‘黄冠梨’钾转运体基因PbKT8的克隆与表达分析
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摘要
[目的]本文旨在克隆‘黄冠梨’钾转运体PbKT8基因,对基因定位及表达特征进行初步分析,为其功能研究提供基础。[方法]从‘黄冠梨’果实中克隆PbKT8基因的cDNA全长序列,利用生物信息学对其同源序列进行分析,构建含GFP载体并通过激光共聚焦显微镜精确定位蛋白位置,实时荧光定量PCR分析不同施钾处理下该基因在果实成熟期的表达特征。[结果]钾转运体PbKT8基因序列全长2 328 bp,编码775个氨基酸,PbKT8蛋白与苹果(Malus domestica)进化关系最相近,同源序列相似性高达99.10%;亚细胞定位结果显示,PbKT8蛋白定位于细胞膜上;PbKT8基因转化酵母突变菌株R5421在低于5 mmol·L~(-1) K~+培养基上恢复生长;qRT-PCR结果表明,PbKT8基因在果实和叶片中相对表达量随施钾水平增加而上升。[结论]PbKT8蛋白定位在质膜上具有转运钾离子的功能,在果实成熟期受高钾响应表达。
[Objectives]The study cloned the potassium transporter gene PbKT8 from'Huangguan'pear fruits and analyzed its genetic mapping and expression characteristics,which provided the basis for further research. [Methods]The whole cDNA sequence PbKT8 gene was cloned from'Huangguan'pear,and its amino acid sequence was analyzed by bioinformatics,we had constructed the subcellular localization vector GFP and analyzed the spatial expression by confocal laser scanning microscope,and then the real-time fluorescence quantitative PCR technology was used to explore its expression pattern in fruit under different potassium concentrations. [Results]The full length of potassium transporter PbKT8 cDNA sequence was 2 328 bp,encoding 775 amino acids. The phylogenetic analysis of amino acid homologous showed that the PbKT8 had the highest similarity(99.10%)with the apples(Malus domestica). PbKT8 was verified to be located in the cytoplasm. The yeast mutant strain R5421 containing PbKT8 gene resumed growth in the 5 mmol·L~(-1) K~+ mediums. qRT-PCR analysis showed that the expression of PbKT8 gene increased with the potassium concentration in fruit and leaf. [Conclusions]PbKT8 is located in the cytoplasm and has a significant impact on potassium absorption,and it can be induced by high potassium at the fruit maturity.
[Objectives]The study cloned the potassium transporter gene PbKT8 from'Huangguan'pear fruits and analyzed its genetic mapping and expression characteristics,which provided the basis for further research. [Methods]The whole cDNA sequence PbKT8 gene was cloned from'Huangguan'pear,and its amino acid sequence was analyzed by bioinformatics,we had constructed the subcellular localization vector GFP and analyzed the spatial expression by confocal laser scanning microscope,and then the real-time fluorescence quantitative PCR technology was used to explore its expression pattern in fruit under different potassium concentrations. [Results]The full length of potassium transporter PbKT8 cDNA sequence was 2 328 bp,encoding 775 amino acids. The phylogenetic analysis of amino acid homologous showed that the PbKT8 had the highest similarity(99.10%)with the apples(Malus domestica). PbKT8 was verified to be located in the cytoplasm. The yeast mutant strain R5421 containing PbKT8 gene resumed growth in the 5 mmol·L~(-1) K~+ mediums. qRT-PCR analysis showed that the expression of PbKT8 gene increased with the potassium concentration in fruit and leaf. [Conclusions]PbKT8 is located in the cytoplasm and has a significant impact on potassium absorption,and it can be induced by high potassium at the fruit maturity.
引文
[1] 李廷强,王昌全.植物钾素营养研究进展[J].四川农业大学学报,2001,19(3):281-285.Li T Q,Wang C Q.Advances in plant potassium nutrition[J].Journal of Sichuan Agricultural University,2001,19(3):281-285(in Chinese with English abstract).
[2] Pettigrew W T.Potassium influences on yield and quality production for maize,wheat,soybean and cotton[J].Physiologia Plantarum,2008,133:670-681.
[3] Shen C W,Wang J,Shi X Q,et al.Transcriptome analysis of differentially expressed genes induced by low and high potassium levels provides insight into fruit sugar metabolism of pear[J].Frontiers in Plant Science,2017,8:938.
[4] 王英珍,张虎平,黄小三,等.钾在梨树内的分配及对梨树生长和叶片光合能力的影响[J].南京农业大学学报,2017,40(1):60-67.DOI:10.7685/jnau.201603054.Wang Y Z,Zhang H P,Huang X S,et al.Effect of potassium supply on plant potassium distribution and growth and leaf photosynthetic capacity of Pyrus pyrifolia[J].Journal of Nanjing Agricultural University,2017,40(1):60-67(in Chinese with English abstract).
[5] Gattward J N,Almeida A A F,Souza J O,et al.Sodium-potassium synergism in Theobroma cacao:stimulation of photosynthesis,wateruse efficiency and mineral nutrition[J].Physiologia Plantarum,2012,146:350-362.
[6] Grabov A.Plant KT/KUP/HAK potassium transporters:single family-multiple functions[J].Annals of Botany,2007,99:1035-1041.
[7] Epstein E,Rains D W,Elzam O E.Resolution of dual mechanisms of potassium absorption by barley roots[J].Proc Natl Acad Sci USA,1963,49:684-692.
[8] Li W H,Xu G H,Abdel A,et al.Plant HAK/KUP/KT K+transporters:function and regulation[J].Seminars in Cell & Developmental Biology,2018,74:133-141.
[9] Wang Y,Wu W H.Genetic approaches for improvement of the crop potassium acquisition and utilization efficiency[J].Curr Opin Plant Biol,2015,25:46-52.
[10] Véry A A,Sentenac H.Molecular mechanisms and regulation of K+transport in higher plants[J].Annual Review of Plant Biology,2003,54:575-603.
[11] Rubio F,Santa-Maria G E,Rodriguez-Navarro A.Cloning of Arabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells[J].Physiologia Plantarum,2000,109(1):34-43.
[12] Gupta M,Qiu X H,Wang L,et al.KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice(Oryza sativa)[J].Molecular Genetics and Genomics,2008,280(5):437-452.
[13] Zhang Z,Zhang J,Chen Y,et al.Genome-wide analysis and identification of HAK potassium transporter gene family in maize(Zea mays L.)[J].Molecular Biology Reports,2012,39(8):8465-8473.
[14] Wang Y,Wu W H.Regulation of potassium transport and signaling in plants[J].Current Opinion in Plant Biology,2017,39:123-128.
[15] Osakabe Y,Arinaga N,Umezawa T,et al.Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis[J].The Plant Cell,2013,25(2):609-624.
[16] Elumalai R P,Nagpal P,Reed J W.A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion[J].The Plant Cell,2002,14:119-131.
[17] Lebaudy A,Véry A A,Sentenac H.K+channel activity in plants:genes,regulations and functions[J].FEBS Letters,2007,581(12):2357-2366.
[18] Wang Y G,Lü J H,Chen D,et al.Genome-wide identification,evolution,and expression analysis of the KT/HAK/KUP family in pear[J].NRC Research Press,2018,61:755-765.
[19] 武晓,申长卫,丁易飞,等.‘黄冠梨’果实和叶片钾素积累特征及其对施钾的响应[J].植物营养与肥料学报,2016,22(5):1425-1432.Wu X,Shen C W,Ding Y F,et al.Potassium accumulation in‘Huangguan’pear fruits and leaves and their response to different potassium application[J].Journal of Plant Nutrition and Fertilizers,2016,22(5):1425-1432(in Chinese with English abstract).
[20] 金昕,刘娜,申长卫,等.杜梨IRT1基因的克隆及表达分析[J].农业生物技术学报,2017,25(5):739-749.Jin X,Liu N,Shen C W,et al.Cloning and expression analysis of IRT1 gene in Pyrus betulaefolia[J].Journal of Agricultural Biotechnology,2017,25(5):739-749(in Chinese with English abstract).
[21] 陈光,高振宇,徐国华,等.植物响应缺钾胁迫的机制及提高钾利用效率的策略[J].植物学报,2017,52(1):89-101.Chen G,Gao Z Y,Xu G H,et al.Adaption of plants to potassium deficiency and strategies to improve potassium use efficiency[J].Chinese Bulletin of Botany,2017,52(1):89-101(in Chinese with English abstract).
[22] Fu H H,Sheng L.AtKUP1:a dual-affinity K+ transporter from Arabidopsis[J].The Plant Cell,1998,10(1):63-73.
[23] Qi Z,Hampton C R,Shin R,et al.The high affinity K+transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis[J].Journal of Experimental Botany,2008,59(3):595-607.
[24] 宋志忠,许建兰,张斌斌,等.叶面喷施钾肥对霞脆桃果实品质及KUP基因表达的影响[J].江苏农业学报,2018,34(5):1107-1112.Song Z Z,Xu J L,Zhang B B,et al.Effect of foliar spraying of potassium fertilizer on Xiacui peach quality and expression of KUP transporter family genes[J].Jiangsu Journal of Agricultural Sciences,2018,34(5):1107-1112(in Chinese with English abstract).
[25] Chen G,Hu Q D,Luo L,et al.Rice potassium transporter OsHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges[J].Plant,Cell and Environment,2015,38(12):2747-2765.
[26] Yang T Y,Zhang S,Hu Y B,et al.The role of a potassium transporter OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels[J].Plant Physiol,2014,166(2):945-959.
[27] Ko C H,Gaber R F.TRKI and TRK2 encode structurally related K+ transporters in Saccharomy cescerevisiae[J].Molecular and Cellular Biology,1991,11(8):4266-4273.
[2] Pettigrew W T.Potassium influences on yield and quality production for maize,wheat,soybean and cotton[J].Physiologia Plantarum,2008,133:670-681.
[3] Shen C W,Wang J,Shi X Q,et al.Transcriptome analysis of differentially expressed genes induced by low and high potassium levels provides insight into fruit sugar metabolism of pear[J].Frontiers in Plant Science,2017,8:938.
[4] 王英珍,张虎平,黄小三,等.钾在梨树内的分配及对梨树生长和叶片光合能力的影响[J].南京农业大学学报,2017,40(1):60-67.DOI:10.7685/jnau.201603054.Wang Y Z,Zhang H P,Huang X S,et al.Effect of potassium supply on plant potassium distribution and growth and leaf photosynthetic capacity of Pyrus pyrifolia[J].Journal of Nanjing Agricultural University,2017,40(1):60-67(in Chinese with English abstract).
[5] Gattward J N,Almeida A A F,Souza J O,et al.Sodium-potassium synergism in Theobroma cacao:stimulation of photosynthesis,wateruse efficiency and mineral nutrition[J].Physiologia Plantarum,2012,146:350-362.
[6] Grabov A.Plant KT/KUP/HAK potassium transporters:single family-multiple functions[J].Annals of Botany,2007,99:1035-1041.
[7] Epstein E,Rains D W,Elzam O E.Resolution of dual mechanisms of potassium absorption by barley roots[J].Proc Natl Acad Sci USA,1963,49:684-692.
[8] Li W H,Xu G H,Abdel A,et al.Plant HAK/KUP/KT K+transporters:function and regulation[J].Seminars in Cell & Developmental Biology,2018,74:133-141.
[9] Wang Y,Wu W H.Genetic approaches for improvement of the crop potassium acquisition and utilization efficiency[J].Curr Opin Plant Biol,2015,25:46-52.
[10] Véry A A,Sentenac H.Molecular mechanisms and regulation of K+transport in higher plants[J].Annual Review of Plant Biology,2003,54:575-603.
[11] Rubio F,Santa-Maria G E,Rodriguez-Navarro A.Cloning of Arabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells[J].Physiologia Plantarum,2000,109(1):34-43.
[12] Gupta M,Qiu X H,Wang L,et al.KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice(Oryza sativa)[J].Molecular Genetics and Genomics,2008,280(5):437-452.
[13] Zhang Z,Zhang J,Chen Y,et al.Genome-wide analysis and identification of HAK potassium transporter gene family in maize(Zea mays L.)[J].Molecular Biology Reports,2012,39(8):8465-8473.
[14] Wang Y,Wu W H.Regulation of potassium transport and signaling in plants[J].Current Opinion in Plant Biology,2017,39:123-128.
[15] Osakabe Y,Arinaga N,Umezawa T,et al.Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis[J].The Plant Cell,2013,25(2):609-624.
[16] Elumalai R P,Nagpal P,Reed J W.A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion[J].The Plant Cell,2002,14:119-131.
[17] Lebaudy A,Véry A A,Sentenac H.K+channel activity in plants:genes,regulations and functions[J].FEBS Letters,2007,581(12):2357-2366.
[18] Wang Y G,Lü J H,Chen D,et al.Genome-wide identification,evolution,and expression analysis of the KT/HAK/KUP family in pear[J].NRC Research Press,2018,61:755-765.
[19] 武晓,申长卫,丁易飞,等.‘黄冠梨’果实和叶片钾素积累特征及其对施钾的响应[J].植物营养与肥料学报,2016,22(5):1425-1432.Wu X,Shen C W,Ding Y F,et al.Potassium accumulation in‘Huangguan’pear fruits and leaves and their response to different potassium application[J].Journal of Plant Nutrition and Fertilizers,2016,22(5):1425-1432(in Chinese with English abstract).
[20] 金昕,刘娜,申长卫,等.杜梨IRT1基因的克隆及表达分析[J].农业生物技术学报,2017,25(5):739-749.Jin X,Liu N,Shen C W,et al.Cloning and expression analysis of IRT1 gene in Pyrus betulaefolia[J].Journal of Agricultural Biotechnology,2017,25(5):739-749(in Chinese with English abstract).
[21] 陈光,高振宇,徐国华,等.植物响应缺钾胁迫的机制及提高钾利用效率的策略[J].植物学报,2017,52(1):89-101.Chen G,Gao Z Y,Xu G H,et al.Adaption of plants to potassium deficiency and strategies to improve potassium use efficiency[J].Chinese Bulletin of Botany,2017,52(1):89-101(in Chinese with English abstract).
[22] Fu H H,Sheng L.AtKUP1:a dual-affinity K+ transporter from Arabidopsis[J].The Plant Cell,1998,10(1):63-73.
[23] Qi Z,Hampton C R,Shin R,et al.The high affinity K+transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis[J].Journal of Experimental Botany,2008,59(3):595-607.
[24] 宋志忠,许建兰,张斌斌,等.叶面喷施钾肥对霞脆桃果实品质及KUP基因表达的影响[J].江苏农业学报,2018,34(5):1107-1112.Song Z Z,Xu J L,Zhang B B,et al.Effect of foliar spraying of potassium fertilizer on Xiacui peach quality and expression of KUP transporter family genes[J].Jiangsu Journal of Agricultural Sciences,2018,34(5):1107-1112(in Chinese with English abstract).
[25] Chen G,Hu Q D,Luo L,et al.Rice potassium transporter OsHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges[J].Plant,Cell and Environment,2015,38(12):2747-2765.
[26] Yang T Y,Zhang S,Hu Y B,et al.The role of a potassium transporter OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels[J].Plant Physiol,2014,166(2):945-959.
[27] Ko C H,Gaber R F.TRKI and TRK2 encode structurally related K+ transporters in Saccharomy cescerevisiae[J].Molecular and Cellular Biology,1991,11(8):4266-4273.