甜橙CsKT1的钾转运功能鉴定及其互作蛋白分析
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摘要
植物K~+的跨膜运输过程是由细胞膜上的钾转运蛋白介导的。分析甜橙基因组序列信息发现,甜橙CsKT1与拟南芥Shaker家族成员中的AKT1同源性最高,具有典型的Shaker钾通道特征。互补钾吸收缺陷酵母的试验结果显示,表达CsKT1的钾吸收缺陷酵母能在K~+浓度为10、1和0.1 mmol·L~(-1)的培养条件下生长。以‘冰糖橙’(Citrus sinensis L. Osbeck‘Bingtangcheng’)为试验材料,利用qRT-PCR方法检测到CsKT1在植株根部的表达量比冠部高,并且低钾(0.1mmol·L~(-1)K~+)胁迫处理24h时植株中CsKT1的转录水平无明显变化。亚细胞定位检测结果显示,CsKT1能定位到细胞质膜上。利用酵母双杂交试验发现,CsKT1蛋白C端能与拟南芥CIPK23互作,还能与甜橙CsCIPK7蛋白(与拟南芥CIPK23同源性最高)互作,同时甜橙CsCIPK7蛋白能与拟南芥CBL1蛋白互作;进一步的蛋白序列相似性分析结果显示,柑橘CBL家族有8个成员,其中CsCBL1与拟南芥CBL1、CBL9的同源性最高。这些结果说明,CsKT1是柑橘中的类AKT1钾转运蛋白。
Potassium transporters play important roles in the transmembrane transport of potassium ions. In this study,it was found that CsKT1 had the characteristics of Shaker potassium channel,and displayed the highest homologous to AKT1 in Shaker family members of Arabidopsis thaliana. The experiments showed that the K~+-uptake deficient yeast containing CsKT1 can grow under the conditions with K~+ concentration of 10,1 and 0.1 mmol · L~(-1). qRT-PCR assays showed that the expression of CsKT1 in root was higher than that in shoot,and the transcription level was not changed after 24 h low potassium treatment(0.1 mmol · L~(-1))in Citrus sinensis plants. CsKT1 exhibited plasma membrane localization specifically. The yeast two hybrid experiments showed that the C-terminal cytosolic region of CsKT1 protein not only interacted with AtCIPK23,but also interacted with CsCIPK7 protein,which displayed the highest homology with AtCIPK23. CsCIPK7 could also interact with AtCBL1. There were 8 members in citrus CBL family,in which CsCBL1 displayed the highest similarity with AtCBL1/AtCBL9. These results suggested that CsKT1 function as an AKT1-like potassium transporter.
Potassium transporters play important roles in the transmembrane transport of potassium ions. In this study,it was found that CsKT1 had the characteristics of Shaker potassium channel,and displayed the highest homologous to AKT1 in Shaker family members of Arabidopsis thaliana. The experiments showed that the K~+-uptake deficient yeast containing CsKT1 can grow under the conditions with K~+ concentration of 10,1 and 0.1 mmol · L~(-1). qRT-PCR assays showed that the expression of CsKT1 in root was higher than that in shoot,and the transcription level was not changed after 24 h low potassium treatment(0.1 mmol · L~(-1))in Citrus sinensis plants. CsKT1 exhibited plasma membrane localization specifically. The yeast two hybrid experiments showed that the C-terminal cytosolic region of CsKT1 protein not only interacted with AtCIPK23,but also interacted with CsCIPK7 protein,which displayed the highest homology with AtCIPK23. CsCIPK7 could also interact with AtCBL1. There were 8 members in citrus CBL family,in which CsCBL1 displayed the highest similarity with AtCBL1/AtCBL9. These results suggested that CsKT1 function as an AKT1-like potassium transporter.
引文
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Cuéllar T,Pascaud F,Verdeil J L,Torregrosa L,Adam-Blondon A F,Thibaud J B,Sentenac H,Gaillard I.2010.A grapevine Shaker inward K+channel activated by the calcineurin B-like calcium sensor1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions.Plant J,61(1):58-69.
Fuchs I,Stolzle S,Ivashikina N,Hedrich R.2005.Rice K+uptake channel OsAKT1 is sensitive to salt stress.Planta,221(2):212-221.
Gierth M,M?ser P,Schroeder J I.2005.The potassium transporter AtHAK5 functions in K+deprivation-induced high-affinity K+uptake and AKT1K+channel contribution to K+uptake kinetics in Arabidopsis roots.Plant Physiol,137(3):1105-1114.
Han M,Wu W,Wu W H,Wang Y.2016.Potassium transporter KUP7 is involved in K+acquisition and translocation in Arabidopsis root under K+-limited conditions.Molecular Plant,9(3):437-446.
Hirsch R E,Lewis B D,Spalding E P,Sussman M R.1998.A role for the AKT1 potassium channel in plant nutrition.Science,280:918-921.
Hong Lin,Wen Ze-fu,Cheng Chang-feng,Liu Zheng-fu,Xie Yong-hong.2012.Effects of different rootstocks on growth and mineral nutrition changes of lemon leaves.Southwest China Journal of Agricultural Sciences,25(5):1827-1833.(in Chinese)洪林,文泽富,程昌凤,刘正富,谢永红.2012.砧木对柠檬幼树生长及叶片矿质元素积累的影响.西南农业学报,25(5):1827-1833.
Kleist T J,Spencley A L,Luan S.2014.Comparative phylogenomics of the CBL-CIPK calcium-decoding network in the moss Physcomitrella,Arabidopsis,and other green lineages.Front Plant Sci,5(5):187.
Kong You-han,Yuan Ping,Wu Juan-juan.2017a.Advances on the potassium nutrition in Citrus.Journal of Natural Science of Hunan Normal University,40(3):37-41.(in Chinese)孔佑涵,苑平,吴娟娟.2017a.柑橘的钾营养研究进展.湖南师范大学自然科学学报,40(3):37-41.
Kong You-han,Yuan Ping,Li Xian-xin,Li Wei-dong,Wu Juan-juan.2017b.Comparison of potassium nutrition efficiency among Poncirus trifoliata.Citrange and Citrus sinensis seedings.Chinese Journal of Tropical Crops,37(3):472-477.(in Chinese)孔佑涵,苑平,李先信,李卫东,吴娟娟.2017b.枳、枳橙和甜橙实生苗的钾利用效率比较.热带作物学报,38(3):472-477.
Kong You-han,Yuan Ping,Zhang Wen,Li Xian-xin,Wu Juan-juan.2017c.Cloning and expression analysis of AtCIPK23-like gene in Citrus sinensis.Molecular Plant Breeding,15(10):3900-3906.(in Chinese)孔佑涵,苑平,张文,李先信,吴娟娟.2017c.甜橙中类AtCIPK23基因的克隆与表达分析.分子植物育种,15(10):3900-3906.
Lan W Z,Lee S C,Che Y F,Jiang Y Q,Luan S.2011.Mechanistic analysis of AKT1 regulation by the CBL-CIPK-PP2CA interactions.Molecular Plant,4(3):527-536.
Lee S C,Lan W Z,Kim B G,Li L,Cheong Y H,Pandey G K,Lu G,Buchanan B B,Luan S.2007.A protein phosphorylation/dephosphorylation network regulates a plant potassium channel.Proc Natl Acad Sci USA,104(40):15959-15964.
Li J,Long Y,Qi G N,Xu Z J,Wu W H,Wang Y.2014.The Os-AKT1 channel is critical for K+uptake in rice roots and is modulated by the rice CBL1-CIPK23 complex.Plant Cell,26(8):3387-3402.
Li L,Kim B G,Cheong Y H,Pandey G K,Luan S.2006.A Ca2+signaling pathway regulates a K+channel for low-K+response in Arabidopsis.Proc Natl Acad Sci USA,103(33):12625-12630.
Mao J,Nuruzzaman Manik S M,Shi S J,Chao J T,Jin Y R,Wang Q,Liu H B.2016.Mechanisms and physiological roles of the CBL-CIPKnetworking system in Arabidopsis thaliana.Genes,7(9):62.
Pilot G,Gaymard F,Mouline K,Cherel I,Sentenac H.2003.Regulated expression of Arabidopsis Shaker K+channel genes involved in K+uptake and distribution in the plant.Plant Mol Biol,51(5):773-787.
Pyo Y J,Gierth M,Schroeder J I,Cho M H.2010.High-affinity K+transport in Arabidopsis:AtHAK5 and AKT1 are vital for seedling establishment and postgermination growth under low-potassium conditions.Plant Physiol,153(2):863-875.
Qi J Q,Sun S M,Yang L,Li M J,Ma F W,Zou Y J.2019.Potassium uptake and transport in apple roots under drought stress.Horticultural Plant Journal,5(1):10-16.
Ragel P,Ródenas R,Garcíamartín E,Andrés Z,Villalta I,Nievescordones M,Rivero R M,Martínez V,Pardo J M,Quintero F J,Rubio F.2015.CIPK23 regulates HAK5-mediated high-affinity K+uptake in Arabidopsis roots.Plant Physiol,169(4):2863-2873.
Rubio F,Aleman F,Nieves-cordines M,Martinez V.2010.Studies on Arabidopsis athak5,atakt1 double mutants disclose the range of concentrations at which AtHAK5,AtAKT1 and unknown systems mediate K+uptake.Physiol Plant,139(2):220-228.
Wang X P,Chen L M,Liu W X,Shen L K,Wang F L,Zhou X,Zhang Z D,Wu W H,Wang Y.2016.AtKC1 and CIPK23 synergistically modulate AKT1-mediated low potassium stress responses in Arabidopsis.Plant Physiol,170(4):2264-2277.
Wang Y,Wu W H.2017.Regulation of potassium transport and signaling in plants.Curr Opin Plant Biol,39:123-128.
Xu J,Li H D,Chen L Q,Wang Y,Liu L L,He L,Wu W H.2006.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+transporter AKT1 in Arabidopsis.Cell,125:1347-1360.
Zhang H,Yin W,Xia X.2010.Shaker-like potassium channels in Populus,regulated by the CBL-CIPK signal transduction pathway,increase tolerance to low-K+stress.Plant Cell Rep,29(9):1007-1012.
Cuéllar T,Pascaud F,Verdeil J L,Torregrosa L,Adam-Blondon A F,Thibaud J B,Sentenac H,Gaillard I.2010.A grapevine Shaker inward K+channel activated by the calcineurin B-like calcium sensor1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions.Plant J,61(1):58-69.
Fuchs I,Stolzle S,Ivashikina N,Hedrich R.2005.Rice K+uptake channel OsAKT1 is sensitive to salt stress.Planta,221(2):212-221.
Gierth M,M?ser P,Schroeder J I.2005.The potassium transporter AtHAK5 functions in K+deprivation-induced high-affinity K+uptake and AKT1K+channel contribution to K+uptake kinetics in Arabidopsis roots.Plant Physiol,137(3):1105-1114.
Han M,Wu W,Wu W H,Wang Y.2016.Potassium transporter KUP7 is involved in K+acquisition and translocation in Arabidopsis root under K+-limited conditions.Molecular Plant,9(3):437-446.
Hirsch R E,Lewis B D,Spalding E P,Sussman M R.1998.A role for the AKT1 potassium channel in plant nutrition.Science,280:918-921.
Hong Lin,Wen Ze-fu,Cheng Chang-feng,Liu Zheng-fu,Xie Yong-hong.2012.Effects of different rootstocks on growth and mineral nutrition changes of lemon leaves.Southwest China Journal of Agricultural Sciences,25(5):1827-1833.(in Chinese)洪林,文泽富,程昌凤,刘正富,谢永红.2012.砧木对柠檬幼树生长及叶片矿质元素积累的影响.西南农业学报,25(5):1827-1833.
Kleist T J,Spencley A L,Luan S.2014.Comparative phylogenomics of the CBL-CIPK calcium-decoding network in the moss Physcomitrella,Arabidopsis,and other green lineages.Front Plant Sci,5(5):187.
Kong You-han,Yuan Ping,Wu Juan-juan.2017a.Advances on the potassium nutrition in Citrus.Journal of Natural Science of Hunan Normal University,40(3):37-41.(in Chinese)孔佑涵,苑平,吴娟娟.2017a.柑橘的钾营养研究进展.湖南师范大学自然科学学报,40(3):37-41.
Kong You-han,Yuan Ping,Li Xian-xin,Li Wei-dong,Wu Juan-juan.2017b.Comparison of potassium nutrition efficiency among Poncirus trifoliata.Citrange and Citrus sinensis seedings.Chinese Journal of Tropical Crops,37(3):472-477.(in Chinese)孔佑涵,苑平,李先信,李卫东,吴娟娟.2017b.枳、枳橙和甜橙实生苗的钾利用效率比较.热带作物学报,38(3):472-477.
Kong You-han,Yuan Ping,Zhang Wen,Li Xian-xin,Wu Juan-juan.2017c.Cloning and expression analysis of AtCIPK23-like gene in Citrus sinensis.Molecular Plant Breeding,15(10):3900-3906.(in Chinese)孔佑涵,苑平,张文,李先信,吴娟娟.2017c.甜橙中类AtCIPK23基因的克隆与表达分析.分子植物育种,15(10):3900-3906.
Lan W Z,Lee S C,Che Y F,Jiang Y Q,Luan S.2011.Mechanistic analysis of AKT1 regulation by the CBL-CIPK-PP2CA interactions.Molecular Plant,4(3):527-536.
Lee S C,Lan W Z,Kim B G,Li L,Cheong Y H,Pandey G K,Lu G,Buchanan B B,Luan S.2007.A protein phosphorylation/dephosphorylation network regulates a plant potassium channel.Proc Natl Acad Sci USA,104(40):15959-15964.
Li J,Long Y,Qi G N,Xu Z J,Wu W H,Wang Y.2014.The Os-AKT1 channel is critical for K+uptake in rice roots and is modulated by the rice CBL1-CIPK23 complex.Plant Cell,26(8):3387-3402.
Li L,Kim B G,Cheong Y H,Pandey G K,Luan S.2006.A Ca2+signaling pathway regulates a K+channel for low-K+response in Arabidopsis.Proc Natl Acad Sci USA,103(33):12625-12630.
Mao J,Nuruzzaman Manik S M,Shi S J,Chao J T,Jin Y R,Wang Q,Liu H B.2016.Mechanisms and physiological roles of the CBL-CIPKnetworking system in Arabidopsis thaliana.Genes,7(9):62.
Pilot G,Gaymard F,Mouline K,Cherel I,Sentenac H.2003.Regulated expression of Arabidopsis Shaker K+channel genes involved in K+uptake and distribution in the plant.Plant Mol Biol,51(5):773-787.
Pyo Y J,Gierth M,Schroeder J I,Cho M H.2010.High-affinity K+transport in Arabidopsis:AtHAK5 and AKT1 are vital for seedling establishment and postgermination growth under low-potassium conditions.Plant Physiol,153(2):863-875.
Qi J Q,Sun S M,Yang L,Li M J,Ma F W,Zou Y J.2019.Potassium uptake and transport in apple roots under drought stress.Horticultural Plant Journal,5(1):10-16.
Ragel P,Ródenas R,Garcíamartín E,Andrés Z,Villalta I,Nievescordones M,Rivero R M,Martínez V,Pardo J M,Quintero F J,Rubio F.2015.CIPK23 regulates HAK5-mediated high-affinity K+uptake in Arabidopsis roots.Plant Physiol,169(4):2863-2873.
Rubio F,Aleman F,Nieves-cordines M,Martinez V.2010.Studies on Arabidopsis athak5,atakt1 double mutants disclose the range of concentrations at which AtHAK5,AtAKT1 and unknown systems mediate K+uptake.Physiol Plant,139(2):220-228.
Wang X P,Chen L M,Liu W X,Shen L K,Wang F L,Zhou X,Zhang Z D,Wu W H,Wang Y.2016.AtKC1 and CIPK23 synergistically modulate AKT1-mediated low potassium stress responses in Arabidopsis.Plant Physiol,170(4):2264-2277.
Wang Y,Wu W H.2017.Regulation of potassium transport and signaling in plants.Curr Opin Plant Biol,39:123-128.
Xu J,Li H D,Chen L Q,Wang Y,Liu L L,He L,Wu W H.2006.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+transporter AKT1 in Arabidopsis.Cell,125:1347-1360.
Zhang H,Yin W,Xia X.2010.Shaker-like potassium channels in Populus,regulated by the CBL-CIPK signal transduction pathway,increase tolerance to low-K+stress.Plant Cell Rep,29(9):1007-1012.