盐地碱蓬高亲和性K~+转运蛋白基因SsHAK2的克隆与表达模式分析
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摘要
盐地碱蓬在高盐生境中可以有效吸收K+,并维持细胞内K+浓度的相对稳定。高亲和性K+转运蛋白KT/HAK/KUP家族成员在植物K+吸收过程中发挥重要作用。本研究从盐地碱蓬中克隆到一个KT/HAK/KUP家族成员HAK2的同源基因SsHAK2,并对其进行生物信息学分析和表达模式分析。SsHAK2编码788个氨基酸,与不同植物的HAK2类蛋白具有较高的同源性(80%~92%)。系统进化分析表明,SsHAK2属于KT/HAK/KUP家族亚族Ⅱ成员,与拟南芥AtKUP2位于同一进化分枝。实时定量qPCR分析显示,SsHAK2在盐地碱蓬的根和叶中均有高丰度表达,且叶中的表达丰度显著高于根中。SsHAK2的表达受外界不同浓度K+(2.5和0.01mmol/L)的诱导。2.5mmol/L K+处理下,根和叶中SsHAK2的表达受25mmol/L NaCl的显著诱导;0.01mmol/L K+处理下,25和150mmol/L NaCl的添加抑制根中SsHAK2的表达,却显著促进其在叶中的表达。以上研究结果表明,SsHAK2可能参与盐地碱蓬K+吸收及转运过程,在根和叶中发挥不同的功能。
Suaeda salsa,a typical salt-accumulating halophyte,is capable of absorbing K+with high efficiency,and thus maintains a relatively stable K+level in cells,and grows well,even in highly saline soil.Members of the KT/HAK/KUP gene family have an important role in K+uptake in plants.In this study,we cloned SsHAK2 in S.salsaand analyzed the expression patterns of SsHAK2 when the plants were exposed to different concentrations of KCl and NaCl.Results revealed that SsHAK2 coded for 788 amino acid residues and shared a high homology(80%-92%)with the identified members of KT/HAK/KUP family from other plants.Phylogenetic analysis showed that SsHAK2 belonged to a sub-group of the family known as group II,and formed a clade with AtKUP2 of Arabidopsisthaliana,indicating close relationship.SsHAK2 was highly expressed in roots and leaves,and was induced by widely differing K+concentrations(2.5and 0.01mmol/L).Under 2.5mmol/L K+conditions,the expression of SsHAK2 in roots and leaves was induced by 25mmol/L Na+application.However,in the medium containing 0.01mmol/L K+,the expression of SsHAK2 in roots was down-regulated by Na+(25and 150mmol/L)application,but up-regulated in leaves.The results therefore indicate that SsHAK2 might mediate K+uptake and transport in S.salsa,and functioned differently in roots and leaves.
Suaeda salsa,a typical salt-accumulating halophyte,is capable of absorbing K+with high efficiency,and thus maintains a relatively stable K+level in cells,and grows well,even in highly saline soil.Members of the KT/HAK/KUP gene family have an important role in K+uptake in plants.In this study,we cloned SsHAK2 in S.salsaand analyzed the expression patterns of SsHAK2 when the plants were exposed to different concentrations of KCl and NaCl.Results revealed that SsHAK2 coded for 788 amino acid residues and shared a high homology(80%-92%)with the identified members of KT/HAK/KUP family from other plants.Phylogenetic analysis showed that SsHAK2 belonged to a sub-group of the family known as group II,and formed a clade with AtKUP2 of Arabidopsisthaliana,indicating close relationship.SsHAK2 was highly expressed in roots and leaves,and was induced by widely differing K+concentrations(2.5and 0.01mmol/L).Under 2.5mmol/L K+conditions,the expression of SsHAK2 in roots and leaves was induced by 25mmol/L Na+application.However,in the medium containing 0.01mmol/L K+,the expression of SsHAK2 in roots was down-regulated by Na+(25and 150mmol/L)application,but up-regulated in leaves.The results therefore indicate that SsHAK2 might mediate K+uptake and transport in S.salsa,and functioned differently in roots and leaves.
引文
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[15]Nieves-Cordones M,Alemán F,Martínez V,et al.The Arabidopsis thaliana HAK5K+transporter is required for plant growth and K+acquisition from low K+solutions under saline conditions.Molecular Plant,2010,3(2):326-333.
[16]Wang Q,Guan C,Wang P,et al.AtHKT1;1and AtHAK5mediate low-affinity Na+uptake in Arabidopsis thaliana under mild salt stress.Plant Growth Regulation,2015,75:615-623.
[17]Zhang J L,Flowers T J,Wang S M.Differentiation of low-affinity Na+uptake pathways and kinetics of the effects of K+on Na+uptake in the halophyte Suaeda maritima.Plant and Soil,2012,368(1-2):629-640.
[18]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,2001,25(4):402-408.
[19]Zhang Z,Zhang J,Chen Y,et al.Genome-wide analysis and identification of HAK potassium transporter gene family in maize(Zea mays L.).Molecular Biology Reports,2012,39(8):8465-8473.
[20]Martinez-Cordero M A,Martinez V,Rubio F.Cloning and functional characterization of the high-affinity K+transporter HAK1of pepper.Plant Molecular Biology,2004,56(3):413-421.
[21]Su H,Golldack D,Zhao C S,et al.The expression of HAK-type K+transporters is regulated in response to salinity stress in common ice plant.Plant Physiology,2002,129(4):1482-1493.
[22]Davies C,Shin R,Liu W,et al.Transporters expressed during grape berry(Vitis vinifera L.)development are associated with an increase in berry size and berry potassium accumulation.Journal of Experimental Botany,2006,57(12):3209-3216.
[23]Nieves-Cordones M,Martinez-Cordero M A,Martinez V,et al.An NH4+-sensitive component dominates high-affinity K+uptake in tomato plants.Plant Science,2007,172(2):273-280.
[24]Véry A A,Nieves-Cordones M,Daly M,et al.Molecular biology of K+transport across the plant cell membrane:What do we learn from comparison between plant species?Journal of Plant Physiology,2014,171(9):748-769.
[25]Gupta M,Qiu X,Wang L,et al.KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice(Oryza sativa).Molecular Genetics and Genomics,2008,280(5):437-452.
[26]Gierth M,Mser P.Potassium transporters in plants——involvement in K+acquisition,redistribution and homeostasis.FEBS Letters,2007,581(12):2348-2356.
[27]Ahn S J,Shin R,Schachtman D P.Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+uptake.Plant Physiology,2004,134(3):1135-1145.
[28]Rigas S,Debrosses G,Haralampidis K,et al.TRH1encodes a potassium transporter required for tip growth in Arabidopsis root hairs.Plant Cell,2001,13(1):139-151.
[29]Quintero F J,Blatt M R.A new family of K+transporters fromArabidopsis that are conserved across phyla.FEBS Letters,1997,415(2):206-211.
[30]Fu H H,Luan S.AtKUP1:a dual-affinity K+transporter fromArabidopsis.Plant Cell,1998,10(1):63-73.
[31]Osakabe Y,Arinaga N,Umezawa T,et al.Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis.Plant Cell,2013,25:609-624.
[32]Li P H,Chen M,Wang B S.Effect of K+nutrition on growth and activity of leaf tonoplast V-H+-ATPase and V-H+-PPase of Suaeda salsa under NaCl stress.Acta Botanica Sinica,2002,44(4):433-440.
[33]Rubio F,Santa-Maria G E,Rodriguez-Navarro A.Cloning of Arabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells.Physiologia Plantarum,2000,109(1):34-43.
[4]周向睿,岳利军,王锁民.钠复合肥提高多浆旱生植物霸王幼苗生长及抗旱性.草业学报,2014,23(6):142-147.
[5]种培芳,李航逸,李毅.荒漠植物红砂根系对干旱胁迫的生理响应.草业学报,2015,24(1):72-80.
[6]李莹,柳参奎.碱茅6-磷酸海藻糖合成酶基因的克隆及其耐逆性分析.草业学报,2015,24(1):99-106.
[32]李平华,陈敏,王宝山.K+营养对NaCl胁迫下盐地碱蓬生长及叶片液泡膜V-H+-ATPase和V-H+-PPase活性的影响.植物学报,2002,44(4):433-440.
[2]Maathuis F J.Physiological functions of mineral macronutrients.Current Opinion in Plant Biology,2009,12(3):250-258.
[3]Kronzucker H J,Coskun D,Schulze L M,et al.Sodium as nutrient and toxicant.Plant and Soil,2013,369(1-2):1-23.
[4]Zhou X R,Yue L J,Wang S M.Sodium compound fertilizer improved growth and drought tolerance of Zygophyllum xanthoxylum seedlings under drought stress.Acta Prataculturae Sinica,2014,23(6):142-147.
[5]Chong P F,Li H Y,Li Y.Physiological responses of seedling roots of the desert plant Reaumuria soongorica to drought stress.Acta Prataculturae Sinica,2015,24(1):72-80.
[6]Li Y,Liu S K.Cloing of a TPS gene and analysis of its function in stress tolerance in Puccinellia tenuiflora.Acta Prataculturae Sinica,2015,24(1):99-106.
[7]Kefu Z,Hai F,Ungar I.Survey of halophyte species in China.Plant Science,2002,163(3):491-498.
[8]Song J,Wang B S.Using euhalophytes to understand salt tolerance and to develop saline agriculture:Suaeda salsa as a promising model.Annals of Botany,2015,115(3):541-553.
[9]Wang S M,Zhang J L,Flowers T J.Low-affinity Na+uptake in the halophyte Suaeda maritima.Plant Physiology,2007,145(2):559-571.
[10]Mori S,Suzuki K,Oda R,et al.Characteristics of Na+and K+absorption in Suaeda salsa(L.)Pall.Soil Science and Plant Nutrition,2011,57(3):377-386.
[11]Nieves-Cordones M,Alemán F,Martínez V,et al.K+uptake in plant roots.The systems involved,their regulation and parallels in other organisms.Journal of Plant Physiology,2014,171(9):688-695.
[12]Shao Q,Han N,Ding T,et al.SsHKT1;1is a potassium transporter of the C3halophyte Suaeda salsa that is involved in salt tolerance.Functional Plant Biology,2014,41(8):790-802.
[13]Duan H R,Ma Q,Zhang J L,et al.The inward-rectifying K+channel SsAKT1is a candidate involved in K+uptake in the halophyte Suaeda salsa under saline condition.Plant and Soil,DOI:10.1007/S11104-015-2539-9.
[14]Santa-Maria G E,Rubio F,Dubcovsky J,et al.The HAK1gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter.Plant Cell,1997,9(12):2281-2289.
[15]Nieves-Cordones M,Alemán F,Martínez V,et al.The Arabidopsis thaliana HAK5K+transporter is required for plant growth and K+acquisition from low K+solutions under saline conditions.Molecular Plant,2010,3(2):326-333.
[16]Wang Q,Guan C,Wang P,et al.AtHKT1;1and AtHAK5mediate low-affinity Na+uptake in Arabidopsis thaliana under mild salt stress.Plant Growth Regulation,2015,75:615-623.
[17]Zhang J L,Flowers T J,Wang S M.Differentiation of low-affinity Na+uptake pathways and kinetics of the effects of K+on Na+uptake in the halophyte Suaeda maritima.Plant and Soil,2012,368(1-2):629-640.
[18]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,2001,25(4):402-408.
[19]Zhang Z,Zhang J,Chen Y,et al.Genome-wide analysis and identification of HAK potassium transporter gene family in maize(Zea mays L.).Molecular Biology Reports,2012,39(8):8465-8473.
[20]Martinez-Cordero M A,Martinez V,Rubio F.Cloning and functional characterization of the high-affinity K+transporter HAK1of pepper.Plant Molecular Biology,2004,56(3):413-421.
[21]Su H,Golldack D,Zhao C S,et al.The expression of HAK-type K+transporters is regulated in response to salinity stress in common ice plant.Plant Physiology,2002,129(4):1482-1493.
[22]Davies C,Shin R,Liu W,et al.Transporters expressed during grape berry(Vitis vinifera L.)development are associated with an increase in berry size and berry potassium accumulation.Journal of Experimental Botany,2006,57(12):3209-3216.
[23]Nieves-Cordones M,Martinez-Cordero M A,Martinez V,et al.An NH4+-sensitive component dominates high-affinity K+uptake in tomato plants.Plant Science,2007,172(2):273-280.
[24]Véry A A,Nieves-Cordones M,Daly M,et al.Molecular biology of K+transport across the plant cell membrane:What do we learn from comparison between plant species?Journal of Plant Physiology,2014,171(9):748-769.
[25]Gupta M,Qiu X,Wang L,et al.KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice(Oryza sativa).Molecular Genetics and Genomics,2008,280(5):437-452.
[26]Gierth M,Mser P.Potassium transporters in plants——involvement in K+acquisition,redistribution and homeostasis.FEBS Letters,2007,581(12):2348-2356.
[27]Ahn S J,Shin R,Schachtman D P.Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+uptake.Plant Physiology,2004,134(3):1135-1145.
[28]Rigas S,Debrosses G,Haralampidis K,et al.TRH1encodes a potassium transporter required for tip growth in Arabidopsis root hairs.Plant Cell,2001,13(1):139-151.
[29]Quintero F J,Blatt M R.A new family of K+transporters fromArabidopsis that are conserved across phyla.FEBS Letters,1997,415(2):206-211.
[30]Fu H H,Luan S.AtKUP1:a dual-affinity K+transporter fromArabidopsis.Plant Cell,1998,10(1):63-73.
[31]Osakabe Y,Arinaga N,Umezawa T,et al.Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis.Plant Cell,2013,25:609-624.
[32]Li P H,Chen M,Wang B S.Effect of K+nutrition on growth and activity of leaf tonoplast V-H+-ATPase and V-H+-PPase of Suaeda salsa under NaCl stress.Acta Botanica Sinica,2002,44(4):433-440.
[33]Rubio F,Santa-Maria G E,Rodriguez-Navarro A.Cloning of Arabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells.Physiologia Plantarum,2000,109(1):34-43.
[4]周向睿,岳利军,王锁民.钠复合肥提高多浆旱生植物霸王幼苗生长及抗旱性.草业学报,2014,23(6):142-147.
[5]种培芳,李航逸,李毅.荒漠植物红砂根系对干旱胁迫的生理响应.草业学报,2015,24(1):72-80.
[6]李莹,柳参奎.碱茅6-磷酸海藻糖合成酶基因的克隆及其耐逆性分析.草业学报,2015,24(1):99-106.
[32]李平华,陈敏,王宝山.K+营养对NaCl胁迫下盐地碱蓬生长及叶片液泡膜V-H+-ATPase和V-H+-PPase活性的影响.植物学报,2002,44(4):433-440.