts K,Schiefelbein J W,Hauser M T,Aeschbacher R A.Root development in Arabidopsis:Four mutants with dramatically altered root morphogenesis.Development,1993,119(1):57-70.
[9]Laurenzio L D,Wysockadiller J,Malamy J E,Pysh L,Helariutta Y,Freshour G,Hahn M G,Feldmann K A,Benfey P N.The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.Cell,1996,86(3):423-433.
[10]倪君.Os IAA23介导的生长素信号胚后维持水稻根静止中心.杭州:浙江大学,2011.Ni J.Os IAA23-mediated auxin signaling defines postembryonic maintenance of QC in primary in rice.Hangzhou:Zhejiang University,2011.(in Chinese with English abstract)
[11]Lim J,Benfey P N.Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems.Discuss Pap,2000,12(8):1307-1318.
[12]Sbabou L,Bucciarelli B,Miller S,Liu J,Berhada F,Filali-Maltouf A,Allan D,Vance C.Molecular analysis of SCARECROW genes expressed in white lupin cluster roots.J Exp Bot,2010,61(5):1351-1363.
[13]Wang J,Anderssongunneras S,Gaboreanu I,Hertzberg M,Tucker M R,Zheng B,Lesniewska J,Mellerowicz E J,Laux T,Sandberg G,Jones B.Reduced expression of the SHORT-ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis.Plo S ONE,2011,6(12):e28878.
[14]Wysockadiller J W,Helariutta Y,Fukaki H,Malamy J E,Benfey P N.Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.Development,2000,127(3):595-603.
[15]霍胜楠.水稻胚胎发生相关基因的表达及其功能鉴定.济南:山东农业大学,2008.Huo S N.Isolation and characterization of rice genes involved in embryo development.Jinan:Shandong Agricultural University,2008.(in Chinese with English abstract)
[16]Cui H,Kong D,Liu X,Hao Y.SCARECROW,SCR-LIKE 23 and SHORT-ROOT control bundle sheath cell fate and function in Arabidopsis thaliana.Plant J Cell&Mol Biol,2014,78(2):319-327.
[17]Gao X R,Wang C L,Cui H C.Identification of bundle sheath cell fate factors provides new tools for C3-to-C4engineering.Plant Signal&Behav,2014,9(6):e29163.
[18]Morikami A.The SCARECROW gene’s role in asymmetric cell divisions in rice plants.Plant J,2003,36(1):45-54.
[19]Lucas M,Swarup R,Paponov I A,Swarup K,Casimiro I,Lake D,Peret B,Zappala S,Mairhofer S,Whitworth M,Wang J H,Ljung K,Marchant A,Sandberg G,Holdsworth M J,Palme K,Pridmore T,Mooney S,Bennett M J.Short-Root regulates primary,lateral,and adventitious root development in Arabidopsis.Plant Physiol,2011,155(1):384-398.
[20]Tian H,Jia Y,Niu T,Yu Q,Ding Z.The key players of the primary root growth and development also function in lateral roots in Arabidopsis.Plant Cell Rep,2014,33(5):745-753.
[21]Goh T,Toyokura K,Wells D M,Swarup K,Yamamoto M,Mimura T,Weijers D,Fukaki H,Laplaze L,Bennett M J,Guyomarc’h S.Quiescent center initiation in the Arabidopsis lateral root primordia is dependent on the SCARECROW transcription factor.Development,1991,143(18):3363.
[22]Lavenus J,Goh T,Guyomarc’h S,Hill K,Lucas M,Vo?U,Kenobi K,Wilson M H,Farcot E,Hagen G,Guilfoyle T J,Fukaki H,Laplaze L,Bennettb M J.Inference of the Arabidopsis lateral root gene regulatory network suggests a bifurcation mechanism that defines primordia flanking and central zones.Plant Cell,2015,27(5):1368-1388.
[23]Bieleski R.Phosphate pools,phosphate transport,and phosphate availability.Ann Rev Plant Physiol,1973,24(1):225-252.
[24]Muchhal U S,Pardo J M,Raghothama K G.Phosphate transporters from the higher plant Arabidopsis thaliana.Proc Natl Acad Sci USA,1996,93(19):10519-105123.
[25]Wang L,Shan L,Ye Z,Li Z,Du X,Liu D.Comparative genetic analysis of Arabidopsis purple acid phosphatases At PAP10,At PAP12,and At PAP26 provides new insights into their roles in plant adaptation to phosphate deprivation.J Integr Plant Biol,2014,56(3):299-314.
[26]Rausch C,Bucher M.Molecular mechanisms of phosphate transport in plants.Planta,2002,216(1):23-37.
[27]Paszkowski U,Kroken S,Roux C,Briggs S P.Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis.Proc Natl Acad Sci USA,2002,99(20):13324-13329.
[28]Liu F,Chang X J,Ye Y,Xie W B,Wu P,Lian X M.Comprehensive sequence and whole-life-cycle expression profile analysis of the phosphate transporter gene family in rice.Mol Plant,2011,4(6):1105-1122.
[29]Zhang F,Sun Y,Pei W,Jain A,Sun R,Cao Y,Wu X N,Jiang T T,Zhang L,Fan X R,Chen A Q,Shen Q R,Xu G H,Sun S B.Involvement of Os Pht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice.Plant J Cell&Mol Biol,2015,82(4):556.
[30]Rubio V,Linhares F,Solano R,Martín A C,Iglesias J,Leyva A,Paz-Ares J.A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae.Genes&Dev,2001,15(16):2122-2133.
[31]Bustos R,Castrillo G,Linhares F,Puga M I,Rubio V,Perez-Perez J,Solano R,Leyva A,Paz-Ares J.A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis.Plo S Genet,2010,6(9):e1001102.
[32]Wu P,Wang X.Role of Os PHR2 on phosphorus homeostasis and root hairs development in rice(Oryza sativa L.).Plant Signal&Behav,2008,3(9):674-675.
[33]Zhou J,Jiao F,Wu Z C,Li Y Y,Wang X M,He X W,Zhong W Q,Wu P.Os PHR2 is involved in phosphatestarvation signaling and excessive phosphate accumulation in shoots of plants.Plant Physiol,2008,146(4):1673-1686.
[34]Raghothama K G,Maggio A,Narasimhan M L,Kononowicz A K,Wang G,D’Urzo M P,Hasegawa P M,Bressanl R A.Tissue-specific activation of the Os Motingene by ABA,C2H4 and Na Cl involves the same promoter region.Plant Mol Biol,1997,34(3):393-402.
[35]Liao H,Rubio G,Yan X,Cao A,Brown K M,Lynch J P.Effect of phosphorus availability on basal root shallowness in common bean.Plant&Soil,2001,232(1):69-79.
[2]Riechmann J L,Heard J,Martin G,Reuber L,Jiang C Z,Keddie J,Adam L,Pineda O,Ratcliffe O J,Samaha R R,Creelman R,Pilgrim M,Broun P,Zhang J Z,Ghandehari D,Sherman B K,Yu G L.Arabidopsis transcription factors:Genome-wide comparative analysis among eukaryotes.Science,2000,290(5499):2105-2110.
[3]Pysh L D,Wysocka-Diller J W,Christine C,David B,Benfey P N.The GRAS gene family in Arabidopsis:Sequence characterization and basic expression analysis of the SCARECROW-LIKE genes.Plant J Cell&Mol Biol,1999,18(1):111.
[4]Cui H,Levesque M P,Vernoux T,Jung J W,Paquette A J,Gallagher K L,Wang J Y,Blilou I,Scheres B,Benfey P N.An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of endodermis in plants.Science,2007,316(5823):421-425.
[5]Dolan L.SCARECROWs at the Border.Science,2007,316(5823):377-378.
[6]Wu S,Lee C M,Hayashi T,Pricea S,Divolb F,Henryb S,Pauluzzib G,Perinb C,Gallaghera K L.A plausible mechanism,based upon short-root movement,for regulating the number of cortex cell layers in roots.Proc Natl Acad Sci USA,2014,111(45):16184-16189.
[7]Benfey P N,Scheres B.Root development.Curr Biol,2000,10(22):813-815.
[8]Benfey P N,Linstead P J,Roberts K,Schiefelbein J W,Hauser M T,Aeschbacher R A.Root development in Arabidopsis:Four mutants with dramatically altered root morphogenesis.Development,1993,119(1):57-70.
[9]Laurenzio L D,Wysockadiller J,Malamy J E,Pysh L,Helariutta Y,Freshour G,Hahn M G,Feldmann K A,Benfey P N.The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.Cell,1996,86(3):423-433.
[10]倪君.Os IAA23介导的生长素信号胚后维持水稻根静止中心.杭州:浙江大学,2011.Ni J.Os IAA23-mediated auxin signaling defines postembryonic maintenance of QC in primary in rice.Hangzhou:Zhejiang University,2011.(in Chinese with English abstract)
[11]Lim J,Benfey P N.Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems.Discuss Pap,2000,12(8):1307-1318.
[12]Sbabou L,Bucciarelli B,Miller S,Liu J,Berhada F,Filali-Maltouf A,Allan D,Vance C.Molecular analysis of SCARECROW genes expressed in white lupin cluster roots.J Exp Bot,2010,61(5):1351-1363.
[13]Wang J,Anderssongunneras S,Gaboreanu I,Hertzberg M,Tucker M R,Zheng B,Lesniewska J,Mellerowicz E J,Laux T,Sandberg G,Jones B.Reduced expression of the SHORT-ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis.Plo S ONE,2011,6(12):e28878.
[14]Wysockadiller J W,Helariutta Y,Fukaki H,Malamy J E,Benfey P N.Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.Development,2000,127(3):595-603.
[15]霍胜楠.水稻胚胎发生相关基因的表达及其功能鉴定.济南:山东农业大学,2008.Huo S N.Isolation and characterization of rice genes involved in embryo development.Jinan:Shandong Agricultural University,2008.(in Chinese with English abstract)
[16]Cui H,Kong D,Liu X,Hao Y.SCARECROW,SCR-LIKE 23 and SHORT-ROOT control bundle sheath cell fate and function in Arabidopsis thaliana.Plant J Cell&Mol Biol,2014,78(2):319-327.
[17]Gao X R,Wang C L,Cui H C.Identification of bundle sheath cell fate factors provides new tools for C3-to-C4engineering.Plant Signal&Behav,2014,9(6):e29163.
[18]Morikami A.The SCARECROW gene’s role in asymmetric cell divisions in rice plants.Plant J,2003,36(1):45-54.
[19]Lucas M,Swarup R,Paponov I A,Swarup K,Casimiro I,Lake D,Peret B,Zappala S,Mairhofer S,Whitworth M,Wang J H,Ljung K,Marchant A,Sandberg G,Holdsworth M J,Palme K,Pridmore T,Mooney S,Bennett M J.Short-Root regulates primary,lateral,and adventitious root development in Arabidopsis.Plant Physiol,2011,155(1):384-398.
[20]Tian H,Jia Y,Niu T,Yu Q,Ding Z.The key players of the primary root growth and development also function in lateral roots in Arabidopsis.Plant Cell Rep,2014,33(5):745-753.
[21]Goh T,Toyokura K,Wells D M,Swarup K,Yamamoto M,Mimura T,Weijers D,Fukaki H,Laplaze L,Bennett M J,Guyomarc’h S.Quiescent center initiation in the Arabidopsis lateral root primordia is dependent on the SCARECROW transcription factor.Development,1991,143(18):3363.
[22]Lavenus J,Goh T,Guyomarc’h S,Hill K,Lucas M,Vo?U,Kenobi K,Wilson M H,Farcot E,Hagen G,Guilfoyle T J,Fukaki H,Laplaze L,Bennettb M J.Inference of the Arabidopsis lateral root gene regulatory network suggests a bifurcation mechanism that defines primordia flanking and central zones.Plant Cell,2015,27(5):1368-1388.
[23]Bieleski R.Phosphate pools,phosphate transport,and phosphate availability.Ann Rev Plant Physiol,1973,24(1):225-252.
[24]Muchhal U S,Pardo J M,Raghothama K G.Phosphate transporters from the higher plant Arabidopsis thaliana.Proc Natl Acad Sci USA,1996,93(19):10519-105123.
[25]Wang L,Shan L,Ye Z,Li Z,Du X,Liu D.Comparative genetic analysis of Arabidopsis purple acid phosphatases At PAP10,At PAP12,and At PAP26 provides new insights into their roles in plant adaptation to phosphate deprivation.J Integr Plant Biol,2014,56(3):299-314.
[26]Rausch C,Bucher M.Molecular mechanisms of phosphate transport in plants.Planta,2002,216(1):23-37.
[27]Paszkowski U,Kroken S,Roux C,Briggs S P.Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis.Proc Natl Acad Sci USA,2002,99(20):13324-13329.
[28]Liu F,Chang X J,Ye Y,Xie W B,Wu P,Lian X M.Comprehensive sequence and whole-life-cycle expression profile analysis of the phosphate transporter gene family in rice.Mol Plant,2011,4(6):1105-1122.
[29]Zhang F,Sun Y,Pei W,Jain A,Sun R,Cao Y,Wu X N,Jiang T T,Zhang L,Fan X R,Chen A Q,Shen Q R,Xu G H,Sun S B.Involvement of Os Pht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice.Plant J Cell&Mol Biol,2015,82(4):556.
[30]Rubio V,Linhares F,Solano R,Martín A C,Iglesias J,Leyva A,Paz-Ares J.A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae.Genes&Dev,2001,15(16):2122-2133.
[31]Bustos R,Castrillo G,Linhares F,Puga M I,Rubio V,Perez-Perez J,Solano R,Leyva A,Paz-Ares J.A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis.Plo S Genet,2010,6(9):e1001102.
[32]Wu P,Wang X.Role of Os PHR2 on phosphorus homeostasis and root hairs development in rice(Oryza sativa L.).Plant Signal&Behav,2008,3(9):674-675.
[33]Zhou J,Jiao F,Wu Z C,Li Y Y,Wang X M,He X W,Zhong W Q,Wu P.Os PHR2 is involved in phosphatestarvation signaling and excessive phosphate accumulation in shoots of plants.Plant Physiol,2008,146(4):1673-1686.
[34]Raghothama K G,Maggio A,Narasimhan M L,Kononowicz A K,Wang G,D’Urzo M P,Hasegawa P M,Bressanl R A.Tissue-specific activation of the Os Motingene by ABA,C2H4 and Na Cl involves the same promoter region.Plant Mol Biol,1997,34(3):393-402.
[35]Liao H,Rubio G,Yan X,Cao A,Brown K M,Lynch J P.Effect of phosphorus availability on basal root shallowness in common bean.Plant&Soil,2001,232(1):69-79.