CN111411113B - 梨保卫细胞钾离子吸收通道基因PbrKAT1及其应用 - Google Patents
梨保卫细胞钾离子吸收通道基因PbrKAT1及其应用 Download PDFInfo
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- CN111411113B CN111411113B CN201811559176.5A CN201811559176A CN111411113B CN 111411113 B CN111411113 B CN 111411113B CN 201811559176 A CN201811559176 A CN 201811559176A CN 111411113 B CN111411113 B CN 111411113B
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Abstract
本发明公开了梨保卫细胞钾离子吸收通道基因PbrKAT1及其应用，该基因为具有如SEQ ID No.1所示核苷酸序列的DNA分子，或与SEQ ID No.1具有85％以上同源性，且编码调节植物保卫细胞中钾离子吸收转运及抗盐相关蛋白的DNA分子。本发明提供的梨保卫细胞钾离子吸收通道基因PbrKAT1，经电生理学功能验证具有吸收转运钾离子及受到胞外钠离子抑制的特性。本发明提供的PbrKAT1基因为调控植物钾离子的吸收转运，调控气孔保卫细胞的运动，提高植物耐盐性，有利于植物抗盐机制的研究，从而降低农业生产成本，提高农业经济效益。
Description
技术领域
本发明属于植物基因工程技术领域，涉及梨保卫细胞钾离子吸收通道基因PbrKAT1及其重组表达载体构建和在调控植物保卫细胞钾离子吸收能力方面的应用。
背景技术
钾离子是植物细胞中含量最丰富的阳离子，它不仅是植物重要的营养物质，而且还参与调节植物生长发育过程中的基本生理特性，如调控保卫细胞的膨压，从而控制细胞扩张和保卫细胞的运动(Szczerba et al.2009)。近十年来，人们对植物根系钾离子吸收和转运的分子机制进行了深入研究，因此，已经在分子水平上鉴定到了许多钾离子通道基因( et al.2001)。其中Shaker家族钾离子吸收转运通道已经被证明参与多种组织和细胞的钾离子吸收转运以及对钾离子特异性选择吸收的功能。它们在植物吸收转运钾离子的过程中扮演了重要的角色，如土壤中钾离子的吸收，根系中钾离子向植物地上部的运输，钾离子的再分配，以及调控气孔中保卫细胞的运动等(Lebaudy et al.2007)。
Shaker钾离子通道是植物研究最早的通道之一，随着电生理技术的出现，研究人员在拟南芥中克隆出第一个钾离子通道基因AKT1，并且研究了其功能特性，发现该基因具有高度的组织表达特异性，主要在根系中表达(Sentenac et al.1992)。此外，在水稻中研究发现，在盐胁迫条件下，OsAKT1通道对钾离子吸收起了重要作用(Ines et al.2005)。随后，人们在玉米(Bauer et al.2000)、烟草(Guo et al.2008)、胡萝卜(Bregante etal.2008)等物种中分别克隆了Shaker家族的钾离子通道，并且对其功能特性进行了研究。KAT1几乎是同时与AKT1从拟南芥中筛选并克隆出的一种向内整流钾离子通道基因，其表达也具有较强的组织特异性，其主要在叶片保卫细胞中表达(Pilot et al.2001)。研究还表明，KAT1主要功能与外向整流钾离子通道GORK协调作用，调节气孔的运动(Eisenach etal.2014)。此外，KAT1在气孔保护细胞中被脱落酸磷酸化，对钾离子的吸收具有高度选择性(Sato et al.2010)。
土壤中高盐度是作物生长发育过程中最重要的非生物胁迫之一。钠离子能够干扰钾离子转运和细胞溶质的正常功能，是这种应激反应的主要组成部分(Qi and Spalding2004)。细胞溶质中保持着高K+:Na+浓度比的能力在植物耐盐方面起着重要作用。研究显示，在水稻细胞中过表达内向钾离子通道基因可能会提高水稻的耐盐性(Obata et al.2007)。然而，钾离子通道基因在耐盐方面的分子机制还尚未了解。
梨作为一种重要的经济作物，生长在温热带地区；是一种非常受欢迎的水果，因为它们果肉细腻香甜。果实中钾离子的含量与糖含量呈正相关，钾肥的施用能够显著提高果实的产量和品质。然而，在梨中还没有确定钾离子吸收转运的相关基因以及梨中钾离子通道基因在植物耐盐性上的作用也没有报道。因此，本研究开展关于果树中钾离子吸收转运的基因克隆以及功能研究，研究结果为梨树钾离子吸收转运的分子机制研究奠定了基础，同时也为以后果树抗盐胁迫能力方面提供了新的见解，对提高果树的经济效益具有重要的意义。
参考文献：
Bauer,C.S.,Hoth,S.,Haga,K.,Philippar,K.,Aoki,N.and Hedrich,R.(2000)Differential expression and regulation of K+channels in the maize coleoptile:molecular and biophysical analysis of cells isolated from cortex andvasculature.Plant Journal 24:139-145.
Bregante,M.,Yang,Y.,Formentin,E.,Carpaneto,A.,Schroeder,J.I.,Gambale,F.,et al.(2008)KDC1,a carrot Shaker-like potassium channel,reveals its roleas a silent regulatory subunit when expressed in plant cells.Plant MolecularBiology 66:61-72.
Eisenach,C.,Papanatsiou,M.,Hillert,E.K.and Blatt,M.R.(2014)Clusteringof the K+channel GORK of Arabidopsis parallels its gating by extracellular K+.Plant Journal 78:203-214.
Guo,Z.K.,Yang,Q.and Yan,P.Q.(2008)Cloning and homology modeling of apotassium channel gene NKC1from Nicotiana rustica.Acta Tabacaria Sinica 14:63-68.
Ines,F.,Sonja,S.,Natalya,I.and Rainer,H.(2005)Rice K+uptake channelOsAKT1is sensitive to salt stress.Planta 221:212-221.
Lebaudy,A.,Very,A.A.and Sentenac,H.(2007)K+channel activity inplants:Genes,regulations and functions.Febs Letters 581:2357-2366.
P.,Thomine,S.,Schroeder,J.I.,Ward,J.M.,Hirschi,K.,Sze,H.,et al.(2001)Phylogenetic relationships within cation transporter families ofArabidopsis.Plant Physiology 126:1646-1667.Obata,T.,Kitamoto,H.K.,Nakamura,A.,Fukuda,A.and Tanaka,Y.(2007)Rice Shaker potassium channel OsKAT1conferstolerance to salinity stress on yeast and rice cells.Plant Physiology 144:1978-1985.
Pilot,G.,Lacombe,B.,Gaymard,F.,Cherel,I.,Boucherez,J.,Thibaud,J.B.,etal.(2001)Guard cell inward K+channel activity in Arabidopsis involvesexpression of the twin channel subunits KAT1and KAT2.Journal of BiologicalChemistry 276:3215-3221.
Qi,Z.and Spalding,E.P.(2004)Protection of plasma membrane K+transportby the salt overly sensitive1Na+-H+antiporter during salinity stress.PlantPhysiology 136:3849-3849.
Sato,A.,Gambale,F.,Dreyer,I.and Uozumi,N.(2010)Modulation of theArabidopsis KAT1 channel by an activator of protein kinase C in Xenopuslaevis oocytes.Febs Journal 277:2318-2328.
Sentenac,H.,Bonneaud,N.,Minet,M.,Lacroute,F.,Salmon,J.M.,Gaymard,F.,et al.(1992)Cloning and expression in yeast of a plant potassium-iontransport-system.Science 256:663-665.Szczerba,M.W.,Britto,D.T.and Kronzucker,H.J.(2009)K+transport in plants:Physiology and molecular biology.Journal ofPlant Physiology 166:447-466.
发明内容
本发明的目的在于提供梨中保卫细胞钾离子吸收通道基因PbrKAT1及其在吸收转运钾离子和抗盐方面的应用。
为了实现上述发明目的，本发明提供以下技术方案：
梨保卫细胞钾离子吸收通道基因PbrKAT1，该基因为具有如SEQ ID No.1所示核苷酸序列的DNA分子，或与SEQ ID No.1具有85％以上同源性，且编码调节植物保卫细胞中钾离子吸收转运及抗盐相关蛋白的DNA分子。该基因具有钾选择性吸收特性和胞外钠离子抑制钾通道活性。
本发明还提供了所述的梨保卫细胞钾离子吸收通道基因PbrKAT1编码的蛋白质。所述蛋白质的氨基酸序列如SEQ ID No.2所示，或将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代且与植物保卫细胞钾离子吸收和耐盐调节相关的由SEQ IDNo.2衍生的蛋白质。
本发明还提供了含有上述梨保卫细胞钾离子吸收通道基因PbrKAT1的重组表达载体、表达盒、转基因细胞系或重组菌。
含有本发明所述基因PbrKAT1的重组表达载体也属于本发明的保护范围，可用现有的植物表达载体构建含有所述基因的重组表达载体。
用于扩增上述梨保卫细胞钾离子吸收通道基因PbrKAT1全长或任一片段的引物对也属于本发明的保护范围，该引物对的正反向引物如SEQ ID No.3和SEQ ID No.4所示。
本发明还提供了所述的梨保卫细胞钾离子吸收通道基因PbrKAT1，所述的蛋白质，所述的重组表达载体、表达盒、转基因细胞系或重组菌中的至少一种在植物育种或/和提高植物抗盐性中的应用。
本发明还提供了所述的梨保卫细胞钾离子吸收通道基因PbrKAT1，所述的蛋白质，所述的重组表达载体、表达盒、转基因细胞系或重组菌中的至少一种在调节植物钾离子吸收和耐盐能力方面的应用。作为一种优选技术方案，所述植物优选为梨，更优选的，所述梨为砀山酥梨。
上述的应用，优选的包括以下步骤：
1)扩增所述的梨保卫细胞钾离子吸收通道基因PbrKAT1；
2)将所述的梨保卫细胞钾离子吸收通道基因PbrKAT1与表达载体连接获得重组载体；
3)将所述的重组载体转入根癌农杆菌中获得重组根癌农杆菌；
4)将获得的重组根癌农杆菌侵染目标植物筛选得到转基因植株。
步骤1)中扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1的过程为：以梨叶片cDNA为模板，进行PCR扩增得到梨保卫细胞钾离子吸收通道基因PbrKAT1；所述扩增特异引物对包括正向引物F1和反向引物R1；所述正向引物F1的序列如SEQ ID No.3所示；所述反向引物R1的序列如SEQ ID No.4所示。
本发明所述的调节可以包括促进也可以是抑制，当应用于促进钾离子吸收时，可以通过异源表达技术或者转基因技术研究基因PbrKAT1的功能；当抑制时，可以通过抑制该基因表达来实现。所述的异源表达和转基因或者抑制基因表达均可通过现有技术常用方法来实现。
优选的，所述异源表达和转基因或者抑制基因表达所采用的动物或植物的材料优选的分别为爪蟾卵母细胞、拟南芥和小叶烟草。
所述植物的材料为烟草时，实验过程包括以下步骤：
1)扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1；
2)将所述梨保卫细胞钾离子吸收通道基因PbrKAT1与表达载体BS-35S-GFP连接获得重组载体；
3)将所述重组载体PbrKAT1-BS-35S-GFP转入根癌农杆菌中获得重组根癌农杆菌；
4)将转入所述重组载体PbrKAT1-BS-35S-GFP的根癌农杆菌侵染烟草，获得PbrKAT1基因在小叶烟草叶片瞬时表达的植株。优选的采用瞬时转化的方法侵染烟草。
所述植物的材料为拟南芥时，实验过程包括以下步骤：
1)扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1的启动子；
2)将所述梨保卫细胞钾离子吸收通道基因PbrKAT1的启动子与表达载体pB35S-GFPXB-4连接获得重组载体；
3)将所述重组载体PbrKAT1-pB35S-GFPXB-4转入根癌农杆菌中获得重组根癌农杆菌；
4)将所述重组载体PbrKAT1-pB35S-GFPXB-4根癌农杆菌侵染拟南芥；从而获得PbrKAT1基因启动子在拟南芥中表达的植株。优选采用浸花序法转化未开花的野生型拟南芥植株。
所述动物的材料为非洲爪蟾卵母细胞时，实验过程包括以下步骤：
1)扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1；
2)将所述梨保卫细胞钾离子吸收通道基因PbrKAT1与表达载体pT7TS连接获得重组载体；
3)将所述重组载体质粒PbrKAT1-pT7TS体外合成cRNA；
4)将所述重组载体质粒PbrKAT1-pT7TS体外合成的cRNA注射到非洲爪蟾卵母细胞中，从而获得PbrKAT1基因异源表达的非洲爪蟾卵母细胞。优选通过显微注射的方法注射到非洲爪蟾卵母细胞中。
上述的实验过程中，扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1的过程为：以梨叶片cDNA为模板，进行PCR扩增得到梨保卫细胞钾离子吸收通道基因PbrKAT1；用于扩增所述PbrKAT1基因的特异引物对包括正向引物F1和反向引物R1；所述正向引物F1的序列见SEQ ID No.3；所述反向引物R1的序列见SEQ ID No.4。
扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1启动子的过程为：以梨叶片DNA为模板，进行PCR扩增得到PbrKAT1基因启动子；用于扩增所述PbrKAT1基因启动子的特异引物对包括正向引物F2和反向引物R2；所述正向引物F2的序列见SEQ ID No.5；所述反向引物R2的序列见SEQ ID No.6。
本发明的有益效果：本发明提供的梨保卫细胞钾离子吸收通道基因PbrKAT1，经电生理学功能验证具有吸收转运钾离子及受到胞外钠离子抑制的特性。本发明提供的PbrKAT1基因为调控植物钾离子的吸收转运，调控气孔保卫细胞的运动，提高植物耐盐性，有利于植物抗盐机制的研究，从而降低农业生产成本，提高农业经济效益。
附图说明
图1为本发明梨保卫细胞钾离子吸收通道基因PbrKAT1扩增电泳图。
图2为本发明梨保卫细胞钾离子吸收通道基因PbrKAT1的克隆、定位和功能验证流程示意图。
图3为实施例1中表达重组载体的构建流程和载体结构图；
其中，图3A为重组表达载体的构建流程，图3B为BS-35S-GFP载体结构图，图3C为pB35S-GFPXB-4载体结构图；图3D为pT7TS载体结构图。
图4为实施例2中本发明梨保卫细胞钾离子吸收通道基因PbrKAT1在不同组织中的表达模式图。其中图4A为拟南芥整个植株；4B为根系；4C为根尖；4D为叶片；4E为叶片气孔放大图。
图5为实施例3和实施例4中本发明梨保卫细胞钾离子吸收通道基因PbrKAT1组织定位和亚细胞定位图。
图6为实施例5中异源表达PbrKAT1进行钾离子通道鉴定电生理图；
其中，图6A表示在10mM钾离子存在的条件下，PbrKAT1通道的电流图。图6B表示PbrKAT1通道在不同浓度钾离子条件下的I-V曲线。图6C表示PbrKAT1对钾离子的亲和力。图6D表示PbrKAT1是钾离子选择性通道。图6E表示PbrKAT1受钾离子通道抑制剂的抑制。图6F表示PbrKAT1通道的活性不依赖钙离子。图6G表示PbrKAT1通道的离子选择性。图6H表示PbrKAT1受胞外pH的调控。
图7为实施例5中异源表达PbrKAT1基因研究钾离子通道活性受钠离子抑制的电生理图；
其中，图7A表示浴液为1mM KCl+99mM LiCl，或1mM KCl+99mM NaCl的条件下所记录到的代表性电流图。图7B表示浴液为10mM KCl+90mM LiCl,或10mM KCl+90mM NaCl的条件下所记录到的电流代表性图。图7C表示PbrKAT1通道的稳态电流受胞外钠离子的抑制。图7D表示PbrKAT1通道的外向电流受钠离子的抑制。图7E表示不同Na/K比对PbrKAT1通道内向电流的影响。图7F表示不同Na/K比对PbrKAT1通道外向电流的影响。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
本发明提供了梨保卫细胞钾离子吸收通道基因PbrKAT1，该基因的核苷酸序列如SEQ ID No.1所示。在本发明中，所述梨保卫细胞钾离子吸收通道基因PbrKAT1优选的来源于砀山酥梨叶片，所述梨保卫细胞钾离子吸收通道基因PbrKAT1包含2316bp的开放阅读框。
在本发明中所述梨保卫细胞钾离子吸收通道基因PbrKAT1通过以下方法获得：从砀山酥梨叶片中提取获得砀山酥梨的RNA；将所述RNA反转录获得cDNA；以所述cDNA为模板，用正向特异引物F1和反向特异引物R1扩增获得所述梨保卫细胞钾离子吸收通道基因PbrKAT1。在本发明中，所述正向引物F1的序列如SEQ ID No.3所示，具体为5’-TCTAGAATGACGTTTTCGTGCACGAAAAACTTCTTC-3’；
所述反向引物R1的序列如SEQ ID No.4所示，具体为5’-GGATCCCTAAAAGGTACAAGTTATATTCATATTATTATAGTC-3’。
在本发明中，所述砀山酥梨叶片优选采用长势幼嫩的叶片，所述砀山酥梨的RNA采用CTAB法进行提取，无其他特殊限定。
本发明在获得砀山酥梨RNA后，反转录获得cDNA；在本发明中所述反转录采用本领域常规的方法即可，优选的采用Thermo反转录试剂盒(美国)进行，具体方法步骤参照所述试剂盒的说明书。
本发明在获得所述cNDA后，以获得的cDNA为模板，用正向引物F1和反向引物R1扩增获得所述梨保卫细胞钾离子吸收通道PbrKAT1的基因序列。在本发明中所述正向引物F1的序列如SEQ ID No.3所示；所述反向引物R1的序列如SEQ ID No.4所示。在本发明具体实施过程中，所述扩增体系优选为50μL体系，所述扩增体系包括100ng模板DNA，I-5TM 2×High-Fidelity Master Mix，10.0μM正向引物和10.0μM的反向引物。本发明中所述的I-5TM2×High-Fidelity Master Mix优选的购自Molecular Cloning Labotratones。本发明中所述扩增反应的程序优选为：98℃预变性2min；35个扩增循环，包括98℃变性10s、60℃退火15s、72℃延伸3min，；循环完成后72℃延伸5min，之后在15℃保温。
本发明在扩增获得所述梨保卫细胞钾离子吸收通道基因PbrKAT1后，优选的将扩增获得的产物进行1％的琼脂糖凝胶电泳、回收后测序验证获得所述梨保卫细胞钾离子吸收通道基因PbrKAT1的核苷酸序列(如图1和图2所示)。
本发明还提供了所述的梨保卫细胞钾离子吸收通道基因PbrKAT1编码的蛋白质，所述蛋白质的氨基酸序列如SEQ ID No.2所示，所述PbrKAT1编码的蛋白质包括772个氨基酸。本发明所述的蛋白质定位在细胞膜，属于膜蛋白(如图4B所示)；该蛋白能够选择吸收钾离子，并且受到钠离子的抑制，从而能够提高植株的耐盐性能。
本发明提供了所述的梨保卫细胞钾离子吸收通道基因PbrKAT1或所述的蛋白质在调控钾离子吸收和抗盐能力方面的应用。
在本发明中，验证所述梨保卫细胞钾离子吸收通道基因PbrKAT1或其所述编码的蛋白质在调控钾离子吸收和抗盐能力的实验中所采用的植物或动物材料为烟草和拟南芥或者非洲爪蟾卵母细胞。
所述植物的材料为烟草时，实验过程包括以下步骤：
1)扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1；
2)将所述梨保卫细胞钾离子吸收通道基因PbrKAT1与表达载体BS-35S-GFP连接获得重组载体；
3)将所述重组载体PbrKAT1-BS-35S-GFP转入根癌农杆菌中获得重组根癌农杆菌；4)将转入所述重组载体PbrKAT1-BS-35S-GFP的根癌农杆菌侵染烟草，获得PbrKAT1基因在小叶烟草叶片瞬时表达的植株。优选的采用瞬时转化的方法侵染烟草。
所述植物的材料为拟南芥时，实验过程包括以下步骤：
1)扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1的启动子；
2)将所述梨保卫细胞钾离子吸收通道基因PbrKAT1的启动子与表达载体pB35S-GFPXB-4连接获得重组载体；
3)将所述重组载体PbrKAT1-pB35S-GFPXB-4转入根癌农杆菌中获得重组根癌农杆菌；
4)将所述重组载体PbrKAT1-pB35S-GFPXB-4根癌农杆菌侵染拟南芥；从而获得PbrKAT1基因启动子在拟南芥中表达的植株。优选采用浸花序法转化未开花的野生型拟南芥植株。
所述动物的材料为非洲爪蟾卵母细胞时，获得PbrKAT1基因异源表达的非洲爪蟾卵母细胞的过程包括以下步骤：
1)扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1；
2)将所述梨保卫细胞钾离子吸收通道基因PbrKAT1与表达载体pT7TS连接获得重组载体；
3)将所述重组载体质粒PbrKAT1-pT7TS体外合成cRNA；
4)将所述重组载体质粒PbrKAT1-pT7TS体外合成的cRNA注射到非洲爪蟾卵母细胞中，从而获得PbrKAT1基因异源表达的非洲爪蟾卵母细胞。优选通过显微注射的方法注射到非洲爪蟾卵母细胞中。
扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1的过程为：以梨叶片cDNA为模板，进行PCR扩增得到梨保卫细胞钾离子吸收通道基因PbrKAT1；用于扩增所述PbrKAT1基因的特异引物对包括正向引物F1和反向引物R1；所述正向引物F1的序列见SEQ ID No.3；所述反向引物R1的序列见SEQ ID No.4。在本发明中所述PCR扩增获得梨保卫细胞钾离子吸收通道基因PbrKAT1的具体方法和步骤参见上述梨保卫细胞钾离子吸收通道基因PbrKAT1的获得方法，在此不再赘述。
扩增所述梨保卫细胞钾离子吸收通道基因PbrKAT1启动子的过程为：以梨叶片DNA为模板，进行PCR扩增得到PbrKAT1基因启动子；用于扩增所述PbrKAT1基因启动子的特异引物对包括正向引物F2和反向引物R2；所述正向引物F2的序列见SEQ ID No.5；所述反向引物R2的序列见SEQ ID No.6。
本发明获得梨保卫细胞钾离子吸收通道基因PbrKAT1，将所述梨保卫细胞钾离子吸收通道基因PbrKAT1与表达载体连接获得重组载体，在本发明中所述表达载体优选BS-35S-GFP、pB35S-GFPXB-4和pT7TS载体作为表达载体，在本发明中所述重组载体的构建方法具体包括以下步骤：
将获得的包含Xba I和BamH I酶切位点的梨保卫细胞钾离子吸收通道基因PbrKAT1，与所述表达载体BS-35S-GFP和pT7TS连接获得重组载体；以及包含HindIII和BamHI酶切位点的PbrKAT1基因的启动子，与所述表达载体pB35S-GFPXB-4连接获得重组载体。在本发明具体实施过程中优选采用诺唯赞公司生产的同源重组连接试剂盒进行，具体方法步骤参照所述试剂盒的说明书。
本发明在获得重组载体后，优选将所述重组载体转入大肠杆菌菌株DH5α中，进行菌落PCR测序验证所述重组表达载体是否构建成功；在本发明中所述菌落PCR测序验证的方法采用本领域常规的菌落PCR测序验证方法；在本发明中PCR验证成功后，菌液经Sanger测序验证。
本发明在获得重组载体后，将所述重组载体PbrKAT1-BS-35S-GFP、PbrKAT1-pB35S-GFPXB-4转入根癌农杆菌GV3101中获得重组根癌农杆菌；以及将所述重组载体质粒PbrKAT1-pT7TS体外合成cRNA。
在本发明中，将所述重组载体转入根癌农杆菌的方法优选为冻融法，以及将所述重组载体质粒PbrKAT1-pT7TS体外合成的cRNA转入到非洲爪蟾卵母细胞中通过显微注射的方法。在本发明中具体冻融法的方法步骤见《分子克隆实验指南》(萨姆布鲁克，黄培堂译，《分子克隆实验指南》第三版，科学出版社，2002年)。
下面结合具体实施例对本发明提供的梨保卫细胞钾离子吸收通达基因PbrKAT1及其在钾离子吸收转运和抗盐能力方面的应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1梨PbrKAT1基因全长cDNA及启动子的克隆
通过筛选梨全基因组cDNA文库筛选到一个调控梨保卫细胞钾离子吸收通道基因PbrKAT1，以及PbrKAT1基因上游的2000bp作为启动子进行克隆，根据同源重组表达载体重组的特点和PbrKAT1基因及其启动子的序列，用Primer premier 5.0设计引物，以‘砀山酥梨’叶片cDNA和DNA为模板进行PCR全长扩增。详细步骤如下：
研究材料砀山酥梨种植在南京农业大学国家梨工程中心，其苗龄为3～5年。挑选生长势健壮、无病虫害的砀山酥梨幼嫩叶片，随机称取500mg样品，立即用液氮速冻。采用CTAB法提取总RNA和DNA，实验前的准备RNA-free的蓝吸头、黄吸头、白吸头和1.5ml离心管；研钵、研棒、小钥匙需提前进行酒精高温灭菌，冷却后使用液氮速冻。CTAB提取缓冲液：2％CTAB(W/V，g/100ml)、2％PVP K-30(W/V，g/100ml)、10mM Tris-HCl(pH 8.0)、25mM EDTA、2MNaCl、0.5g/L亚精胺。
提取完成后，用1％的琼脂糖凝胶电泳检测提取的RNA和DNA的质量，并用NanoDrop2000分光光度计检测RNA和DNA的浓度和质量。
cDNA第一条链的合成参照Thermo Scientific RevertAid First Strand cDNASynthesis Kit的操作手册进行。所得的第一链cDNA用于PbrKAT1基因的扩增以及以DNA为模板进行PbrKAT1基因启动子的扩增。PCR按以下程序完成：98℃预变性2min；35个扩增循环，包括98℃变性10s，60℃退火15s，72℃延伸3min，循环完成后72℃延伸5min，之后在15℃下保温。扩增完成后，经1％的琼脂糖凝胶电泳检测到单一目的条带的PCR产物，按照胶回收试剂盒(购自康为世纪，中国)使用说明提取步骤，回收特异目的条带。
实施例2砀山酥梨不同组织中梨保卫细胞钾离子吸收通道基因PbrKAT1的qRT-PCR分析
为了分析砀山酥梨中PbrKAT1基因在梨不同组织中的表达模式，使用Real-timePCR技术对PbrKAT1基因的表达模式进行分析。根据PbrKAT1基因的编码区序列，按照一般设计引物的原则用Primer primier 5.0软件设计出扩增基因整个编码区的上、下游PCR引物。采用试剂盒提取RNA，cDNA第一条链的合成参照Thermo Scientific RevertAid FirstStrand cDNA Synthesis Kit的操作手册进行。20μL的反应体系包括：10μL SYBR Green，5μL灭菌超纯水，1μL cDNA，2μL正向引物，F3：5’-GATCTAGCACAACAGCAAGG-3’(SEQ ID No.7)，2μL反向引物，R3：5’-GATACAGGTGATCACCATCTCGG-3’(SEQ ID No.8)。(以Actin为内参，序列如下：Actin-F4:5’-CAATGTGCCTGCCATGTATG-3’(SEQ ID No.9)；Actin-R4:5’-CCAGCAGCTTCCATTCCAAT-3’(SEQ ID No.10))。
qRT-PCR的程序如下：94℃预变性5min；94℃变性3s，60℃退火10s，72℃延伸30s，45个循环；72℃延伸3min，20℃保温30s。
以砀山酥梨不同组织为材料，PbrKAT1基因的相对表达量表现出了不同组织表达的特性，如图4所示。表明了PbrKAT1基因主要在叶片中表达。
实施例3梨调控钾离子吸收基因PbrKAT1的组织定位
1.植物转化载体构建
根据同源重组的特点和PbrKAT1基因启动子的序列(PbrKAT1基因上游2000bp)，利用梨叶片DNA为模板进行克隆。PCR产物经1％琼脂糖凝胶电泳后，利用凝胶试剂盒回收目的条带。回收纯化的扩增片段与表达载体进行重组，采用热击法转化到大肠杆菌感受态DH5α中。将转化后的菌液经过PCR检测，PCR鉴定呈阳性的菌液送去测序。得到正确的重组目的载体命名为PbrKAT1-pB35S-GFPXB-4。应用冻融法(参照萨姆布鲁克，黄培堂译，《分子克隆实验指南》第三版，科学出版社，2002年)将重组载体PbrKAT1-pB35S-GFPXB-4导入到农杆菌GV3101中。
2.农杆菌介导的拟南芥遗传转化步骤如下：
(1)农杆菌培养：取超低温冰箱(-80℃)中保存的根癌农杆菌菌液，在添加了卡那霉素50mg/L和利福平50mg/L的LB的平板培养基上划线，挑取单克隆，在卡那霉素50mg/L和利福平50mg/L的LB液体培养基中振荡培养，28～30℃，220-240rpm/min振荡培养16-24h。
(2)待OD600至0.6-0.8时，收集菌体，5000rpm/min，离心10min。
(3)配制诱导转化液，如下表1：
表1拟南芥浸花法诱导液配制方法
(4)菌体重悬于同等体积的的转化介质中。
(5)将待转化的拟南芥植株剪去角果和已经开放的花。
(6)把拟南芥苗倒扣至100ml烧杯中，使整个花序浸泡在侵染液中，抽真空至380mmHg，侵染时间5～10min，优选7min。
(7)所述侵染后优选的为暗培养12h，随后进行正常光照培养16h/8h。所述侵染后的培养温度22～25℃，正常管理，等待收取拟南芥种子。
3.转基因阳性苗的的筛选
按照上述方法得到转PbrKAT1基因启动子的T0代拟南芥种子，以潮霉素抗性(20μg/mL)为表型标记性状，同时含有抗生素羧卞(100μg/mL)、特美汀(200μg/mL)的MS培养基筛选种子，并筛选出带有PbrKAT1基因启动子的转基因植株。
3.1转基因拟南芥的种植
(1)MS培养基配制方法如下表2：
表2 MS培养基配制方法
经过121℃，高温灭菌20min，待培养基温度冷却至40℃时(触摸不烫手)，加入抗生素，浓度如下表3：
表3 MS培养基抗生素配制方法
(2)拟南芥种子种植
拟南芥种子用70％酒精消毒1min，无菌水冲洗2次；8.33％的次氯酸钠消毒5min，无菌水冲洗5次，均匀播种在含有潮霉素(20μg/ml)、羧卞(100μg/ml)和特美汀(200μg/ml)的MS固体培养基表面。4℃黑暗条件下进行春化处理48h，正常光照培养16h/8h。经过两周时间左右，将能够正常生长出的拟南芥幼苗移栽到营养钵里，22～25℃温度，16h/8h光照培养。以营养土和蛭石的体积比为1：1.5的配方进行栽培。移栽后覆盖塑料透明盖保温保湿，待缓苗2～3天后，移除塑料透明盖，正常培养。
3.2拟南芥叶片DNA的提取
转基因拟南芥植株叶片为材料，采用CTAB法提取DNA。以上已做详细描述，在此不再叙述。
4.PbrKAT1组织定位分析
为了鉴定PbrKAT1基因的组织定位情况，将侵染PbrKAT1基因启动子的拟南芥在培养室中正常管理，培养室温度优选在22～25℃，光照优选为16h/8h。选取生长7天的拟南芥幼苗，将其放入含有GUS染料的10mL离心管中，37℃，避光孵育3小时，之后分别用30％、50％、80％和100％无水乙醇进行脱色，每次脱色时间为30分钟。然后分别用佳能相机和显微镜观察并拍照拟南芥不同组织(幼苗、根、叶和气孔)的GUS染色情况。结果如图4A-E所示。
实施例4梨调控钾离子吸收通道PbrKAT1蛋白的亚细胞定位
根据PbrKAT1基因的核苷酸序列和表达载体图谱，在基因序列前后分别加入Xba I和BamH I酶切位点。酶切位点的序列如下所示：
Xba I：TCTAGA
BamH I：GGATCC
以砀山酥梨叶片的cDNA为模板，用加入酶切位点的引物扩增，所用PCR程序为：98℃预变性2min；98℃变性10s，60℃退火15s，72℃延伸3min，35个循环；72℃延伸5min。酶切位点的引物序列如下所示：
F1：5’-TCTAGAATGACGTTTTCGTGCACGAAAAACTTCTTC-3’(SEQ ID No.3)
R1：5’-GGATCCCTAAAAGGTACAAGTTATATTCATATTATTATAGTC-3’(SEQ ID No.4)
基因3′端去除了终止密码子TAG，目的是让基因与GFP融合。PCR产物经1％琼脂糖凝胶电泳后，利用凝胶试剂盒回收目的条带。回收纯化的扩增片段与表达载体进行重组，采用热击法转化到大肠杆菌感受态DH5α中。将转化后的菌液用PCR检测，对PCR鉴定呈阳性的菌液进行测序，得到正确的重组目的载体命名为PbrKAT1-BS-35S-GFP。采用冻融法(参照萨姆布鲁克，黄培堂译，《分子克隆实验指南》第三版，科学出版社，2002年)将重组载体PbrKAT1-BS-35S-GFP导入到农杆菌GV3101中。
采用烟草叶片瞬时转化法，诱导液配制如下表4:
表4烟草叶片瞬时转化中诱导液配制方法
具体操作方法：
1)从-80℃冰箱中取出保存的农杆菌液，在相应抗性(一般为卡那和利福平)的LB固体培养基上划线，倒扣放置在30℃培养箱，活化菌液；
2)待长出单克隆，在卡那和利福平抗生素的LB液体培养基中进行扩培，28～30℃摇床，220～240rpm，振荡培养过夜；
3)收集菌液，一般使用10mL离心管收集两次即可，看管底菌量而定，可多收集；
4)倒掉上清，沥干液体，加入2mL诱导液；
5)涡旋混匀，在摇床上进行上下摇晃诱导3h以上；
6)通过注射侵染烟草，从本生烟草背面注射；2-3天后通过激光共聚焦扫描显微镜观察荧光情况，在观察前使用真空泵或注射器将叶片抽真空，以便更清楚观察。
通过将PbrKAT1-BS-35S-GFP和对照空载BS-35S-GFP的质粒分别注射到烟草的表皮细胞中，通过检测叶片表皮细胞中的GFP荧光位置来确定PbrKAT1蛋白定位的定位情况，结果如图4所示，其中图4F为对照空载体的定位，荧光布满细胞膜、细胞质和细胞核；图4G为PbrKAT1-BS-35S-GFP在烟草表皮细胞中的瞬时表达，绿色荧光分布在细胞膜上，而未在其他地方发现荧光。结果表明，PbrKAT1蛋白定位在细胞膜，是一个膜定位蛋白。
实施例5 PbrKAT1异源表达对钾离子吸收特性的研究
1.cRNA的体外合成
1)高纯度质粒的提取
采用大量高纯度质粒提取试剂盒(维格拉斯)将含有PbrKAT1基因表达载体pT7TS的DNA从大肠杆菌中提取出来。具体方法参照试剂盒说明书。
2)质粒的线性化：吸取总量6-7μg的质粒，加入4μL限制性内切酶XbaⅠ和2μL10ⅹbuffer，用ddH2O定容到50μL，37℃酶切5个小时。之后用1％的琼脂糖凝胶电泳检测线性化是否完全。
3)线性化质粒的纯化：向线性化质粒中加入20μL 1％SDS(g/100mL)，0.4μL蛋白酶K(20mg/ml)，随后50℃温浴30min；用无RNA酶水补充到100μL，再加100μL酚/氯仿溶液。轻轻混匀，常温离心5min，13000rpm。吸取上清到新的无RNA酶的离心管中，并加入10μL醋酸钠(3M)溶液和200μL 95％乙醇，放在-20℃冷冻30min。随后4℃，13000rpm离心30min，去掉上清，用70％的乙醇清洗沉淀的线性化质粒两次，阴干离心管里的乙醇，并加入无RNA酶水使其溶解，并使其最终浓度为0.5ug/uL左右。
4)cRNA的体外合成：
按照以下体系加入线性化质粒及反应液:
线性化质粒--------------------------1.2μg
Enzyme Mix-------------------------2μL
2 x NTP/Cap-------------------------10μL
10 ⅹ buffer--------------------------2μL
RNase free H2O----------------------to 20μL
37℃反应3h。
之后加入115μL无RNA酶水，15μL醋酸钠(3M)，150μL酚/氯仿溶液，漩涡混匀，室温离心3min，13000rpm。吸取上清到新的无RNA酶离心管中，注意不要吸到下层溶液，之后再重复步骤(3)(4)(5)。向上清溶液中加入等体积的异丙醇，-20℃沉淀30min，再离心30min，4℃，13000rpm。吸干上清溶液，保留沉淀，并用70％的乙醇洗脱两次。用无RNA酶水溶解cRNA，使其终浓度定为1ug/uL，并用新配的1％的琼脂糖凝胶电泳检测cRNA质量。最后将cRNA进行分装，保存到-80℃，待用。
2.爪蟾卵母细胞的分离与消化
选取颜色较深、体型健硕成熟的非洲爪蟾一只，将其埋在冰里一个小时，使其休眠，随后解刨取卵。将卵母细胞放入Ca2+-free溶液(82.5mM NaCl，2mM KCl，1mM MgCl2，5mMHEPES，pH 7.4)中，并用该溶液清洗5-6次。随后加入1mg/ml胶原酶(Collagenase A，Roche)在20-24℃条件下消化1-2小时，至大部分卵母细胞呈单个细胞状态。再用加入1mg/ml庆大酶素(Gentamycin)的ND96溶液(96mM NaCl，2mM KCl，1mM MgCl2，1.8mM CaCl2，5mM HEPES，pH 7.4)洗涤卵母细胞6-8次，在显微镜下用吸管挑选大小一致，表面光滑的卵母细胞待用。
3.显微注射
首先用微电极拉制仪拉制玻璃毛细管，使其尖端直径为20μm。先向毛细管内注入矿物油，并确保没有气泡产生，将微注射管安装到微注射仪上(PLI-100型,美国制造)。吸取1μL水(RNase free)，调节注射时间，使1μL水(RNase free)注射次数在19-21次之间,反复标定2-3次。由于cRNA比较粘稠，所以，同样1μL cRNA注射次数会比1μL水少1-2次。注射时应在卵母细胞的白色区域进行注射，用注水的爪蟾卵母细胞作为对照。
4.爪蟾卵母细胞的培养
将注射cRNA及水的爪蟾卵母细胞放在含有100mg/L链霉素和60mg/L青霉素的ND96中培养，并分装到48孔培养板中，每孔2-3个卵母细胞。随后放到18℃恒温培养箱中，培养2-3天，其间每天更换两次ND96溶液，并将坏掉的爪蟾卵母细胞及时剔除掉。
5.电压钳记录与数据分析
表达完的卵母细胞采用双电极电压钳技术记录卵母细胞的电流，同时采用Axoclamp 900A(Axon Instruments,Foster City,CA,USA)电压钳放大器。首先向两个电极玻璃管内注入3mol/L的KCl，充分赶去尖端的气泡后分别安装在电压钳和电流钳的2个探头上，将细胞置于记录槽内，然后将电压及电流电极插入细胞，通过电压极的读数判断卵母细胞状态是否良好。能钳制在-39.6mV以上说明细胞状态良好，可进行脉冲激活和电流记录。标准溶液包含5mM HEPES(pH 7.5),100mM NaCl,1.8mM CaCl2,1mM MgCl2。为了平衡检测液的渗透压，我们用山梨醇调节缓冲液的渗透压，使其达到220mos-mol/kg。最后数据的采集与数据的分析分别使用pClampfit 10.3和Sigmaplot 12.5软件进行处理。
在不同浓度钾离子以及一些钾离子通道抑制剂的条件下检测PbrKAT1通道的特性，结果如图6所示。其中，图6A表示在10mM钾离子存在的条件下，PbrKAT1通道的电流图。图6B表示PbrKAT1通道在不同浓度钾离子条件下的I-V曲线。图6C表示PbrKAT1对钾离子的亲和力。图6D表示PbrKAT1是钾离子选择性通道。图6E表示PbrKAT1受钾离子通道抑制剂的抑制。图6F表示PbrKAT1通道的活性不依赖钙离子。图6G表示PbrKAT1通道的离子选择性。图6H表示PbrKAT1受胞外PH的调控。PbrKAT1基因在非洲爪蟾卵母细胞中异源表达的结果显示，PbrKAT1通道对钾离子具有较高的选择性，是一个低亲和性钾通道(图6)。此外，图7结果显示，PbrKAT1钾通道受胞外钠离子的抑制，其中图7A表示浴液为1mM KCl+99mM LiCl或1mMKCl+99mM NaCl的条件下所记录到的代表性电流图。图7B表示浴液为10mM KCl+90mM LiCl或10mM KCl+90mM NaCl的条件下所记录到的电流代表性图。图7C表示PbrKAT1通道的稳态电流受胞外钠离子的抑制。图7D表示PbrKAT1通道的外向电流受钠离子的抑制。图7E表示不同Na/K比对PbrKAT1通道内向电流的影响。图7F表示表示不同Na/K比对PbrKAT1通道外向电流的影响。总之，该结果说明在外界低钾浓度时，钠离子能够抑制钾离子通道的活性，抑制其钾离子的吸收转运，使气孔的开度减小，进而影响作物的蒸腾速率，减少Na+从地下部向地上部的转移，从而可能增加梨树的抗盐特性(图7)。
序列表
<110> 南京农业大学
<120> 梨保卫细胞钾离子吸收通道基因PbrKAT1及其应用
<160> 10
<170> SIPOSequenceListing 1.0
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atgacgtttt cgtgcacgaa aaacttcttc cggaggttct gtattgatga ataccaaatg 60
gacactgttg ctcagagcag cttcttctct actgatcttc tgccttccct tggagccaga 120
atcaaccagt ctactaagct caggaaatac attatatcgc cgtataatcc tcgttacagg 180
gcttggggga tgctacttgt tcttctagtc atctactcag cgtggatttg cccatttgag 240
tttgcatttc tgccttacaa gcgggatgct cttttcgtca ttgacaacat tgtcaacggc 300
ttctttggca ttgacatcat cctcactttc tttgttgcct atctcgacag ccgctcttac 360
cttcttgttg acaatccaaa gcaaatcgca ataaggtact tatcaacctg gtttcttttc 420
gacgtgtgtt ccactgcacc atttcagtct attagcctcc tcttgacaaa tcacagcagc 480
gaacttgagt ttaaagtact caatatgctc cgcctctggc gcctccgacg agtcagctcc 540
ctctttgcaa gactggagaa ggacatccga ttcaactact tctggattcg ctgcacgaag 600
ctcatttctg ttaccctttt cgcagtgcac tgcgcaggat gtttcaacta tctgatcgca 660
gatcggtatc ctgacccgaa aagaacatgg atcggcgctg tgtacccgga tttcaaacaa 720
gatagtctct ggaatagata tgttacttca atgtactggt caatcacaac gctaaccacc 780
actggctatg gagatctgca tgctgagaac cctagagaga tgctgtttga tattttctac 840
atgctcttca acttgggatt gacatcttac ctcattggaa acatgacaaa tcttgtagtt 900
cactggacca gcagaaccag aatctttagg gacacagtga gagctgcatc agaatttgca 960
gcaagaaacg acttgccccc cacgattcaa gaccagatgt tgtcacacat atgcctcaag 1020
tttaagacag aaggactgaa acagcaagag accttaaatg gtctcccgaa agcccttcgt 1080
tccagcattg cccaacatct cttcttcccc gtcgttcaaa acatctacct ctttcaagga 1140
gtttctcatg atttcctctt tcaattggtt ccagaaatag atgcagagta ttttccaccc 1200
aaggaagatg taattctgca aaacgaggct ccgaccgatc tttatatact ggtttccggt 1260
gcggcggagt taatatctga cttgacatac tctaaacagt tcatacgaaa ggcaactgcg 1320
ggggatactt tgggagaaat cggagtatta tgtcataggc cacagccttt caccgttcgg 1380
acaaccgaac tttcccagat actacgactc cgcaaaagtt cactcatgac caccatagaa 1440
gcaaataagg acgacgagca aattatcatg aacaacattt ttcagaaact gaaggaacaa 1500
gaagggttgg gctgtgaata tccacatacc gaaggatgct gttcttgcgc tggatgtaaa 1560
gacaactcac gtcaaaatcc atcaatggag gaagcaagga acgacttgtt tacaggttca 1620
gaggctacaa aaaagagtga aataggcaga gctgataatt caacgagatg tgcaatggat 1680
gtctgcatga tggctgagga tggccaaaca gctcttccca ctgctgttca tcggggacat 1740
ctggaaatgg tcaaaatttt ggtcgaagga ggagcaaatg taaacaaacc agatgctaga 1800
ggatggacgc cgaaagatct agcacaacag caaggaaaca agagcataac tgacctatta 1860
cgaagatatg agaataggag aacagatgaa catagaatag agtttattga accggaaaca 1920
tctgaaataa ccaggaattg taaaggaaat tccaaaagac acgagggggc ccaattttcc 1980
caatctcacc agagaaaagt acccattaag tcctacccga gcaattctat ccctgataaa 2040
gaatggatga gatcaatcaa caagagagta actatccaca tgcattttca aaatggaagt 2100
gcattggaga ggcagcttgc gaagttaata atcctacctg attcgatgga agagcttctc 2160
agagtggctg gtgagaagtt tgaaggatac aaacctacaa aagtcgttaa tgaagaaaat 2220
gcagaaatag atgacataag tgttgtccga gatggtgatc acctgtatct tcttcacaac 2280
gactataata atatgaatat aacttgtacc ttttag 2316
<210> 2
<211> 771
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Met Thr Phe Ser Cys Thr Lys Asn Phe Phe Arg Arg Phe Cys Ile Asp
1 5 10 15
Glu Tyr Gln Met Asp Thr Val Ala Gln Ser Ser Phe Phe Ser Thr Asp
20 25 30
Leu Leu Pro Ser Leu Gly Ala Arg Ile Asn Gln Ser Thr Lys Leu Arg
35 40 45
Lys Tyr Ile Ile Ser Pro Tyr Asn Pro Arg Tyr Arg Ala Trp Gly Met
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Leu Leu Val Leu Leu Val Ile Tyr Ser Ala Trp Ile Cys Pro Phe Glu
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Phe Ala Phe Leu Pro Tyr Lys Arg Asp Ala Leu Phe Val Ile Asp Asn
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Ile Val Asn Gly Phe Phe Gly Ile Asp Ile Ile Leu Thr Phe Phe Val
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Ala Tyr Leu Asp Ser Arg Ser Tyr Leu Leu Val Asp Asn Pro Lys Gln
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Ile Ala Ile Arg Tyr Leu Ser Thr Trp Phe Leu Phe Asp Val Cys Ser
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Thr Ala Pro Phe Gln Ser Ile Ser Leu Leu Leu Thr Asn His Ser Ser
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Glu Leu Glu Phe Lys Val Leu Asn Met Leu Arg Leu Trp Arg Leu Arg
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Arg Val Ser Ser Leu Phe Ala Arg Leu Glu Lys Asp Ile Arg Phe Asn
180 185 190
Tyr Phe Trp Ile Arg Cys Thr Lys Leu Ile Ser Val Thr Leu Phe Ala
195 200 205
Val His Cys Ala Gly Cys Phe Asn Tyr Leu Ile Ala Asp Arg Tyr Pro
210 215 220
Asp Pro Lys Arg Thr Trp Ile Gly Ala Val Tyr Pro Asp Phe Lys Gln
225 230 235 240
Asp Ser Leu Trp Asn Arg Tyr Val Thr Ser Met Tyr Trp Ser Ile Thr
245 250 255
Thr Leu Thr Thr Thr Gly Tyr Gly Asp Leu His Ala Glu Asn Pro Arg
260 265 270
Glu Met Leu Phe Asp Ile Phe Tyr Met Leu Phe Asn Leu Gly Leu Thr
275 280 285
Ser Tyr Leu Ile Gly Asn Met Thr Asn Leu Val Val His Trp Thr Ser
290 295 300
Arg Thr Arg Ile Phe Arg Asp Thr Val Arg Ala Ala Ser Glu Phe Ala
305 310 315 320
Ala Arg Asn Asp Leu Pro Pro Thr Ile Gln Asp Gln Met Leu Ser His
325 330 335
Ile Cys Leu Lys Phe Lys Thr Glu Gly Leu Lys Gln Gln Glu Thr Leu
340 345 350
Asn Gly Leu Pro Lys Ala Leu Arg Ser Ser Ile Ala Gln His Leu Phe
355 360 365
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370 375 380
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Leu Lys Glu Gln Glu Gly Leu Gly Cys Glu Tyr Pro His Thr Glu Gly
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Cys Cys Ser Cys Ala Gly Cys Lys Asp Asn Ser Arg Gln Asn Pro Ser
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530 535 540
Lys Ser Glu Ile Gly Arg Ala Asp Asn Ser Thr Arg Cys Ala Met Asp
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Val Cys Met Met Ala Glu Asp Gly Gln Thr Ala Leu Pro Thr Ala Val
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His Arg Gly His Leu Glu Met Val Lys Ile Leu Val Glu Gly Gly Ala
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Asn Val Asn Lys Pro Asp Ala Arg Gly Trp Thr Pro Lys Asp Leu Ala
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Gln Gln Gln Gly Asn Lys Ser Ile Thr Asp Leu Leu Arg Arg Tyr Glu
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Asn Arg Arg Thr Asp Glu His Arg Ile Glu Phe Ile Glu Pro Glu Thr
625 630 635 640
Ser Glu Ile Thr Arg Asn Cys Lys Gly Asn Ser Lys Arg His Glu Gly
645 650 655
Ala Gln Phe Ser Gln Ser His Gln Arg Lys Val Pro Ile Lys Ser Tyr
660 665 670
Pro Ser Asn Ser Ile Pro Asp Lys Glu Trp Met Arg Ser Ile Asn Lys
675 680 685
Arg Val Thr Ile His Met His Phe Gln Asn Gly Ser Ala Leu Glu Arg
690 695 700
Gln Leu Ala Lys Leu Ile Ile Leu Pro Asp Ser Met Glu Glu Leu Leu
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Arg Val Ala Gly Glu Lys Phe Glu Gly Tyr Lys Pro Thr Lys Val Val
725 730 735
Asn Glu Glu Asn Ala Glu Ile Asp Asp Ile Ser Val Val Arg Asp Gly
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Asp His Leu Tyr Leu Leu His Asn Asp Tyr Asn Asn Met Asn Ile Thr
755 760 765
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<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgacgtttt cgtgcacgaa aaacttcttc 30
<210> 4
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctaaaaggta caagttatat tcatattatt atagtc 36
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agtgtaccga aatgcgcgta cc 22
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctgagcaaca gtgtccattt ggt 23
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gatctagcac aacagcaagg 20
<210> 8
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gatacaggtg atcaccatct cgg 23
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
caatgtgcct gccatgtatg 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccagcagctt ccattccaat 20
Claims (5)
1.梨保卫细胞钾离子吸收通道基因PbrKAT1，其特征在于，该基因的核苷酸序列如SEQID No.1所示。
2.权利要求1所述的梨保卫细胞钾离子吸收通道基因PbrKAT1编码的蛋白质。
3.根据权利要求2所述的蛋白质，其特征在于，所述蛋白质的氨基酸序列如SEQ IDNo.2所示。
4.含有权利要求1所述梨保卫细胞钾离子吸收通道基因PbrKAT1的重组表达载体、表达盒、转基因细胞系或重组菌。
5.用于扩增权利要求1所述梨保卫细胞钾离子吸收通道基因PbrKAT1全长或任一片段的引物对，其特征在于，该引物对的正反向引物如SEQ ID No.3和 SEQ ID No.4所示。
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