梨钾通道基因PbAKT1的克隆及其表达分析
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摘要
AKT1 (Arabidopsis potassium transport 1)是重要的钾通道蛋白基因,参与植物钾吸收和转运过程。为了探究梨(Pyrus betulifolia) PbAKT1基因对钾的响应及表达模式,本研究采用不同浓度K+处理杜梨幼苗,对PbAKT1基因进行克隆并对其所编码的蛋白进行组织特异性分析。结果表明,克隆获得的PbAKT1基因(GenBank No. MN150549) cDNA全长2 646 bp,编码881个氨基酸,有Shaker家族典型的S1～S6的跨膜结构和1个loop环;氨基酸序列的同源关系分析表明,梨PbAKT1基因与苹果(Malus domestica) MdAKT1基因的亲缘关系最近;亚细胞定位发现,该基因主要定位于细胞膜上,为亲水性稳定蛋白;PbAKT1基因在梨苗根、茎、叶片中都有表达,不存在组织特异性;缺钾条件下,该基因有明显响应。综上所述,梨PbAKT1的表达受到缺钾胁迫的诱导,可能参与梨树K+吸收和转运过程。本研究结果为进一步揭示梨树耐低钾分子机制提供基础了资料。
AKT1(Arabidopsis potassium transport 1) is an important potassium channel protein gene involved in plant potassium absorption and transport. In order to study the response and expression pattern of PbAKT1 gene in Pyrus betulifolia to potassium, this study adopted K+at different concentrations(0.1 mmol/L and 3 mmol/L K+) to treat pear seedlings, cloned PbAKT1 gene and analyzed the tissue specificity of its encoded protein. Results showed that the PbAKT1 gene(GenBank No. MN150549) cDNA was 2 646 bp in length,encoding 881 amino acids. The gene had a transmembrane structure with a typical S1～S6 of the Shaker family and a loop ring. The homology analysis of amino acid sequence indicated that there was a close phylogenetic relationship between PbAKT1 gene and MdAKT1 gene(Malus domestica). Subcellular localization showed that the gene mainly presented in the cell membrane and was a hydrophilic stable protein. PbAKT1 gene was found no tissue specificity in roots, stems and leaves of pear seedlings and its expression obviously responded to low potassium stress. It was indicated that the expression of PbAKT1 was induced by potassium supply and might be involved in K+absorption and transport of pear trees. It provides basic information for further research of the molecular mechanism to low potassium stress in pear.
AKT1(Arabidopsis potassium transport 1) is an important potassium channel protein gene involved in plant potassium absorption and transport. In order to study the response and expression pattern of PbAKT1 gene in Pyrus betulifolia to potassium, this study adopted K+at different concentrations(0.1 mmol/L and 3 mmol/L K+) to treat pear seedlings, cloned PbAKT1 gene and analyzed the tissue specificity of its encoded protein. Results showed that the PbAKT1 gene(GenBank No. MN150549) cDNA was 2 646 bp in length,encoding 881 amino acids. The gene had a transmembrane structure with a typical S1～S6 of the Shaker family and a loop ring. The homology analysis of amino acid sequence indicated that there was a close phylogenetic relationship between PbAKT1 gene and MdAKT1 gene(Malus domestica). Subcellular localization showed that the gene mainly presented in the cell membrane and was a hydrophilic stable protein. PbAKT1 gene was found no tissue specificity in roots, stems and leaves of pear seedlings and its expression obviously responded to low potassium stress. It was indicated that the expression of PbAKT1 was induced by potassium supply and might be involved in K+absorption and transport of pear trees. It provides basic information for further research of the molecular mechanism to low potassium stress in pear.
引文
龙雨.2014.水稻钾离子通道OsAKT1生理功能及其调控机制的电生理学研究[D].博士学位论文,中国农业大学,导师:武维华,pp 60-62.(Long Y.2014.Electrophysiological study on physiological function and regulation mechanism of potassium channel OsAKT1 in rice[D].Thesis for Ph.D.,Agricultural University of China,Supervisor:Wu W H,pp 60-62.)
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王毅,武维华.2009.植物钾营养高效分子遗传机制[J].植物学报,44(1):27-36.(Wang Y,Wu W H.2009.High-efficiency molecular genetic mechanism of plant potassium nutrition[J].Journal of Integrative,44(1):27-36.)
吴娟娟,苑平,李卫东,等.2019.甜橙CsKT1的钾转运功能鉴定及其互作蛋白分析[J].园艺学报,46(02):16-27.(Identification of potassium transport function and analysis of interaction proteins for CsKT1in Citrus sinensis[J].Horticultural plant Journal,46(02):16-27.)
徐赫韩,侯国媛,李洋,等.2018.植物钾离子通道AKT1的研究进展[J].生物技术,28(2):200.(Xu C H,Hou GY,Li Y,et al.2018.Research progress of potassium ion channel AKT1 in plants[J].Biotechnology,28(2):200.)
张晨光.2017.不同砧木类型苹果幼树养分吸收特性及对钾营养的响应研究[D].硕士学位论文,中国农业科学院,导师:程存刚,pp 1-7.(Zhang C G.2017.Studies on nutrient absorption characteristics and response to potassium nutrition of young apple trees with different rootstocks[D].Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Cheng C G,pp 1-7.)
张烨,邹雪,吴明阳,等.2013.异源表达AtCIPK23基因对马铃薯试管薯结薯及耐低钾抗性的影响[J].四川农业大学学报,31(3):249-253.(Zhang Y,Zou X,Wu M Y,et al.2013.Effects of heterologous expression of AtCIPK23 gene on potato in vitro and resistance to low potassium[J].Journal of Sichuan Agricultural University,31(3):249-253.)
Basset M,Conejero G,Lepetit M,et al.1995.Organization and expression of the gene coding for the potassium transport system AKT1 of Arabidopsis thaliana[J].Plant Molecular Biology,29(5):947-958.
Becker D,Dreyer I,Hoth S,et al.1996.Changes in voltage activation,Cs+sensitivity,and ion permeability in H5mutants of the plant K+channel KAT1[J].Proceedings of the National Academy of Sciences of the USA.93(15):8123-8128.
Cuéllar T,Pascaud F,Verdeil J L,et al.2010.A grapevine Shaker inward K+channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[J].Plant Journal,61(1):58-69.
Epstein E,Rains D W,Elzam O E.1963.Resolution of dual mechanisms of potassium absorption by barley roots[J].Proceedings of the National Academy of Sciences of the USA,49(5):684-692.
Fuchs I,StLzle S,Ivashikina N,et al.2005.Rice K+uptake channel OsAKT1 is sensitive to salt stress[J].Planta(Berlin),221(2):212-221.
Formentin E,Varotto S,Costa A,et al.2004.DKT1,a novel K+channel from carrot,forms functional heteromeric channels with KDC1[J].FEBS Letters,573(1):61-67.
Hartje S,Zimmermann S,Klonus D,et al.2000.Functional characterisation of LKT1,a K+uptake channel from tomato root hairs,and comparison with the closely related potato inwardly rectifying K+channel SKT1 after expression in X enopusoocytes[J].Planta,210(5):723-731.
Hirsch R E,Lewis B D,Spalding E P,et al.1998.A role for the AKT1 potassium channel in plant nutrition.Science,280(5365):918-921.
Lebaudy A,AnneAliénor Véry,HervéSentenac,et al.2007.K+channel activity in plants:Genes,regulations and functions[J].FEBS Letters,581(12):2357-2366.
Li L,Kim B G,Cheong Y H,et al.2006.A Ca2+signaling pathway regulates a K+channel for low-K response in Arabidopsis[J].Proceedings of the National Academy of Sciences of the USA,103(33):12625-12630.
Li Y,Peng L,Xie C,et al.2018.Genome-wide identification,characterization,and expression analyses of the HAK/KUP/KT,potassium transporter gene family reveals their involvement in K+,deficient and abiotic stress responses in pear rootstock seedlings[J].Genome,2018,61(10):755-765
Philippar K,Kai Büchsenschütz,Edwards D,et al.2006.The auxin-induced K+channel gene Zmk1 in maize functions in coleoptile growth and is required for embryo development[J].Plant Molecular Biology,61(4-5):757-768.
Pilot G,Gaymard F,Mouline K,et al.2003.Regulated expression of Arabidopsis Shaker K+channel genes involved in K+uptake and distribution in the plant[J].Plant Molecular Biology,51(5):773-787.
Rubio F,Nieves-Cordones M,Fernando Alemán,et al.2009.Relative contribution of AtHAK5 and AtAKT1 to K+uptake in the high-affinity range of concentrations[J].Physiologia Plantarum,134(4):598-608.
Sentenac H,Bonneaud N,Minet M,et al.1992.Cloning and expression in yeast of a plant potassium ion transport system[J].Science,256:663-666.
Shen C,Li Y,Wang J,et al.2018.Potassium Influences expression of key genes involved in sorbitol metabolism and its assimilation in pear leaf and fruit[J].Journal of Plant Growth Regulation,37(3):883-895.
Wang,Hao-Dong,Jiang,et al.2010.Potassium channelα-subunit AtKC1 negatively regulates AKTl-mediated K+uptake in Arabidopsis roots under low-K+stress[J].Cell research,(7):826-837.
Wu J,Wang Z,Shi Z,et al.2013.The genome of the pear(Py?rus bretschneideri Rehd.)[J].Genome Research,23(2):396-408.
Xu J,Li H D,Chen L Q et al.2006.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+transporter AKT1 in Arabidopsis[J].Cell,125(7):1347-1360.
Xu J,Tian X,Egrinya E A,et al.2014.Functional characterization of GhAKT1,a novel Shaker-like K+channel gene involved in K+uptake from cotton(Gossypium hirsutum)[J].Gene,545(1):61-71.
Zimmermann S,Talke I,Ehrhardt T,et al.1998.Characterization of SKT1,an inwardly rectifying potassium channel from potato,by heterologous expression in insect cells[J].Plant Physiology,116(3):879-890.
彭莉润,谢昶琰,刘慧冉,等.2018.不同梨砧木幼苗钾营养效率的差异[J].南京农业大学学报,41(3):465-472.(Peng L R,Xie C Y,Liu H R,et al.2018.Difference of potassium nutrient efficiency in different pear rootstocks[J].Journal of Nanjing Agricultural University,41(3):465-472.)
王毅,武维华.2009.植物钾营养高效分子遗传机制[J].植物学报,44(1):27-36.(Wang Y,Wu W H.2009.High-efficiency molecular genetic mechanism of plant potassium nutrition[J].Journal of Integrative,44(1):27-36.)
吴娟娟,苑平,李卫东,等.2019.甜橙CsKT1的钾转运功能鉴定及其互作蛋白分析[J].园艺学报,46(02):16-27.(Identification of potassium transport function and analysis of interaction proteins for CsKT1in Citrus sinensis[J].Horticultural plant Journal,46(02):16-27.)
徐赫韩,侯国媛,李洋,等.2018.植物钾离子通道AKT1的研究进展[J].生物技术,28(2):200.(Xu C H,Hou GY,Li Y,et al.2018.Research progress of potassium ion channel AKT1 in plants[J].Biotechnology,28(2):200.)
张晨光.2017.不同砧木类型苹果幼树养分吸收特性及对钾营养的响应研究[D].硕士学位论文,中国农业科学院,导师:程存刚,pp 1-7.(Zhang C G.2017.Studies on nutrient absorption characteristics and response to potassium nutrition of young apple trees with different rootstocks[D].Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Cheng C G,pp 1-7.)
张烨,邹雪,吴明阳,等.2013.异源表达AtCIPK23基因对马铃薯试管薯结薯及耐低钾抗性的影响[J].四川农业大学学报,31(3):249-253.(Zhang Y,Zou X,Wu M Y,et al.2013.Effects of heterologous expression of AtCIPK23 gene on potato in vitro and resistance to low potassium[J].Journal of Sichuan Agricultural University,31(3):249-253.)
Basset M,Conejero G,Lepetit M,et al.1995.Organization and expression of the gene coding for the potassium transport system AKT1 of Arabidopsis thaliana[J].Plant Molecular Biology,29(5):947-958.
Becker D,Dreyer I,Hoth S,et al.1996.Changes in voltage activation,Cs+sensitivity,and ion permeability in H5mutants of the plant K+channel KAT1[J].Proceedings of the National Academy of Sciences of the USA.93(15):8123-8128.
Cuéllar T,Pascaud F,Verdeil J L,et al.2010.A grapevine Shaker inward K+channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[J].Plant Journal,61(1):58-69.
Epstein E,Rains D W,Elzam O E.1963.Resolution of dual mechanisms of potassium absorption by barley roots[J].Proceedings of the National Academy of Sciences of the USA,49(5):684-692.
Fuchs I,StLzle S,Ivashikina N,et al.2005.Rice K+uptake channel OsAKT1 is sensitive to salt stress[J].Planta(Berlin),221(2):212-221.
Formentin E,Varotto S,Costa A,et al.2004.DKT1,a novel K+channel from carrot,forms functional heteromeric channels with KDC1[J].FEBS Letters,573(1):61-67.
Hartje S,Zimmermann S,Klonus D,et al.2000.Functional characterisation of LKT1,a K+uptake channel from tomato root hairs,and comparison with the closely related potato inwardly rectifying K+channel SKT1 after expression in X enopusoocytes[J].Planta,210(5):723-731.
Hirsch R E,Lewis B D,Spalding E P,et al.1998.A role for the AKT1 potassium channel in plant nutrition.Science,280(5365):918-921.
Lebaudy A,AnneAliénor Véry,HervéSentenac,et al.2007.K+channel activity in plants:Genes,regulations and functions[J].FEBS Letters,581(12):2357-2366.
Li L,Kim B G,Cheong Y H,et al.2006.A Ca2+signaling pathway regulates a K+channel for low-K response in Arabidopsis[J].Proceedings of the National Academy of Sciences of the USA,103(33):12625-12630.
Li Y,Peng L,Xie C,et al.2018.Genome-wide identification,characterization,and expression analyses of the HAK/KUP/KT,potassium transporter gene family reveals their involvement in K+,deficient and abiotic stress responses in pear rootstock seedlings[J].Genome,2018,61(10):755-765
Philippar K,Kai Büchsenschütz,Edwards D,et al.2006.The auxin-induced K+channel gene Zmk1 in maize functions in coleoptile growth and is required for embryo development[J].Plant Molecular Biology,61(4-5):757-768.
Pilot G,Gaymard F,Mouline K,et al.2003.Regulated expression of Arabidopsis Shaker K+channel genes involved in K+uptake and distribution in the plant[J].Plant Molecular Biology,51(5):773-787.
Rubio F,Nieves-Cordones M,Fernando Alemán,et al.2009.Relative contribution of AtHAK5 and AtAKT1 to K+uptake in the high-affinity range of concentrations[J].Physiologia Plantarum,134(4):598-608.
Sentenac H,Bonneaud N,Minet M,et al.1992.Cloning and expression in yeast of a plant potassium ion transport system[J].Science,256:663-666.
Shen C,Li Y,Wang J,et al.2018.Potassium Influences expression of key genes involved in sorbitol metabolism and its assimilation in pear leaf and fruit[J].Journal of Plant Growth Regulation,37(3):883-895.
Wang,Hao-Dong,Jiang,et al.2010.Potassium channelα-subunit AtKC1 negatively regulates AKTl-mediated K+uptake in Arabidopsis roots under low-K+stress[J].Cell research,(7):826-837.
Wu J,Wang Z,Shi Z,et al.2013.The genome of the pear(Py?rus bretschneideri Rehd.)[J].Genome Research,23(2):396-408.
Xu J,Li H D,Chen L Q et al.2006.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+transporter AKT1 in Arabidopsis[J].Cell,125(7):1347-1360.
Xu J,Tian X,Egrinya E A,et al.2014.Functional characterization of GhAKT1,a novel Shaker-like K+channel gene involved in K+uptake from cotton(Gossypium hirsutum)[J].Gene,545(1):61-71.
Zimmermann S,Talke I,Ehrhardt T,et al.1998.Characterization of SKT1,an inwardly rectifying potassium channel from potato,by heterologous expression in insect cells[J].Plant Physiology,116(3):879-890.
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