基于iTRAQ技术筛选东乡野生稻根系低温响应蛋白
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摘要
为揭示东乡野生稻的耐冷机理,本研究以东乡野生稻为材料,分别进行6℃(低温)和25℃(对照)处理3 d,采用iTRAQ技术开展东乡野生稻苗期根系的差异蛋白质组分析。结果表明,利用iTRAQ技术共鉴定到146个差异表达蛋白质(p<0.05且差异倍数>1.2或<0.83),其中,上调表达48个,下调表达98个。GO分析表明,差异表达蛋白质主要参与代谢过程、细胞过程、单生物过程和刺激反应等,涉及到细胞、细胞成分、细胞器、细胞器成分和膜等细胞组分,主要发挥结合和催化活性功能。KEGG富集分析显示,差异表达蛋白质主要参与代谢途径和次级代谢产物的生物合成。48个表达上调的蛋白质主要涉及逆境胁迫与信号传导、呼吸作用与能量代谢以及生长发育等功能,可以作为东乡野生稻耐冷性研究的候选蛋白。这些鉴定到的差异蛋白质为进一步阐明东乡野生稻的耐冷机理提供理论依据。
In order to reveal the chilling tolerant mechanism of Dongxiang wild rice, using Dongxiang wild rice as material, differential proteome analysis of Dongxiang wild rice roots in seedling stage was carried out based on iTRAQ technique at 6℃(low temperature) and 25℃(control) for 3 d, respectively. The results indicated that a total of 146 differentially expressed proteins(fold change ratio>1.2 or <0.83, p<0.05) were identified using iTRAQ technique, in which 48 were up-regulated and 98 were down-regulated. GO analysis indicated that the differentially expressed proteins mainly participated in the metabolic process, cellular process, single-organism process and response to stimulus, etc.. They involved in cell, cell part, organelle, organelle part and membrane and other cell components, mainly serving binding and catalytic activity function. KEGG enrichment analysis showed that differentially expressed proteins mainly participated in metabolic pathways and biosynthesis of secondary metabolites. The 48 up-regulated proteins primarily involved functions of adversity stress, signal transduction,respiration, energy metabolism and growth and development, which could be used as candidate proteins of studies on cold tolerance of Dongxiang wild rice. These identified differentially expressed proteins could provide theoretical foundation for further elucidating the chilling tolerant mechanism of Dongxiang wild rice.
In order to reveal the chilling tolerant mechanism of Dongxiang wild rice, using Dongxiang wild rice as material, differential proteome analysis of Dongxiang wild rice roots in seedling stage was carried out based on iTRAQ technique at 6℃(low temperature) and 25℃(control) for 3 d, respectively. The results indicated that a total of 146 differentially expressed proteins(fold change ratio>1.2 or <0.83, p<0.05) were identified using iTRAQ technique, in which 48 were up-regulated and 98 were down-regulated. GO analysis indicated that the differentially expressed proteins mainly participated in the metabolic process, cellular process, single-organism process and response to stimulus, etc.. They involved in cell, cell part, organelle, organelle part and membrane and other cell components, mainly serving binding and catalytic activity function. KEGG enrichment analysis showed that differentially expressed proteins mainly participated in metabolic pathways and biosynthesis of secondary metabolites. The 48 up-regulated proteins primarily involved functions of adversity stress, signal transduction,respiration, energy metabolism and growth and development, which could be used as candidate proteins of studies on cold tolerance of Dongxiang wild rice. These identified differentially expressed proteins could provide theoretical foundation for further elucidating the chilling tolerant mechanism of Dongxiang wild rice.
引文
Brandvold K.R.,and Morimoto R.I.,2015,The chemical biology of molecular chaperones-Implications for modulation of proteostasis,J.Mol.Biol.,427(18):2931-2947
Chen N.,Pan L.J.,Chi X.Y.,Chen M.N.,Wang T.,Wang M.,Yang Z.,Hu D.Q.,Wang D.Y.,and Yu S.L.,2014,Cloning and expression of fructose-1,6-bisphosphate aldolase gene AhFBA1 in peanut(Arachis hypogaea L.),Zuowu Xuebao(Acta Agronomica Sinica),40(5):934-941(陈娜,潘丽娟,迟晓元,陈明娜,王通,王冕,杨珍,胡冬青,王道远,禹山林,2014,花生果糖-1,6-二磷酸醛缩酶基因AhFBA1克隆与表达,作物学报,40(5):934-941)
Chen S.N.,Zou X.J.,and Liang B.,1997,Electronmicroscope observation on membrane system of leave cells of some varieties of rice seedlings with different cold-resistance,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),33(3):191-194(陈善娜,邹晓菊,梁斌,1997,水稻不同抗冷品种幼苗叶细胞膜系统的电镜观察,植物生理学通讯,33(3):191-194)
Colás N.A.,García A.P.,Puig S.,and Pe觡arrubia L.,2010,Deregulated copper transport affects Arabidopsis development especially in the absence of environmental cycles,Plant Physiol.,(153):170-184
Cui S.X.,Huang F.,Wang J.,Ma X.,Cheng Y.S.,and Liu J.Y.,2005,A proteomic analysis of cold stress responses in rice seedling,Proteomics,5(12):3162-3172
Cui X.Y.,Tong S.S.,Fan B.,Wang K.,Hu G.Y.,and Liu X.G.,2014,Cloning and plant expression vector construction of AtAAP1 gene from Arabidopsis thaliana,Jilin Nongye Daxue Xuebao(Journal of Jilin Agricultural University),36(5):564-569(崔喜艳,佟珊珊,范贝,王阔,胡广宇,刘相国,2014,拟南芥AAP1基因的克隆及植物表达载体的构建,吉林农业大学学报,36(5):564-569)
Dahan J.,Tcherkez G.,Macherel D.,Benamar A.,Belcram K.,Quadrado M.,Arnal N.,and Mireau H.,2014,Disruption of the cytochrome coxidase dificient 1 gene leads to cytochrome coxidase depletion and reorchestrated respiratory metabolism in Arabidopsis,Plant Physiol.,(166):1788-1802
Hashimoto M.,and Komatsu S.,2007,Proteomic analysis of rice seedlings during cold stress,Proteomics,7(8):1293-1302
Hu D.D.,Zhang F.,Huang L.Y.,Zhuo D.L.,Zhang F.,Zhou Y.L.,Shi Y.Y.,and Li Z.K.,2015,Stress-activated protein kinase OsSAPK2 involved in regulating resistant response to Xanthomonas oryzae pv.oryzae in rice,Zuowu Xuebao(Acta A-gronomica Sinica),41(8):1191-1200(胡丹丹,张帆,黄立钰,卓大龙,张帆,周永力,石英尧,黎志康,2015,胁迫相关蛋白激酶基因OsSAPK2调控水稻抗白叶枯病反应,作物学报,41(8):1191-1200)
Jin Z.X.,Yang J.,Qian C.R.,Liu H.Y.,Jin X.Y.,and Qiu T.Q.,2005,Effects of temperature during grain filling period on activities of key enzymes for starch synthesis and rice grain quality,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),19(4):377-380(金正勋,杨静,钱春荣,刘海英,金学泳,秋太权,2005,灌浆成熟期温度对水稻籽粒淀粉合成关键酶活性及品质的影响,中国水稻科学,19(4):377-380)
Kaur G.,Singh S.,Singh H.,Chawla M.,Dutta T.,Kaur H.,Bender K.,Snedden W.A.,Kapoor S.,Pareek A.,and Singh P.,2015,Characterization of peptidyl-prolyl cis-trans isomeraseand calmodulin-binding activity of a cytosolic arabidopsis thaliana cyclophilin At Cyp19-3,PLoS One,10(8):e0136692
Komatsu S.,Yamada E.,and Furukawa K.,2009,Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths,Amino Acids,36(1):115-123
Kulik A.,Wawer I.,Krzywinska E.,Bucholc M.,and Dobrowolska G.,2011,SnRK2 protein kinases-key regulators of plant response to abiotic stress,Joumal of Integrative Biology,15(12):859-872
Landry A.P.,Duan X.W.,Huang H.,and Ding H.G.,2011,Iron-sulfur proteins are the major source of protein-bound dinitrosyl iron compleses formed in escherichia coli cells under nitric oxide stress,Free Radic Biol.Med.,50(11):1582-1590
Larkindale J.,and Vierling E.,2008,Core genome responses involved in acclimation to high temperature,Plant Physiol.,146(2):748-761
Lee D.G.,Ahsan N.,Lee S.H.,Kang K.Y.,Lee J.J.,and Lee B.H.,2007,An approach to identify cold-induced low-abundant proteins in rice leaf,C.R.Biologies,330(3):215-225
Li J.L.,Huo Z.G.,Wu L.J.,Zhu Q.H.,and Hu F.,2014,Effects of low temperature on grain yield of rice and its physiological mechanism at the booting stage,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),28(3):277-288(李健陵,霍治国,吴丽姬,朱庆华,胡飞,2014,孕穗期低温对水稻产量的影响及其生理机制,中国水稻科学,28(3):277-288)
McDowell S.C.,Lopez-Marques R.L.,Poulsen L.R.,Palmgren M.G.,and Harper J.F.,2013,Loss of the Arabidopsis thaliana P4-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative,pollen,and ovule development,PLo S One,8(5):e62577
McLoughlin F.,Galvan-Ampudia C.S.,Julkowska M.M.,Caarls L.,Van Der Does D.,Laurière C.,Munnik T.,Haring M.A.,and Testerink C.,2012,The snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress,Plant J.,(72):436-449
Neilson K.A.,Mariani M.,and Haynes P.A.,2011,Quantitative proteomic analysis of cold-responsive proteins in rice,Proteomics,11(9):1696-1706
Peng X.J.,Zhao Y.,Cao J.G.,Zhang W.,Jiang H.Y.,Li X.Y.,Ma Q.,Zhu S.W.,and Cheng B.J.,2012,CCCH-type zinc finger family in maize:genome-wide identification,classification and expression profiling under Abscisic acid and drought treatments,PLoS One,7(7):e40120
Walker A.M.,Sattler S.A.,Regner M.,Jones J.P.,Ralph J.,Vermerris W.,Sattler S.E.,and Kang C.,2016,The strcuture and catalytic mechanism of sorghum bicolor Caffeoyl-CoAO-Methyltransferase,Plant Physiol.,172(1):78-92
Wang L.,Xu Y.Y.,Zhang C.,Ma Q.B.,Joo S.H.,Kim S.K.,Xu Z.H.,and Chong K.,2008,Os LIC,a novel CCCH-type zinc finger protein with transcription activation,mediates rice architecture via brassinosteroids signaling,PLoS One,3(10):e3521
Wormit A.,Trentmann O.,Feifer I.,Lohr C.,Tjaden J.,Meyer S.,Schmidt U.,Martinoia E.,and Neuhaus E.,2006,Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport,The Plant Cell,(18):3476-3490
Wu J.N.,Wang Y.M.,Kim S.T.,Kim S.G.,and Kang K.Y.,2013,Characterization of a newly identified rice chitinase-like protein(OsCLP)homologous to xylanase inhibitor,BMC Biotechnol.,13(4):1-8
Xiao N.,Huang W.N.,Zhang X.X.,Gao Y.,Li A.H.,Dai Y.,Yu L.,Liu G.Q.,Pan C.H.,Li Y.H.,Dai Z.Y.,and Chen J.M.,2014,Fine mapping of qRC10-2,a quantitative trait locus for cold tolerance of rice roots at seedling and mature stages,PLoS One,9(5):e96046
Zhang R.R.,Ma J.,Cai G.Z.,and Sun Y.J.,2012,Effects of low temperature stress during flowering stage on flowering and seed setting of rice in Panxi region,Sichuan province,Zuowu Xuebao(Acta Agronomica Sinica),38(9):1734-1742(张荣萍,马均,蔡光泽,孙永健,2012,开花期低温胁迫对四川攀西稻区水稻开花结实的影响,作物学报,38(9):1734-1742)
Zhang X.,Zhang H.,and Zhang F.C.,2015,Dehydrogenase gene from Halostachys caspica under salt stress,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),35(7):1283-1288(张霞,张桦,张富春,2015,盐胁迫下盐穗木甘油醛-3-磷酸脱氢酶基因的表达与亚细胞定位分析,西北植物学报,35(7):1283-1288)
Zhao X.Q.,Zhang T.,Wang W.S.,Zhang F.,Zhu L.H.,Fu B.Y.,and Li Z.K.,2013,Tiem-course metabolic profiling in rice under low temperature treatment,Zuowu Xuebao(Acta A-gronomica Sinica),39(4):720-726(赵秀琴,张婷,王文生,张帆,朱苓华,傅彬英,黎志康,2013,水稻低温胁迫不同时间的代谢物谱图分析,作物学报,39(4):720-726)
Zhong P.,and Huang H.,2017,Recent progress in the research of cold-inducible RNA-binding protein,Future Sci.OA,3(4):FSO246
Chen N.,Pan L.J.,Chi X.Y.,Chen M.N.,Wang T.,Wang M.,Yang Z.,Hu D.Q.,Wang D.Y.,and Yu S.L.,2014,Cloning and expression of fructose-1,6-bisphosphate aldolase gene AhFBA1 in peanut(Arachis hypogaea L.),Zuowu Xuebao(Acta Agronomica Sinica),40(5):934-941(陈娜,潘丽娟,迟晓元,陈明娜,王通,王冕,杨珍,胡冬青,王道远,禹山林,2014,花生果糖-1,6-二磷酸醛缩酶基因AhFBA1克隆与表达,作物学报,40(5):934-941)
Chen S.N.,Zou X.J.,and Liang B.,1997,Electronmicroscope observation on membrane system of leave cells of some varieties of rice seedlings with different cold-resistance,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),33(3):191-194(陈善娜,邹晓菊,梁斌,1997,水稻不同抗冷品种幼苗叶细胞膜系统的电镜观察,植物生理学通讯,33(3):191-194)
Colás N.A.,García A.P.,Puig S.,and Pe觡arrubia L.,2010,Deregulated copper transport affects Arabidopsis development especially in the absence of environmental cycles,Plant Physiol.,(153):170-184
Cui S.X.,Huang F.,Wang J.,Ma X.,Cheng Y.S.,and Liu J.Y.,2005,A proteomic analysis of cold stress responses in rice seedling,Proteomics,5(12):3162-3172
Cui X.Y.,Tong S.S.,Fan B.,Wang K.,Hu G.Y.,and Liu X.G.,2014,Cloning and plant expression vector construction of AtAAP1 gene from Arabidopsis thaliana,Jilin Nongye Daxue Xuebao(Journal of Jilin Agricultural University),36(5):564-569(崔喜艳,佟珊珊,范贝,王阔,胡广宇,刘相国,2014,拟南芥AAP1基因的克隆及植物表达载体的构建,吉林农业大学学报,36(5):564-569)
Dahan J.,Tcherkez G.,Macherel D.,Benamar A.,Belcram K.,Quadrado M.,Arnal N.,and Mireau H.,2014,Disruption of the cytochrome coxidase dificient 1 gene leads to cytochrome coxidase depletion and reorchestrated respiratory metabolism in Arabidopsis,Plant Physiol.,(166):1788-1802
Hashimoto M.,and Komatsu S.,2007,Proteomic analysis of rice seedlings during cold stress,Proteomics,7(8):1293-1302
Hu D.D.,Zhang F.,Huang L.Y.,Zhuo D.L.,Zhang F.,Zhou Y.L.,Shi Y.Y.,and Li Z.K.,2015,Stress-activated protein kinase OsSAPK2 involved in regulating resistant response to Xanthomonas oryzae pv.oryzae in rice,Zuowu Xuebao(Acta A-gronomica Sinica),41(8):1191-1200(胡丹丹,张帆,黄立钰,卓大龙,张帆,周永力,石英尧,黎志康,2015,胁迫相关蛋白激酶基因OsSAPK2调控水稻抗白叶枯病反应,作物学报,41(8):1191-1200)
Jin Z.X.,Yang J.,Qian C.R.,Liu H.Y.,Jin X.Y.,and Qiu T.Q.,2005,Effects of temperature during grain filling period on activities of key enzymes for starch synthesis and rice grain quality,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),19(4):377-380(金正勋,杨静,钱春荣,刘海英,金学泳,秋太权,2005,灌浆成熟期温度对水稻籽粒淀粉合成关键酶活性及品质的影响,中国水稻科学,19(4):377-380)
Kaur G.,Singh S.,Singh H.,Chawla M.,Dutta T.,Kaur H.,Bender K.,Snedden W.A.,Kapoor S.,Pareek A.,and Singh P.,2015,Characterization of peptidyl-prolyl cis-trans isomeraseand calmodulin-binding activity of a cytosolic arabidopsis thaliana cyclophilin At Cyp19-3,PLoS One,10(8):e0136692
Komatsu S.,Yamada E.,and Furukawa K.,2009,Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths,Amino Acids,36(1):115-123
Kulik A.,Wawer I.,Krzywinska E.,Bucholc M.,and Dobrowolska G.,2011,SnRK2 protein kinases-key regulators of plant response to abiotic stress,Joumal of Integrative Biology,15(12):859-872
Landry A.P.,Duan X.W.,Huang H.,and Ding H.G.,2011,Iron-sulfur proteins are the major source of protein-bound dinitrosyl iron compleses formed in escherichia coli cells under nitric oxide stress,Free Radic Biol.Med.,50(11):1582-1590
Larkindale J.,and Vierling E.,2008,Core genome responses involved in acclimation to high temperature,Plant Physiol.,146(2):748-761
Lee D.G.,Ahsan N.,Lee S.H.,Kang K.Y.,Lee J.J.,and Lee B.H.,2007,An approach to identify cold-induced low-abundant proteins in rice leaf,C.R.Biologies,330(3):215-225
Li J.L.,Huo Z.G.,Wu L.J.,Zhu Q.H.,and Hu F.,2014,Effects of low temperature on grain yield of rice and its physiological mechanism at the booting stage,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),28(3):277-288(李健陵,霍治国,吴丽姬,朱庆华,胡飞,2014,孕穗期低温对水稻产量的影响及其生理机制,中国水稻科学,28(3):277-288)
McDowell S.C.,Lopez-Marques R.L.,Poulsen L.R.,Palmgren M.G.,and Harper J.F.,2013,Loss of the Arabidopsis thaliana P4-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative,pollen,and ovule development,PLo S One,8(5):e62577
McLoughlin F.,Galvan-Ampudia C.S.,Julkowska M.M.,Caarls L.,Van Der Does D.,Laurière C.,Munnik T.,Haring M.A.,and Testerink C.,2012,The snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress,Plant J.,(72):436-449
Neilson K.A.,Mariani M.,and Haynes P.A.,2011,Quantitative proteomic analysis of cold-responsive proteins in rice,Proteomics,11(9):1696-1706
Peng X.J.,Zhao Y.,Cao J.G.,Zhang W.,Jiang H.Y.,Li X.Y.,Ma Q.,Zhu S.W.,and Cheng B.J.,2012,CCCH-type zinc finger family in maize:genome-wide identification,classification and expression profiling under Abscisic acid and drought treatments,PLoS One,7(7):e40120
Walker A.M.,Sattler S.A.,Regner M.,Jones J.P.,Ralph J.,Vermerris W.,Sattler S.E.,and Kang C.,2016,The strcuture and catalytic mechanism of sorghum bicolor Caffeoyl-CoAO-Methyltransferase,Plant Physiol.,172(1):78-92
Wang L.,Xu Y.Y.,Zhang C.,Ma Q.B.,Joo S.H.,Kim S.K.,Xu Z.H.,and Chong K.,2008,Os LIC,a novel CCCH-type zinc finger protein with transcription activation,mediates rice architecture via brassinosteroids signaling,PLoS One,3(10):e3521
Wormit A.,Trentmann O.,Feifer I.,Lohr C.,Tjaden J.,Meyer S.,Schmidt U.,Martinoia E.,and Neuhaus E.,2006,Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport,The Plant Cell,(18):3476-3490
Wu J.N.,Wang Y.M.,Kim S.T.,Kim S.G.,and Kang K.Y.,2013,Characterization of a newly identified rice chitinase-like protein(OsCLP)homologous to xylanase inhibitor,BMC Biotechnol.,13(4):1-8
Xiao N.,Huang W.N.,Zhang X.X.,Gao Y.,Li A.H.,Dai Y.,Yu L.,Liu G.Q.,Pan C.H.,Li Y.H.,Dai Z.Y.,and Chen J.M.,2014,Fine mapping of qRC10-2,a quantitative trait locus for cold tolerance of rice roots at seedling and mature stages,PLoS One,9(5):e96046
Zhang R.R.,Ma J.,Cai G.Z.,and Sun Y.J.,2012,Effects of low temperature stress during flowering stage on flowering and seed setting of rice in Panxi region,Sichuan province,Zuowu Xuebao(Acta Agronomica Sinica),38(9):1734-1742(张荣萍,马均,蔡光泽,孙永健,2012,开花期低温胁迫对四川攀西稻区水稻开花结实的影响,作物学报,38(9):1734-1742)
Zhang X.,Zhang H.,and Zhang F.C.,2015,Dehydrogenase gene from Halostachys caspica under salt stress,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),35(7):1283-1288(张霞,张桦,张富春,2015,盐胁迫下盐穗木甘油醛-3-磷酸脱氢酶基因的表达与亚细胞定位分析,西北植物学报,35(7):1283-1288)
Zhao X.Q.,Zhang T.,Wang W.S.,Zhang F.,Zhu L.H.,Fu B.Y.,and Li Z.K.,2013,Tiem-course metabolic profiling in rice under low temperature treatment,Zuowu Xuebao(Acta A-gronomica Sinica),39(4):720-726(赵秀琴,张婷,王文生,张帆,朱苓华,傅彬英,黎志康,2013,水稻低温胁迫不同时间的代谢物谱图分析,作物学报,39(4):720-726)
Zhong P.,and Huang H.,2017,Recent progress in the research of cold-inducible RNA-binding protein,Future Sci.OA,3(4):FSO246