玉米蔗糖转运蛋白基因ZmSUT4克隆及其低温胁迫下的表达模式
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摘要
【目的】克隆玉米蔗糖转运蛋白基因ZmSUT4,并明确其组织表达特性及在低温胁迫下的表达模式,为深入研究SUT4基因响应低温胁迫的作用机理提供理论依据。【方法】以玉米自交系黄早四幼苗为材料,采用RT-PCR克隆其ZmSUT4基因,分析其生物学信息,构建系统发育进化树,并采用实时荧光定量PCR(qRT-PCR)检测其组织表达特异性及低温胁迫(4℃)下不同组织中的表达模式。【结果】克隆获得的ZmSUT4基因(GenBank登录号MK541991)全长为1621 bp,开放阅读框(ORF)长度为1506 bp,编码501个氨基酸,编码蛋白的分子量53.36 kD,理论等电点(pI)8.84,具有12个跨膜结构,定位于细胞膜上,既属于易化扩散载体(MFS)家族成员,也属于蔗糖/H+共转运体(GPH)超家族成员,其中第25～447位氨基酸是MFS家族蛋白的保守结构域,第17～484位氨基酸是GPH超家族成员的蔗糖运转子保守结构域GPH-sucrose。ZmSUT4蛋白的氨基酸序列与单子叶植物SUT4蛋白同源性较高,为86%～96%,聚集在同一分支上;与双子叶植物SUT4蛋白同源性较低,为62%～65%,说明该类蛋白在不同物种间高度保守。ZmSUT4基因在玉米的根、茎和叶中均有表达,以根中的表达量最高,其次是叶,茎中的表达量最低。低温胁迫下,ZmSUT4基因在不同组织中表达量模式不同,根和叶中ZmSUT4基因表达量均在胁迫24 h达最大值,分别是低温胁迫前(0 h)表达量的2.21和2.62倍,茎中的ZmSUT4基因表达量在胁迫6 h达最大值,是低温胁迫前表达量的3.01倍。【结论】ZmSUT4基因受低温胁迫诱导表达,推测其是调控玉米响应低温胁迫的关键基因。
【Objective】The sucrose transporter gene ZmSUT4 was cloned from maize genome,its tissue expression characteristic and expression patterns in different tissues under low temperature stress were analyzed to lay the foundation for studying the mechanism of SUT4 gene in response to low temperature stress.【Method】Reverse transcription-PCR(RTPCR)method was performed to clone the ZmSUT4 gene from the maize inbred line Huangzaosi. Its bioinformatics was analyzed and phylogenetic tree was established. Tissue expression specificity of gene ZmSUT4 and its expression patterns in different tissues under low temperature stress(4 ℃)were analyzed by real-time fluorescence quantitative PCR(qRT-PCR).【Result】ZmSUT4 gene was cloned(GenBank accession number MK541991)with full length of 1621 bp and harboured a open reading frame(ORF)of 1506 bp in length that encoded 501 amino acids with a molecular weight of 53.36 kD and an theoretical isoelectric point(pI)of 8.84. The ZmSUT4 protein had 12 trans-membrane spanning domains and located in cytomembrane,belonging to the major facilitator superfamily(MFS)and the sucrose/H+symporter(glycoside-pentosidehexuronide,GPH)family. Among them,the 25 thto the 447 thamino acids belonged to the conservative domain of MFS,while the 17 thto th 484 thamino acids were the conservative domain of GPH-sucrose. Amino acid sequence of Zm SUT4 had high identity(86-96%)with SUTs proteins in monocotyledon plants and belonged to the same branch,while shared low similarity(62%-65%)with SUTs proteins in dicotyledonous plant,illustrating that the amino acid sequences of SUT4 family proteins in different species were highly conservative. The relative expression level of ZmSUT4 could be detected in the leaves,stems and roots during seedling stage. Among them,the expression was the highest in the roots,followed by the leaves,and the lowest in the stems. Furthermore,the expression patterns of ZmSUT4 in various tissues were different. Under 24 h cold treatment,the expression in leaves and roots reached the peak and was as 2.21 times and 2.62 times as that at0 h,respectively. The expression level in stems reached its maximum at 6 h,which was as 3.01 times as that before cold stress(0 h).【Conclusion】The expression of ZmSUT4 is induced by low temperature stress. It is inferred that ZmSUT4 is a crucial gene in maize for responding to low temperature stress.
【Objective】The sucrose transporter gene ZmSUT4 was cloned from maize genome,its tissue expression characteristic and expression patterns in different tissues under low temperature stress were analyzed to lay the foundation for studying the mechanism of SUT4 gene in response to low temperature stress.【Method】Reverse transcription-PCR(RTPCR)method was performed to clone the ZmSUT4 gene from the maize inbred line Huangzaosi. Its bioinformatics was analyzed and phylogenetic tree was established. Tissue expression specificity of gene ZmSUT4 and its expression patterns in different tissues under low temperature stress(4 ℃)were analyzed by real-time fluorescence quantitative PCR(qRT-PCR).【Result】ZmSUT4 gene was cloned(GenBank accession number MK541991)with full length of 1621 bp and harboured a open reading frame(ORF)of 1506 bp in length that encoded 501 amino acids with a molecular weight of 53.36 kD and an theoretical isoelectric point(pI)of 8.84. The ZmSUT4 protein had 12 trans-membrane spanning domains and located in cytomembrane,belonging to the major facilitator superfamily(MFS)and the sucrose/H+symporter(glycoside-pentosidehexuronide,GPH)family. Among them,the 25 thto the 447 thamino acids belonged to the conservative domain of MFS,while the 17 thto th 484 thamino acids were the conservative domain of GPH-sucrose. Amino acid sequence of Zm SUT4 had high identity(86-96%)with SUTs proteins in monocotyledon plants and belonged to the same branch,while shared low similarity(62%-65%)with SUTs proteins in dicotyledonous plant,illustrating that the amino acid sequences of SUT4 family proteins in different species were highly conservative. The relative expression level of ZmSUT4 could be detected in the leaves,stems and roots during seedling stage. Among them,the expression was the highest in the roots,followed by the leaves,and the lowest in the stems. Furthermore,the expression patterns of ZmSUT4 in various tissues were different. Under 24 h cold treatment,the expression in leaves and roots reached the peak and was as 2.21 times and 2.62 times as that at0 h,respectively. The expression level in stems reached its maximum at 6 h,which was as 3.01 times as that before cold stress(0 h).【Conclusion】The expression of ZmSUT4 is induced by low temperature stress. It is inferred that ZmSUT4 is a crucial gene in maize for responding to low temperature stress.
引文
贺红霞,陈亮,康爽,林春晶,李楠,柳青,刘丹,薛晶,孙瑞聪,赵慧敏.2015.玉米ZmSUT4-J基因的克隆与植物表达载体构建[J].吉林农业科学,40(3):18-22.[He H X,Chen L,Kang S,Lin C J,Li N,Liu Q,Liu D,Xue J,Sun R C,Zhao H M.2015.Cloning of sucrose transporter ZmSUT4-J from Zea mays and construction of plant expression vector[J].Journal of Jilin Agricultural Sciences,40(3):18-22.]
李馨园,杨晔,张丽芳,左师宇,李丽杰,焦健,李晶.2017.外源ABA对低温胁迫下玉米幼苗内源激素含量及Asr1基因表达的调节[J].作物学报,43(1):141-148.[Li X Y,Yang Y,Zhang L F,Zuo S Y,Li L J,Jiao J,Li J.2017.Regulation on contents of endogenous hormones and Asr1 gene expression of maize seedling by exogenous ABA under low-temperature stress[J].Acta Agronomica Sinica,43(1):141-148.]
马小龙,刘颖慧,袁祖丽,石云素,宋燕春,王天宇,黎裕.2009.玉米蔗糖转运蛋白基因ZmERD6 cDNAs的克隆与逆境条件下的表达[J].作物学报,35(8):1410-1417.[Ma X L,Liu Y H,Yuan Z L,Shi Y S,Song Y C,Wang T Y,Li Y.2009.Cloning of cDNAs for a novel sugar transporter gene,ZmERD6,from maize and its expression analysis under abiotic stresses[J].Acta Agronomica Sinica,35(8):1410-1417.]
齐素坤,侯夫云,秦桢,李爱贤,王庆美,张立明.2016.植物蔗糖转运蛋白研究进展[J].山东农业科学,48(2):149-153.[Qi S K,Hou F Y,Qin Z,Li A X,Wang Q M,Zhang LM.2016.Research progress of sucrose transporters in plant[J].Shandong Agricultural Sciences,48(2):149-153.]
Aoki N,Hirose T,Furbank R T.2012.Sucrose transport in higher plants:From source to sink[J].Advances in Photosynthesis and Respiration,34:703-729.
Chincinska I A,Liesche J,Krügel U,Michalska J,Geigenberger P,Grimm B,Kühn C.2008.Sucrose transporter StSUT4 from potato affects flowering,tuberization,and shade avoidance response[J].Plant Physiology,146(2):515-528.
Endler A,Meyer S,Schelbert S,Schneider T,Weschke W,Peters S W,Keller F,Baginsky S,Martinoia E,Schmidt UG.2006.Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach[J].Plant Physiology,141(1):196-207.
Eom J S,Cho J I,Reinders A,Lee S W,Yoo Y,Tuan P Q,Choi S B,Bang G,Park Y I,Cho M H,Bhoo S H,An G,Hahn T R,Ward J M,Jeon J S.2011.Impaired function of the tonoplast-localized sucrose transporter in rice,OsSUT2,limits the transport of vacuolar reserve sucrose and affects plant growth[J].Plant Physiology,157(1):109-119.
Frost C J,Nyamdari B,Tsai C J,Harding S A.2012.The tonoplast-localized sucrose transporter in Populus(PtaS-UT4)regulates whole-plant water relations,responses to water stress,and photosynthesis[J].PLoS One,7(8):e44467.
Gong X,Liu M L,Zhang L J,Ruan Y Y,Ding R,Ji Y Q,Zhang N,Zhang S B,Farmer J,Wang C.2015.Arabidopsis AtSUC2 and AtSUC4,encoding sucrose transporters,are required for abiotic stress tolerance in an ABA-dependent pathway[J].Physiologia Plantarum,153(1):119-136.
Gupta A K,Kaur N.2005.Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants[J].Journal of Biosciences,30(5):761-776.
Hackel A,Schauer N,Carrari F,Fernie A R,Grimm B,Kühn C.2006.Sucrose transporter LeSUTl and LeSUT2 inhibition affects tomato fruit development in different ways[J].The Plant Journal,45(2):180-192.
Hirose T,Zhang Z J,Miyao A,Hirochika H,Ohsugi R,Terao T.2010.Disruption of a gene for rice sucrose transporter,OsSUT1,impairs pollen function but pollen maturation is unaffected[J].Journal of Experimental Botany,61(13):3639-3646.
Hofmann J,Wieczorek K,Bl?chl A,Grundler F M.2007.Sucrose supply to nematode-induced syncytia depends on the apoplasmic and symplasmic pathways[J].Journal of Experimental Botany,58(7):1591-601.
Ibraheem O,Dealtry G,Roux S,Bradley G.2011.The effect of drought and salinity on the expressional levels of sucrose transporters in rice(Oryza sativa Nipponbare)cultivar plants[J].Plant Omics Journal,4(2):68-74.
Jia W Q,Zhang L J,Wu D,Liu S,Gong X,Cui Z H,Cui N,Cao H Y,Rao L B,Wang C.2015.Sucrose transporter AtSUC9 mediated by a low sucrose level is involved in Arabidopsis abiotic stress resistance by regulating sucrose distribution and ABA accumulation[J].Plant Cell Physiology,56(8):1574-1587.
Kühn C,Grof C P L.2010.Sucrose transporters of higher plants[J].Current Opinion in Plant Biology,13(3):288-298.
Ma Q J,Sun M H,Liu Y J,Lu J,Hu D G,Hao Y J.2016.Molecular cloning and functional characterization of the apple sucrose transporter gene MdSUT2[J].Plant Physiology and Bochemistry,109:442-451.
Noiraud N,Delrot S,Lemoine R.2000.The sucrose transporter of celery.Identification and expression during salt stress[J].Plant Physiology,122(4):1447-1455.
Reinders A,Schulze W,Kühn C,Barker L,Schulz A,Ward JM,Frommer W B.2002.Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element[J].The Plant Cell,14(7):1567-1577.
Reinders A,Sivitz A B,Starker C G,Gantt J S,Ward J M.2008.Functional analysis of LjSUT4,a vacuolar sucrose transporter from Lotus japonicus[J].Plant Molecular Biology,68(3):289-299.
Rolland N,Ferro M,Seigneurin-Berny D,Garin J,Douce R,Joyard J.2003.Proteomics of chloroplast envelope membranes[J].Photosynthesis Research,78(3):205-230.
Sauer N.2007.Molecular physiology of higher plant sucrose transporters[J].FEBS Letters,581(12):2309-2317.
Schulze W X,Reinders A,Ward J,Lalonde S,Frommer W B.2003.Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system[J].BMCBiochemistry,4(1):1-10.
Sun A J,Xu H L,Gong W K,Zhai H L,Meng K,Wang Y Q,Wei X L,Xiao G F,Zhu Z.2008.Cloning and expression analysis of rice sucrose transporter genes OsSUT2Mand OsSUT5Z[J].Journal of Integrative Plant Biology,50(1):62-75.
Weise A,Barker L,Kühn C,Lalonde S,Buschmann H,Frommer W B,Ward J M.2000.A new subfamily of sucrose transporters,SUT4,with low affinity/high capacity localized in enucleate sieve elements of plants[J].The Plant Cell,12(8):1345-1355.
Xue G P,Mclntyre C L,Glassop D,Shorter R.2008.Use of expression analysis to dissect alterations in carbohydrate metabolism in wheat leaves during drought stress[J].Plant Molecular Biology,67(3):197-214.
Yan N.2013.Structural advances for the major facilitator superfamily(MFS)transporters[J].Trends in Biochemical Sciences,38(3):151-159.
Zanon L,Falchi R,Hackel A,Kühn C,Vizzotto G.2015.Expression of peach sucrose transporters in heterologous systems points out their different physiological role[J].Plant Sciene,238:262-272.
Zhao J F,Sun Z F,Zheng J,Guo X Y,Dong Z G,Huai J L,Gou M Y,He J G,Jin Y S,Wang J H,Wang G Y.2009.Cloning and characterization of a novel CBL-interacting protein kinase from maize[J].Plant Molecular Biology,69(6):661-674.
李馨园,杨晔,张丽芳,左师宇,李丽杰,焦健,李晶.2017.外源ABA对低温胁迫下玉米幼苗内源激素含量及Asr1基因表达的调节[J].作物学报,43(1):141-148.[Li X Y,Yang Y,Zhang L F,Zuo S Y,Li L J,Jiao J,Li J.2017.Regulation on contents of endogenous hormones and Asr1 gene expression of maize seedling by exogenous ABA under low-temperature stress[J].Acta Agronomica Sinica,43(1):141-148.]
马小龙,刘颖慧,袁祖丽,石云素,宋燕春,王天宇,黎裕.2009.玉米蔗糖转运蛋白基因ZmERD6 cDNAs的克隆与逆境条件下的表达[J].作物学报,35(8):1410-1417.[Ma X L,Liu Y H,Yuan Z L,Shi Y S,Song Y C,Wang T Y,Li Y.2009.Cloning of cDNAs for a novel sugar transporter gene,ZmERD6,from maize and its expression analysis under abiotic stresses[J].Acta Agronomica Sinica,35(8):1410-1417.]
齐素坤,侯夫云,秦桢,李爱贤,王庆美,张立明.2016.植物蔗糖转运蛋白研究进展[J].山东农业科学,48(2):149-153.[Qi S K,Hou F Y,Qin Z,Li A X,Wang Q M,Zhang LM.2016.Research progress of sucrose transporters in plant[J].Shandong Agricultural Sciences,48(2):149-153.]
Aoki N,Hirose T,Furbank R T.2012.Sucrose transport in higher plants:From source to sink[J].Advances in Photosynthesis and Respiration,34:703-729.
Chincinska I A,Liesche J,Krügel U,Michalska J,Geigenberger P,Grimm B,Kühn C.2008.Sucrose transporter StSUT4 from potato affects flowering,tuberization,and shade avoidance response[J].Plant Physiology,146(2):515-528.
Endler A,Meyer S,Schelbert S,Schneider T,Weschke W,Peters S W,Keller F,Baginsky S,Martinoia E,Schmidt UG.2006.Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach[J].Plant Physiology,141(1):196-207.
Eom J S,Cho J I,Reinders A,Lee S W,Yoo Y,Tuan P Q,Choi S B,Bang G,Park Y I,Cho M H,Bhoo S H,An G,Hahn T R,Ward J M,Jeon J S.2011.Impaired function of the tonoplast-localized sucrose transporter in rice,OsSUT2,limits the transport of vacuolar reserve sucrose and affects plant growth[J].Plant Physiology,157(1):109-119.
Frost C J,Nyamdari B,Tsai C J,Harding S A.2012.The tonoplast-localized sucrose transporter in Populus(PtaS-UT4)regulates whole-plant water relations,responses to water stress,and photosynthesis[J].PLoS One,7(8):e44467.
Gong X,Liu M L,Zhang L J,Ruan Y Y,Ding R,Ji Y Q,Zhang N,Zhang S B,Farmer J,Wang C.2015.Arabidopsis AtSUC2 and AtSUC4,encoding sucrose transporters,are required for abiotic stress tolerance in an ABA-dependent pathway[J].Physiologia Plantarum,153(1):119-136.
Gupta A K,Kaur N.2005.Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants[J].Journal of Biosciences,30(5):761-776.
Hackel A,Schauer N,Carrari F,Fernie A R,Grimm B,Kühn C.2006.Sucrose transporter LeSUTl and LeSUT2 inhibition affects tomato fruit development in different ways[J].The Plant Journal,45(2):180-192.
Hirose T,Zhang Z J,Miyao A,Hirochika H,Ohsugi R,Terao T.2010.Disruption of a gene for rice sucrose transporter,OsSUT1,impairs pollen function but pollen maturation is unaffected[J].Journal of Experimental Botany,61(13):3639-3646.
Hofmann J,Wieczorek K,Bl?chl A,Grundler F M.2007.Sucrose supply to nematode-induced syncytia depends on the apoplasmic and symplasmic pathways[J].Journal of Experimental Botany,58(7):1591-601.
Ibraheem O,Dealtry G,Roux S,Bradley G.2011.The effect of drought and salinity on the expressional levels of sucrose transporters in rice(Oryza sativa Nipponbare)cultivar plants[J].Plant Omics Journal,4(2):68-74.
Jia W Q,Zhang L J,Wu D,Liu S,Gong X,Cui Z H,Cui N,Cao H Y,Rao L B,Wang C.2015.Sucrose transporter AtSUC9 mediated by a low sucrose level is involved in Arabidopsis abiotic stress resistance by regulating sucrose distribution and ABA accumulation[J].Plant Cell Physiology,56(8):1574-1587.
Kühn C,Grof C P L.2010.Sucrose transporters of higher plants[J].Current Opinion in Plant Biology,13(3):288-298.
Ma Q J,Sun M H,Liu Y J,Lu J,Hu D G,Hao Y J.2016.Molecular cloning and functional characterization of the apple sucrose transporter gene MdSUT2[J].Plant Physiology and Bochemistry,109:442-451.
Noiraud N,Delrot S,Lemoine R.2000.The sucrose transporter of celery.Identification and expression during salt stress[J].Plant Physiology,122(4):1447-1455.
Reinders A,Schulze W,Kühn C,Barker L,Schulz A,Ward JM,Frommer W B.2002.Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element[J].The Plant Cell,14(7):1567-1577.
Reinders A,Sivitz A B,Starker C G,Gantt J S,Ward J M.2008.Functional analysis of LjSUT4,a vacuolar sucrose transporter from Lotus japonicus[J].Plant Molecular Biology,68(3):289-299.
Rolland N,Ferro M,Seigneurin-Berny D,Garin J,Douce R,Joyard J.2003.Proteomics of chloroplast envelope membranes[J].Photosynthesis Research,78(3):205-230.
Sauer N.2007.Molecular physiology of higher plant sucrose transporters[J].FEBS Letters,581(12):2309-2317.
Schulze W X,Reinders A,Ward J,Lalonde S,Frommer W B.2003.Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system[J].BMCBiochemistry,4(1):1-10.
Sun A J,Xu H L,Gong W K,Zhai H L,Meng K,Wang Y Q,Wei X L,Xiao G F,Zhu Z.2008.Cloning and expression analysis of rice sucrose transporter genes OsSUT2Mand OsSUT5Z[J].Journal of Integrative Plant Biology,50(1):62-75.
Weise A,Barker L,Kühn C,Lalonde S,Buschmann H,Frommer W B,Ward J M.2000.A new subfamily of sucrose transporters,SUT4,with low affinity/high capacity localized in enucleate sieve elements of plants[J].The Plant Cell,12(8):1345-1355.
Xue G P,Mclntyre C L,Glassop D,Shorter R.2008.Use of expression analysis to dissect alterations in carbohydrate metabolism in wheat leaves during drought stress[J].Plant Molecular Biology,67(3):197-214.
Yan N.2013.Structural advances for the major facilitator superfamily(MFS)transporters[J].Trends in Biochemical Sciences,38(3):151-159.
Zanon L,Falchi R,Hackel A,Kühn C,Vizzotto G.2015.Expression of peach sucrose transporters in heterologous systems points out their different physiological role[J].Plant Sciene,238:262-272.
Zhao J F,Sun Z F,Zheng J,Guo X Y,Dong Z G,Huai J L,Gou M Y,He J G,Jin Y S,Wang J H,Wang G Y.2009.Cloning and characterization of a novel CBL-interacting protein kinase from maize[J].Plant Molecular Biology,69(6):661-674.
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