水稻OsNHX5基因的亚细胞定位及表达分析
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摘要
采用PCR方法从水稻中克隆出1个Na~+/H~+逆转运蛋白基因OsNHX5,构建了该基因的亚细胞定位表达载体,利用注射烟草法对OsNHX5进行亚细胞定位,利用荧光定量PCR技术研究OsNHX5基因在盐胁迫下的表达模式。结果表明,OsNHX5与拟南芥AtNHX5和AtNHX6的亲缘关系较近,该蛋白定位于细胞内膜系统上;荧光定量PCR结果表明,叶中OsNHX5基因的表达受KCl胁迫诱导上调表达,而根中OsNHX5基因的表达变化不明显。OsNHX5是一个细胞内膜系统上的K~+/H~+逆转运蛋白,可为耐盐水稻的分子育种提供理论依据。
Salt stress suppresses growth and development of rice. Cloning salt tolerant related genes in rice for analysis of their functions might lay a foundation for research in rice salt tolerance mechanism and for salinity resistance breeding. A Na~+/H~+ antiporter gene OsNHX5 was cloned from Oryza sativa by polymerase chain reaction(PCR) method, and its localization expressing vector was constructed and observed through tobacco injection mediated by Agrobacterium tumefaciens.The expression patterns of OsNHX5 under salinity were analyzed by using real-time PCR. Phylogenetic analysis revealed that OsNHX5 was homologous to Arabidopsis AtNHX5 and AtNHX6. The results showed that OsNHX5 protein was localized in the intracellular membranes. The OsNHX5 gene was induced by KCl stress and its expression was upregulated in leaves but was not significantly different in roots under NaCl or KCl stress. Our results illustrated that OsNHX5 was an endosomal K~+/H~+ antiporter, and this finding would give a theoretical basis for molecular breeding of salt tolerant rice in the future.
Salt stress suppresses growth and development of rice. Cloning salt tolerant related genes in rice for analysis of their functions might lay a foundation for research in rice salt tolerance mechanism and for salinity resistance breeding. A Na~+/H~+ antiporter gene OsNHX5 was cloned from Oryza sativa by polymerase chain reaction(PCR) method, and its localization expressing vector was constructed and observed through tobacco injection mediated by Agrobacterium tumefaciens.The expression patterns of OsNHX5 under salinity were analyzed by using real-time PCR. Phylogenetic analysis revealed that OsNHX5 was homologous to Arabidopsis AtNHX5 and AtNHX6. The results showed that OsNHX5 protein was localized in the intracellular membranes. The OsNHX5 gene was induced by KCl stress and its expression was upregulated in leaves but was not significantly different in roots under NaCl or KCl stress. Our results illustrated that OsNHX5 was an endosomal K~+/H~+ antiporter, and this finding would give a theoretical basis for molecular breeding of salt tolerant rice in the future.
引文
[1] APSE M P,BLUMWALD E.Na+ transport in plants [J].FEBS Letters,2007,581(12):2247-2254.
[2] MAATHUIS J M.The role of monovalent cation transporters in plant responses to salinity [J].Journal of Experimental Botany,2006,57:1137-1147.
[3] ZHOU Y,YIN X,DUAN R,et al.SpAHA1 and SpSOS1 coordinate in transgenic yeast to improve salt tolerance [J].PLoS One,2015,10:e0137447.
[4] APSE M P,AHARON G S,SNEDDEN W A,et al.Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiporter in Arabidopsis [J].Science,1999,285(5341):1256-1258.
[5] ZHOU Y,YANG C,HU Y,et al.The novel Na+/H+ antiporter gene SpNHX1 from Sesuvium portulacastrum confers enhanced salt tolerance to transgenic yeast [J].Acta Physiologiae Plantarum,2018,40:61.
[6] RODRIGUEZ-ROSALES M P,GALVEZ F J,HUERTAS R,et al.Plant NHX cation/proton antiporters [J].Plant Signaling Behavior,2009,4:265-276.
[7] BLUMWLAD E,AHARON G S,APSE M P.Sodium transport in plant cells [J].Biochimica et Biophysica Acta,2000,1465(12):140-151.
[8] LEIDI E O,BARRAGAN V,RUBIO L,et al.The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato [J].The Plant Journal,2010,61(3):495-506.
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[11] 徐璐,郭善利,尹海波.Na+/H+逆向转运蛋白与植物耐盐性研究[J].湖北农业科学,2016,55(11):2727-2730.
[12] YOKOI S,QUINTERO F J,CUBERO B.Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporter in the salt stress response [J].The Plant Journal,2002,30(5):529-539.
[13] 邱全胜.拟南芥NHX5和NHX6:离子平衡与蛋白持运输 [J].中国科学:生命科学,2017,47(8):839-846.
[14] BARRAGAN V,LEIDI E O,ANDRES Z,et al.Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis[J].The Plant Cell,2012,24:1127-1142.
[15] 安静,侯蕾,孔祥强,等.AtNHX5基因过量表达对拟南芥耐盐性的影响 [J].西北植物学报,2012,32(6):1106-1111.
[16] 林小洁,李洪清.转拟南芥AtNHX5基因烟草的耐盐性研究 [J].北方园艺,2011,9:123-126.
[17] 李静,刘明,孙晶,等.Na+(K+)/H+转运蛋白NHX基因的研究进展 [J].大豆科学,2011,30(6):1035-1039.
[18] FUKUDA A ,NAKAMURA A ,HARA N ,et al.Molecular and functional analyses of rice NHX-type Na+/H+ antiporter genes[J].Planta,2010,233(1):175-188.
[19] FUKUDA A,NAKAMURA A,Tagin A,et al.Function,intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice[J].Plant Cell Physiology,2004,45:146-159.
[20] 邱生平,周国安,陆驹飞,等.一个新的水稻液泡膜Na+/H+逆向转运蛋白基因的克隆及表达特征 [J].中国水稻科学,2006,20(2):119-124.
[21] LIU S P,ZHENG L Q,Xue Y H,et al.Overexpression of OsVP1 and OsNHX1 increases tolerance to drought and salinity in rice [J].Journal of Plant Biology,2010,53:444-452.
[22] 彭廷,文慧丽,赵亚帆,等.盐、干旱胁迫下水稻相关miRNA的鉴定及表达分析 [J].华北农学报,2018,33(2):20-27.
[23] 卜华虎.玉米Na+/H+质子泵ZmNHX1功能的初步研究 [D].北京:中央民族大学,2011,.
[24] XU Y Y,ZHOU Y,HONG S,et al.Functional characterization of a wheat NHX antiporter gene TaNHX2 that encodes a K+/H+ exchanger[J].PLoS One,2013,8:e78098.
[25] VENEMA K,QUINTERO F J,PARDO J M,et al.The Arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes [J].The Journal of Biological Chemistry,2002,277:2413-2418.
[26] APSE M P,SOTTOSANTO J B,BLUMWALD E.Vacuolar cation/H+ exchange,ion homeostasis,and leaf development are altered in a T-DNA insertional mutant of AtNHX1,the Arabidopsis thaliana vacuolar Na+/H+ antiporter [J].The Plant Journal,2003,36:229-239.
[27] VENEMA K,BELVER A,MARíN-MANZANO M C,et al.A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants [J].The Journal of Biological Chemistry,2003,278:22453-22459.
[2] MAATHUIS J M.The role of monovalent cation transporters in plant responses to salinity [J].Journal of Experimental Botany,2006,57:1137-1147.
[3] ZHOU Y,YIN X,DUAN R,et al.SpAHA1 and SpSOS1 coordinate in transgenic yeast to improve salt tolerance [J].PLoS One,2015,10:e0137447.
[4] APSE M P,AHARON G S,SNEDDEN W A,et al.Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiporter in Arabidopsis [J].Science,1999,285(5341):1256-1258.
[5] ZHOU Y,YANG C,HU Y,et al.The novel Na+/H+ antiporter gene SpNHX1 from Sesuvium portulacastrum confers enhanced salt tolerance to transgenic yeast [J].Acta Physiologiae Plantarum,2018,40:61.
[6] RODRIGUEZ-ROSALES M P,GALVEZ F J,HUERTAS R,et al.Plant NHX cation/proton antiporters [J].Plant Signaling Behavior,2009,4:265-276.
[7] BLUMWLAD E,AHARON G S,APSE M P.Sodium transport in plant cells [J].Biochimica et Biophysica Acta,2000,1465(12):140-151.
[8] LEIDI E O,BARRAGAN V,RUBIO L,et al.The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato [J].The Plant Journal,2010,61(3):495-506.
[9] BRETT C L,DONOWITZ M,Rao R.Evolutionary origins of eukaryotic sodium/proton exchangers [J].American Journal of Physiology-cell Physiology,2005,288:223-239.
[10] PARDO J M,CUBERO B,LEIDI E O,et al.Alkali cation exchangers:roles in cellular homeostasis and stress tolerance [J].Journal of Experiment Botany,2006,57:1181-1199.
[11] 徐璐,郭善利,尹海波.Na+/H+逆向转运蛋白与植物耐盐性研究[J].湖北农业科学,2016,55(11):2727-2730.
[12] YOKOI S,QUINTERO F J,CUBERO B.Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporter in the salt stress response [J].The Plant Journal,2002,30(5):529-539.
[13] 邱全胜.拟南芥NHX5和NHX6:离子平衡与蛋白持运输 [J].中国科学:生命科学,2017,47(8):839-846.
[14] BARRAGAN V,LEIDI E O,ANDRES Z,et al.Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis[J].The Plant Cell,2012,24:1127-1142.
[15] 安静,侯蕾,孔祥强,等.AtNHX5基因过量表达对拟南芥耐盐性的影响 [J].西北植物学报,2012,32(6):1106-1111.
[16] 林小洁,李洪清.转拟南芥AtNHX5基因烟草的耐盐性研究 [J].北方园艺,2011,9:123-126.
[17] 李静,刘明,孙晶,等.Na+(K+)/H+转运蛋白NHX基因的研究进展 [J].大豆科学,2011,30(6):1035-1039.
[18] FUKUDA A ,NAKAMURA A ,HARA N ,et al.Molecular and functional analyses of rice NHX-type Na+/H+ antiporter genes[J].Planta,2010,233(1):175-188.
[19] FUKUDA A,NAKAMURA A,Tagin A,et al.Function,intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice[J].Plant Cell Physiology,2004,45:146-159.
[20] 邱生平,周国安,陆驹飞,等.一个新的水稻液泡膜Na+/H+逆向转运蛋白基因的克隆及表达特征 [J].中国水稻科学,2006,20(2):119-124.
[21] LIU S P,ZHENG L Q,Xue Y H,et al.Overexpression of OsVP1 and OsNHX1 increases tolerance to drought and salinity in rice [J].Journal of Plant Biology,2010,53:444-452.
[22] 彭廷,文慧丽,赵亚帆,等.盐、干旱胁迫下水稻相关miRNA的鉴定及表达分析 [J].华北农学报,2018,33(2):20-27.
[23] 卜华虎.玉米Na+/H+质子泵ZmNHX1功能的初步研究 [D].北京:中央民族大学,2011,.
[24] XU Y Y,ZHOU Y,HONG S,et al.Functional characterization of a wheat NHX antiporter gene TaNHX2 that encodes a K+/H+ exchanger[J].PLoS One,2013,8:e78098.
[25] VENEMA K,QUINTERO F J,PARDO J M,et al.The Arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes [J].The Journal of Biological Chemistry,2002,277:2413-2418.
[26] APSE M P,SOTTOSANTO J B,BLUMWALD E.Vacuolar cation/H+ exchange,ion homeostasis,and leaf development are altered in a T-DNA insertional mutant of AtNHX1,the Arabidopsis thaliana vacuolar Na+/H+ antiporter [J].The Plant Journal,2003,36:229-239.
[27] VENEMA K,BELVER A,MARíN-MANZANO M C,et al.A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants [J].The Journal of Biological Chemistry,2003,278:22453-22459.