CN115322248A - 一种钾离子通道蛋白AlAKT1、其编码基因及其应用 - Google Patents
一种钾离子通道蛋白AlAKT1、其编码基因及其应用 Download PDFInfo
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- CN115322248A CN115322248A CN202210551792.6A CN202210551792A CN115322248A CN 115322248 A CN115322248 A CN 115322248A CN 202210551792 A CN202210551792 A CN 202210551792A CN 115322248 A CN115322248 A CN 115322248A
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Abstract
本发明公开一种钾离子通道蛋白AlAKT1、其编码基因及其应用，属于生物技术领域。本发明获得的编码钾离子通道蛋白的基因AlAKT1具有K+吸收功能，可提高转基因酵母在1mM K+和100μM K+条件下的K+吸收能力，可通过基因工程技术应用于植物中，从而应对日益严重的土壤低钾和盐渍化问题，在植物高效利用钾离子和耐盐方面具有应用价值，可减少钾肥的施用，从而降低生产成本。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种钾离子通道蛋白AlAKT1、其编码 基因及该基因在培育高效利用钾离子和/或耐盐转基因植物。
背景技术
钾是植物生长所必需的大量营养元素之一，广泛分布于植物的各组织和器官，是植物细胞内含量最丰富的一价阳离子。钾在植物众多生理功能中发挥着重要的作用， 如维持细胞离子平衡、调节细胞膨压、调节各种酶的活性以及参与蛋白质合成等，维 持高并且相对稳定的钾离子含量对植物生长至关重要。植物缺钾会出现明显的缺钾症 状，表现为易倒伏，叶片易失水，耐旱耐盐性降低，叶片发黄和组织坏死等。耕地土 壤缺钾会导致农作物品质和产量大幅下降。因此，维持植物生长过程中钾营养供给十 分重要。
同时，土壤盐渍化是一个全球性的生态问题。过量的钠离子对植物的成长具有毒害作用，大部分农作物在盐胁迫状态下不能正常生长，表现为发育迟缓，发芽率降低， 器官生长和分化受到抑制，新陈代谢减缓等症状。土壤盐渍化不仅会造成粮食减产， 也是土地荒漠化和耕地退化的主要原因之一，严重制约着农业生产。
研究表明，不同种类的植物或同种植物的不同品种对土壤缺钾和盐胁迫的生理反应及对钾的吸收利用效率存在明显差异，并且这种差异性是可以遗传的，说明植物这 种性状是由遗传基因控制的。在高盐环境下仍具有钾吸收能力，进而维持高的K+/Na+比是决定植物的耐盐性的关键因素。因此，植物钾吸收能力与其耐盐性息息相关。自 从20世纪90年代以来，许多负责植物钾吸收及转运的钾离子通道基因和钾离子转运 蛋白基因被相继克隆和鉴定。其中，钾离子通道在植物钾吸收中起着重要的作用，研 究钾离子通道基因，为我们深入认识和理解作物体内钾的吸收及利用机制，进而提高 钾的有效利用率具有积极的作用。目前，已有一些植物的钾离子通道被克隆并鉴定出 来，如OsAKT1(水稻)，AKT1(拟南芥)，ZMK1(玉米)，MIRK(甜瓜)和MKT1(冰叶日 中花)等。然而这些基因大部分来自甜土植物，并且多数对Na+敏感。在土壤既缺钾又 有盐渍化的情况下，只有对Na+不敏感的钾通道蛋白才能正常发挥功能，使转基因作 物既耐低钾又耐盐，因此分离获得对Na+不敏感的钾通道蛋白基因显得尤为重要。
獐茅(Aeluropus littoralis)是一种单子叶禾本科的盐生植物，与小麦同族，主要 分布于中国的山东、辽宁、河北和江苏等省。獐茅高约5-25cm，具有发达的根状茎 和匍匐茎，并且可依靠匍匐茎进行无性繁殖，是防风固沙的优良植被；作为优良牧草 之一，獐茅具有很强的耐盐能力，然而，从獐茅中分离对Na+不敏感的钾通道蛋白基 因的相关研究还未见报道。
发明内容
针对现有技术中存在的上述问题，本发明的目的是提供一种钾离子通道蛋白AlAKT1、其编码基因及该基因在培育高效利用钾离子和/或耐盐转基因植物。该基因 是从獐茅中分离出来的，具有K+吸收功能，编码一种双亲和性钾通道蛋白，转入该基 因能够提高酵母在缺钾及盐胁迫条件的K+吸收能力，这种能够提高转基因酵母的耐低 钾和耐盐的特性，可通过基因工程技术应用于植物上，从而应对日益严重的土壤缺钾 和盐渍化问题，在农作物高效利用钾离子方面具有应用价值，可减少钾肥的施用，从 而降低生产成本。
本发明目的是通过以下技术方案实现：
本发明的目的之一在于提供了一种钾离子通道蛋白AlAKT1，其氨基酸序列如SEQID NO:2所示；其是由764个氨基酸残基组成的蛋白质。
本发明进一步提供所述钾离子通道蛋白的衍生蛋白质，即由序列表中SEQ ID NO:2 所示的氨基酸残基经过一个或几个氨基酸残基取代、缺失或添加而产生的具有相同的生物 学功能的衍生蛋白质。
本发明的目的之二在于提供编码上述的钾离子通道蛋白AlAKT1的基因，其核苷酸序列如SEQ ID NO:1所示，其序列由2292个核苷酸组成，自5′端第1到第2292 位残基为该基因的开放阅读框序列。
本发明进一步提供具有与上述的SEQ ID NO:1所示的核苷酸序列95％以上的同源性，且编码与SEQ ID NO:1所示的核苷酸序列编码的蛋白质具有相同生物学功能的核 苷酸序列。
本发明的目的之三在于提供一种重组表达载体，重组表达载体上都含有上文所述的 碱基序列。本发明将来源于獐茅的钾离子通道蛋白基因AlAKT1与可诱导外源基因表达的 载体pYES 2.0及PTF101相连获得AlAKT1基因重组表达载体。酵母表达载体pYES2.0是带有Amp和Ura筛选标记的穿梭质粒，在原核生物如大肠杆菌中，该载体可以利用T7启 动子启动下游基因的表达；在真核微生物如酿酒酵母中，该载体则可以利用被半乳糖高诱 导的GAL启动子启动下游基因的表达。在大肠杆菌内，载体pYES2.0由于具有Amp抗性 所以可进行氨苄霉素筛选，而在真核微生物酿酒酵母CY162中，氨苄霉素对其生长不会 造成影响，因此在酵母中无法用该方法筛选。酿酒酵母CY162的基因型显示，其Ura基 因突变，尿嘧啶合成功能受到阻碍，因此不能在缺少Ura的培养基中生长，可据此进行有 效筛选。PTF101作为植物表达载体，含有除草剂的抗性基因Bar基因，可以利用除草剂 对转基因植株进行筛选。
本发明的目的之四在于提供一种含有上文所述重组表达载体的宿主细胞。在本发明的技术方案中，所述的宿主细胞优选采用酿酒酵母K+吸收缺陷型菌株CY162 (Matα，ura3-52，his3D200，his44-15，trkD1，trkD2，pcK64)及根癌农杆菌EHA105 菌株。
本发明的目的之五在于提供上文所述基因在培育低钾和盐胁迫条件下仍能良好生长的植物中的应用。本发明的AlAKT1基因所编码的AlAKT1蛋白具有K+吸收和对 Na+不敏感的特性，本发明成功得到该基因的转基因真核微生物(酿酒酵母)，并证 明转基因微生物在低钾和高盐(Na+)浓度下的K+吸收功能；然后成功得到该基因 的转基因植物(烟草)，并证明转基因烟草在低钾和高盐(Na+)浓度下的K+吸收功 能。
本发明相对于现有技术具有的有益效果如下：
本发明的基因AlAKT1具有钾离子通道蛋白的功能，本发明的实施例证明它能够提高钾离子缺陷型酵母在1mM K+、100μM K+以及盐胁迫条件下的K+吸收能力，进而 提高钾离子缺陷型酵母的钾利用效率及耐盐性。同时也能够提高转基因烟草在1mM K+、100μM K+以及盐胁迫条件下的K+吸收能力，进而提高转基因烟草的钾利用效率 及耐盐性。因而本发明的基因可应用于培育在低钾且盐胁迫环境下仍能良好生长的植 物，为耐盐性植物的优良种质资源的开发提供了新途径。
附图说明
为了更清楚地说明本发明实施例，下面将对实施例涉及的附图进行简单地介绍。
图1为AlAKT1基因克隆PCR电泳图，其中，A为保守区部分片段克隆；B为3’ 端部分片段克隆；C为5’端部分片段克隆；D为ORF全长PCR电泳图；M1：DL 2000 Marker；M2：DL5000Marker。
图2为本发明的AlAKT1基因编码的蛋白的结构示意图。
图3为本发明的钾通道蛋白AlAKT1疏水性分析图。
图4为本发明的AlAKT1基因编码的蛋白的进化分析图。
图5为不同胁迫条件下采用实时定量PCR的方法分析AlAKT1基因在地上部表达 的结果图。
图6为不同胁迫条件下采用实时定量PCR的方法分析AlAKT1基因在地下部表达 的结果图。
图7为转基因酵母的功能互补实验结果图；A1为50mM K+浓度下酵母的生长情 况图；A2为50mM K+及200mM Na+浓度下酵母的生长情况图；B1为1mM K+浓度下 酵母的生长情况图；B2为1mM K+及200mM Na+浓度下酵母的生长情况图；C1为 0.1mM K+浓度下酵母的生长情况图；C2为0.1mM K+及200mM Na+浓度下酵母的生长 情况图。
图8为转基因酵母在1mM K+浓度下的离子耗竭实验结果。
图9为转基因酵母在0.1mM K+浓度下的离子耗竭实验结果。
图10为转基因酵母在1mM K+、200mM Na+浓度下的离子耗竭实验结果。
图11为转基因酵母在0.1mM K+、200mM Na+浓度下的离子耗竭实验结果。
图12为转基因酵母在50mM K+及不同Na+浓度下的生长情况比较实验结果。
图13为转基因酵母在1mM K+及不同Na+浓度下的生长情况比较实验结果。
图14为转基因酵母在0.1mM K+及不同Na+浓度下的生长情况比较实验结果。
图15为转基因烟草在10mM、1mM及0.1mM K+浓度下的鲜重。
图16为转基因烟草在10mM、1mM及0.1mM K+浓度下的干重。
图17为转基因烟草在150mM Na+与10mM、1mM及0.1mM K+浓度下的鲜重。
图18为转基因烟草在150mM Na+与10mM、1mM及0.1mM K+浓度下的干重。
具体实施方式
下面结合实施例对本发明进行详细的说明，但本发明的实施方式不限于此，显而易见 地，下面描述中的实施例仅是本发明的部分实施例，对于本领域技术人员来讲，在不付出 创造性劳动性的前提下，获得其他的类似的实施例均落入本发明的保护范围。
下面实施例中未注明具体实验条件的方法，通常按照常规条件或分子克隆中所述条件，或按照产品说明书上所提供的条件。下面实施例中所用的材料、试剂等如无特 殊说明，均可从商业途径得到。
试验中使用的试剂盒以及试剂：无缝连接试剂盒：Trangene公司，pEASY-UniSeamless Cloning and Aseembly Kit；质粒提取试剂盒：Sangon Biotech公司，SanPrep柱式质粒DNA小量抽提试剂盒。
实施例1AlAKT1基因cDNA的克隆与分析
(1)獐茅总RNA提取
称取50mg新鲜獐茅叶片组织，于液氮中研磨成粉末，将研磨好的材料迅速转移至含有1mL预冷的Trizol试剂的离心管中，震荡混匀，室温静置10min；加入0.2mL氯仿， 剧烈震荡15s，室温放置5min；4℃ 12000r/min，10-15min，取上清；将上清转移至新 的离心管中；加入0.5mL异丙醇，混匀，室温10min；4℃，12000r/min，离心10min； 弃上清，加入1mL冰预冷的70％乙醇(新鲜配制)，洗涤沉淀；4℃，7500r/min，5min； 弃上清，室温晾干(勿太干)，加入20μL RNase-free ddH2O，充分溶解RNA，-70℃ 保存备用。采用1％的琼脂糖凝胶电泳检测RNA提取的质量。结果呈现了28S、18S、 5S三种典型的RNA带型，各条带清晰，无明显的拖尾现象，表明提取的RNA无明显降 解，可进一步用于RT-PCR实验。
(2)单链cDNA的获得
以提取的獐茅总RNA为模板(500ng)，按照宝生物Reverse Transcriptase M-mLV反转录说明书进行反转录反应，得到的单链cDNA可直接用于2nd-Strand cDNA的合 成或者PCR扩增等。
(3)獐茅AlAKT1基因部分编码区的获得
①简并引物设计：根据NCBI上提供的冰叶日中花、烟草、拟南芥、水稻和大 麦等植物的AKT1家族基因序列进行同源性比对，在其高度保守区设计一对简并引物 AKF(SEQ IDNO:3)/AKR(SEQ ID NO:4)。此简并引物中Y＝C/T，D＝A/G，W＝A/T， R＝A/G。
②PCR扩增：以反转录获得的单链cDNA为模板，AKF(SEQ ID NO:3)和AKR (SEQ IDNO:4)为引物，PCR扩增AlAKT1通道蛋白基因部分编码区，将PCR产物经 1％琼脂糖凝胶电泳分离(结果如图1A)，至溴酚蓝指示带位于凝胶2/3处，在紫外灯 下切下750bp区域的凝胶放入1.5mL离心管中，用TaKaRa公司(大连)的MiniBEST Agarose Gel DNA Extraction KitVer.3.0回收试剂盒进行回收。
③回收DNA片段连接、转化与测序：采用宝生物pMD18-T vector试剂盒，将回 收后的PCR产物直接与T载体相连，连接产物用于大肠杆菌转化，PCR检测阳性的克隆 送测序，测序后即可确定AlAKT1基因的部分片段信息。将测序结果通过NCBI在线数 据库上进行Blast-N比对显示：该序列与来自小米、玉米、小麦、二穗短柄草和大麦的 钾离子通道基因序列的同源性达到91％，89％，88％，87％和87％。因此初步推断获 得的基因序列是獐茅钾离子通道蛋白基因保守区的部分序列。
(4)RACE法克隆獐茅AlAKT1基因3’cDNA
①3’RACE特异引物的设计：根据已获得的獐茅AlAKT1基因部分cDNA片段 以及TaKaRa(大连)公司的3’-full RACE Kit提供的引物3’RACE Outer Primer/3’ RACE InnerPrime设计PCR引物(表1)：3′正向特异性外侧引物3’O(SEQ ID NO:5)， 巢式特异性内侧引物3’I(SEQ ID NO:6)。
②单链cDNA的获得。按照Takara 3′RACE试剂盒操作，反转录所得单链 cDNA作为巢式PCR模版。
③巢式PCR反应：Outer PCR反应用外侧引物3’O(SEQ ID NO:3)和3’RACE OuterPrimer进行外侧扩增。Inner PCR反应以外侧PCR产物为模板，用内侧引物3’I (SEQ ID NO:6)和3’RACE Inner Primer引物进行巢式扩增。PCR产物经1％琼脂糖凝 胶电泳分离(结果如图1B)、切胶回收、连接克隆载体、转化大肠杆菌感受态、PCR 及酶切检测(方法同上)，选取长度为2000bp的阳性克隆送测序。测序结果通过NCBI 数据库做Blast-N比对，结果显示：该序列和来自小米、二穗短柄草、玉米和小麦的钾 离子通道蛋白基因序列的同源性分别为85％、83％、81％和80％。初步推断，该基因序 列是獐茅钾离子通道蛋白基因的3’端序列。
(5)RACE法克隆AlAKT1基因5’cDNA
本实施例中AlAKT1 5’cDNA序列采用RACE方法获得。步骤如下：根据已得到 的AlAKT1基因部分序列设计3条特异性引物，以獐茅总RNA为模板，首先以GSP1 (SEQ ID NO:7)为引物进行逆转录反应得到第一链cDNA，再使用末端转移酶(TdT) 在cDNA第一链的3′末端加上寡聚胞嘧啶[Oligo d(C)]尾，利用两条反向引物GSP1 (SEQ ID NO:7)、GSP2(SEQ IDNO:8)与锚定引物AAP对加尾产物进行半巢式 PCR扩增，对PCR产物进行电泳，切取目标条带回收。回收片段连接pMD18-T载体， 转化大肠杆菌DH5α感受态，对阳性克隆进行鉴定、筛选和测序。
①cDNA第一链的合成：以提取的獐茅总RNA为模板，GSP1为引物进行逆转 录，获得第一链cDNA，用于后续反应。
②加尾反应：将以上PCR产物进行加尾反应。
③巢式PCR扩增：以上述加尾产物为模板，锚定引物AAP为正向引物，先后 以特异性巢式引物5-GSP1(SEQ ID NO:7)、5-GSP2(SEQ ID NO:8)为反向引物进 行巢式PCR反应。PCR产物经1％琼脂糖凝胶电泳分离(图1C)、切胶回收，连接 克隆载体，转化大肠杆菌感受态，PCR及酶切检测片段长度(方法同上)，检测长度 范围为500～750bp的阳性克隆送测序。通过NCBI数据库对测序结果进行Blast-N比 对，结果显示：该序列和来自甘蔗、玉米、小麦和大麦的AKT1型钾离子通道蛋白基 因的5′端序列同源性分别为87％、86％、85％和84％，初步推断该序列是獐茅钾离 子通道蛋白基因AlAKT1的5′端序列。
(6)AlAKT1基因全长cDNA获得
将上述AlAKT1通道蛋白基因保守区、3’端和5’端片段进行拼接，通过NCBI ORFFinder软件获得AlAKT1基因cDNA编码区全序列2292bp，核苷酸序列如SEQ ID NO:1 所示。即自SEQ ID NO:1序列的5’端1位起始密码子ATG，至2292位终止密码子TAA 的碱基序列，为2292bp的SeAKT1编码区，其编码一个由763个氨基酸构成的蛋白， 将此钾离子通道蛋白命名为AlAKT1(SEQ ID NO:2)；以獐茅cDNA为模板，根据拼 接所得全长序列设计编码区全长引物AlAKT1-F(SEQ ID NO:9)/AlAKT1-R(SEQ ID NO:10)，PCR获得该基因完整ORF序列，长度为2292bp(如图1D)。PCR产物测 序后长度为2292bp的开放阅读框序列。测序结果表明此全长序列与拼接序列完全相 同。
(7)生物信息学分析
利用在线expasy软件(http://web.expasy.org/protscale/)对AlAKT1进行疏水性分 析，结果表明AlAKT1具有多个跨膜结构域，是一个跨膜蛋白(图2)。系统发育树 分析结果表明AlAKT1是典型的Shaker家族内向整流型钾离子通道(图4)，与已报 道的小麦、甘蔗、玉米和水稻等植物钾离子通道蛋白的氨基酸序列同源性在79％～83％ 之间。
表1.AlAKT1基因PCR扩增的引物
实施例2不同处理条件下AlAKT1表达量分析
本实验所用的獐茅材料从辽宁省大连市营城子海滨盐田采集，种植于大连理工大学生物工程学院，采用压条法进行无性繁殖，以保证其遗传背景的一致性。选取长势 一致的植株通过水培分别进行缺钾、盐、缺钾及盐胁迫处理。本实验采用不含钾的营 养液用于诱导缺钾胁迫，采用含200mM NaCl的营养液用于盐胁迫诱导。无胁迫处理 的植株作为对照组。各处理(缺钾、盐胁迫和缺钾与盐胁迫)和对照组均设三个平行， 24h后取样，分别收集其根和叶片并混合均匀提取总RNA，反转录获得cDNA，方法 同实施例1。以eEF为内参基因，进行qRT-PCR，引物碱基序列见表2。
表2.qPCR扩增引物
| 名称 | 引物碱基序列(5’--3’) |
| q-F1(SEQ ID NO:11) | GAGGTCGTCTGGATTTG |
| q-R1(SEQ ID NO:12) | ATTGTTATCTGATTCGTTTG |
| eEF-S(SEQ ID NO:13) | AGCAAAACGACCCAGAGGAG |
| eEF-A(SEQ ID NO:14) | GGTGATGCTGGTATGGTGAAGA |
结果表明，相对于对照组，地上部在缺钾胁迫下AlAKT1显著上调表达，在盐胁 迫下AlAKT1呈上调表达(图5)。地下部在缺钾胁迫、盐胁迫、缺钾及盐胁迫下AlAKT1 都呈现上调表达(图6)。
实施例3转基因酵母的功能互补实验和耗竭实验
(1)转基因酵母的筛选
将用EcoRⅠ和NotⅠ双酶切得到的pMD18-T-AlAKT1和线性化后pYES 2.0进行 连接，转化大肠杆菌DH5α，并进行PCR检测和测序。测序正确后得到连接有AlAKT1 基因ORF序列的重组质粒，命名为pYES2.0-AlAKT1。将质粒pYES 2.0-AlAKT1转入 到K+吸收缺陷型酵母菌株CY162中，在选择性培养基SC-Ura中进行筛选，得到阳 性转化株。转入的质粒中由于含有编码URA基因，所以菌株可以在筛选培养基上正 常生长，而没有转化成功的菌株则无法生长，然后对转化子进行PCR鉴定。
由于K+吸收缺陷型菌株CY162很难在低钾(低于7mM)环境中生长，酵母功能 互补实验可用来鉴定K+通道蛋白行使钾离子吸收转运功能。由于重组质粒中的插入基 因可被载体上GAL1启动子诱导表达，因此选用半乳糖做为糖原进行实验。
(2)转基因酵母的功能互补实验
①将上述转化后的酵母分别接种于50mL(含50mM K+)的YPD液体培养基 中，过夜震荡培养12-14h；
②取1mL菌液转接到100mL SC-Ura+Gal+50mM K+的液体诱导培养基中， 28℃震荡培养48h，诱导重组蛋白表达；
③测量菌液的OD600值，通过计算取适当菌液，低速离心，用不含K+的液体培 养基洗涤2-3次，并重悬菌体使OD600值均为1.0，再对菌液进行梯度稀释(10倍、 100倍和1000倍)；
④各取1μL菌液分别点在SC-Ura+Gal+1mM K+、SC-Ura+Gal+0.1mM K+、 SC-Ura+Gal+1mM K++200mM Na+和SC-Ura+Gal+0.1mM K++200mM Na+的固体培养 基上，正置1h使菌液吸收；
⑤倒置于28℃恒温培养箱中培养3-4d，观察结果。
如图7所示，转入重组质粒pYES 2.0-AlAKT1的酵母菌株在图7中用AlAKT1表 示，转入空载体的酵母菌株则用pYES2.0表示，图7中的1、10-1、10-2和10-3为菌 液梯度稀释的倍数；在不含Na+的培养基中，当培养基含有50mM K+时，酵母均可以 生长(图7A1)；当培养基含有1mM K+(图7B1)或0.1mM K+(图7C1)时，转入 AlAKT1的酵母可以生长，而转空载体酵母的生长则受到了抑制。在含有200mM Na+的培养基中，含有50mM K+(图7A2)、含有1mM K+(图7B2)及0.1mM K+(图 7C2)的培养基中转入AlAKT1的转基因酵母均可以生长，而转空载体的酵母的生长均 受到了抑制。图7的结果表明，AlAKT1蛋白具有双亲和性的K+吸收能力，是一种钾 离子通道蛋白，同时也具有一定的耐盐能力。
(3)转基因酵母的K+耗竭实验
①将转化后的酵母分别接种于50mL(含50mM K+)的YPD液体培养基中， 过夜震荡培养12-14h；
②取1mL菌液转接到100mL SC-Ura+Gal+50mM K+的液体诱导培养基中， 28℃震荡培养48h，诱导重组蛋白表达；
③4℃，3000rpm离心5min，收集酵母细胞，用SC-Ura+Gal+0mM K+的液体培 养基洗涤并重悬，28℃震荡培养5h；
④分别测量菌液的OD600值，通过计算取适当菌液，低速离心，分别用 SC-Ura+Gal+1mM K+、SC-Ura+Gal+0.1mM K+、SC-Ura+Gal+1mM K++200mM Na+和SC-Ura+Gal+0.1mM K++200mM Na+的液体培养基洗涤并重悬于100mL该液体培 养基中，使酵母OD600值均为0.4，继续振荡培养；
⑤前60min每隔20min取150μL菌液，之后每隔1h取样一次，12000r/min离 心10min，弃去沉淀保留上清，同时回补150μL的耗竭所用液体培养基。用原子吸 收分光光度计测定上清中的K+含量。
如图8～图11所示，随着时间变化，培养基中的K+浓度逐渐下降。与转入空载的 酵母相比，转AlAKT1酵母所在的培养基中K+浓度下降的更快，即K+得到更高效地 吸收。结果表明，AlAKT1具有双亲和性的K+吸收能力，是一种钾离子通道蛋白，其 也具有一定的耐盐能力。
实施例4转基因酵母在不同K+/Na+浓度下生长分析
将CY162-AlAKT1和CY162-pYES2.0分别接种于SD液体培养基(含Gal+100mM K+)中，28℃震荡培养(225r/min)过夜。取其中500μL菌液接种于50mL分别含 0、50、100、150、200、250、300mM NaCl的SD(含有50mM、1mM、0.1mM K+) 液体培养基中28℃震荡培养(225r/min)20h。
以含50mM K+的SD液体培养基作参比，在620nm波长下测定各培养物的光密 度，以光密度为纵坐标，以NaCl浓度为横坐标作图。
在50mM K+介质中，随着Na+的浓度的增加，与转入空载的酵母相比，转AlAKT1 酵母生长情况较好(图12)。在1mM K+介质中，随着Na+浓度的增加，转AlAKT1 酵母生长情况明显优于转入空载的酵母(图13)。在0.1mM K+介质中，随着Na+浓 度的增加，转AlAKT1酵母的生长显著优于CY162-pYES2.0(图14)。结果表明，AlAKT1 具有双亲和性的K+吸收能力，是一种钾离子通道蛋白，其也具有一定的耐盐能力。
实施例5烟草的转化及功能分析
(1)AlAKT1基因植物表达载体构建
①植物表达载体PTF101的获得：采用生工生物工程有限公司质粒小提试剂盒提取PTF101植物表达载体的质粒，具体方法见说明书。利用SmaI限制性内切酶对pTF101-35s 植物表达载体进行酶切反应，使其线性化，采用琼脂糖凝胶电泳对酶切后的产物检测，切 下目标区域处的凝胶并用胶回收试剂盒进行回收纯化。
②AlAKT1编码区的获得：以反转录所得单链cDNA为模板，分别以AlAKT-F(SEQ IDNO:9)及AlAKT1-R(SEQ ID NO:10)为正反向引物，扩增AlAKT1基因编码区。利用 1％的琼脂糖凝胶电泳检测PCR结果，切下目标区域处的凝胶并用胶回收试剂盒进行回收 纯化。
③连接：利用无缝克隆试剂盒将回收纯化后的植物表达载体PTF101与AlAKT1基因编码区进行连接。
④采用热击法将连接液转化大肠杆菌DH5α，PCR检测筛选出阳性克隆。
⑤阳性重组质粒转化农杆菌：提取PTF101-AlAKT1质粒，转化农杆菌EHA105感 受态细胞。根癌农杆菌EHA105感受态细胞的制备方法如下：挑取EHA105单菌落于含有 100mg/L利福平和100mg/L卡那霉素的YEP液体培养基中，28℃，180rpm振荡培养过夜。 取过夜培养的菌体按1:100的比例接种到50mL YEP液体培养基中，28℃，180rpm振荡培 养3-4h至细菌生长对数期OD600＝0.5-0.6左右。取5mL菌液于4℃，4000rpm离心10min， 沉淀用5mL预冷的TE(pH7.5)洗涤一次，加1mL新鲜的YEP培养基，重新悬浮，分装， -70℃保存。
⑥采用冻融法将质粒PTF101-AlAKT1导入农杆菌，方法如下：取一管(0.2mL)根癌农杆菌(Agribecterium tumefaciens)EHA105菌株感受态细胞置冰上融化，加入1μg质粒PTF101-AlAKT1，混匀，然后依次在冰上、液氮中及37℃水浴中放置5min，用YEP液体 培养基稀释至1mL，于28℃，180rpm振荡培养2-4h；取适量菌液涂布于含有100mg/L利 福平、100mg/L卡那霉素和50mg/L壮观霉素的YEP平板培养基上，28℃培养36h左右长 出抗性菌落，PCR确定阳性克隆。
(2)叶盘转化法转化烟草
a.转化用烟草叶片的制备：取生长二十天左右的无菌烟草叶片去除主脉，用无菌剪刀 将其剪成1cm2的小块作为侵染用外植体。
b.转化用农杆菌菌液的制备：将从-80℃冰箱中取出来的含有重组表达载体的农杆菌在 固体的YEP培养基(YEB+100mg/L Rif+100mg/L Kan+50mg/L Spec)中进行划线培养。挑取 生长状态良好的单菌落接种于5ml含有上述抗生素的YEP液体培养基中，28℃，180rpm 过夜摇菌20h左右。将过夜活化的农杆菌按照1:50的比例加入到不含抗生素的液体YEP 中，28℃振荡培养4～6h至OD600约为0.5～0.6。
c.转化：将剪好的烟草叶片放入上述农杆菌菌液中5-10min，期间缓慢摇晃2-3次。侵 染结束后将叶片置于无菌的滤纸上以吸干残余的菌液，远轴面向上置于分化培养基(MS+6-BA 0.5mg/L+NAA 0.1mg/L)上28℃暗培养2d。
d.抗性芽的筛选及转基因植株的生根培养：共培养两天后，叶盘周围会长出清晰可见 的农杆菌菌落，此时用灭菌水将材料清洗一次，转移到含有双丙氨膦和头孢霉素的筛选培 养基上进行筛选，两周左右即可形成愈伤组织。将烟草愈伤组织转移到新的筛选培养基上 继续培养至长出幼芽，用灭菌手术刀切下2cm以上的幼芽，置于生根培养基 (MS+NAA0.1mg/L+500mg/L头孢+双丙氨膦)上进行生根培养。
实施例6转基因烟草在低钾及盐胁迫下的生理检测
(1)转基因烟草的K+营养实验
将经PCR和RT-PCR检测阳性的转AlAKT1基因烟草(AlAKT1-1和AlAKT1-2)以及 野生型烟草种子点种在含10mM KCl的1/2MS培养皿中培养一周，挑选表型一致的烟草 幼苗各9棵(每3棵为一组，共3次生物学重复)，分别在含有10mM、1mM及0.1mM K+的1/2MS培养基中再培养54d，分别测量烟草地上部分和地下部分的生物量。
实验结果如图15-16所示，在10mM K+浓度的情况下，转基因烟草的生理指标与野生 型无显著差别；在1mM K+浓度的条件下，AlAKT1-1烟草的鲜重、干重均显著高于野生 型烟草；在0.1mM K+浓度低的条件下，转基因烟草的鲜重与野生型无显著差别，转基因 烟草的干重均显著高于野生型。
(2)盐胁迫下转基因烟草的K+营养实验
将经PCR和RT-PCR检测阳性的转AlAKT1基因烟草(AlAKT1-1和AlAKT1-2)及野 生型烟草种子点种在含10mM KCl的1/2MS培养皿中培养一周，挑选表型一致的烟草幼 苗各9棵(每3棵为一组，共3次生物学重复)，分别在含有NaCl含量为150mM且KCl 含量为10mM、1mM及0.1mM的1/2MS培养基中再培养54d，分别测量烟草地上部分和 地下部分的生物量。
实验结果如17-18所示，当NaCl浓度为150mM时，在10mM K+浓度的情况下，AlAKT1-1烟草的生物量指标均显著低于野生型，AlAKT1-2的生物量指标与野生型无显 著差别；在1mM K+浓度的条件下，转基因烟草的鲜重和干重均显著高于野生型。在0.1mM K+浓度低的条件下，AlAKT1-2烟草的鲜重显著高于野生型(图17-18)。结果表明AlAKT1 蛋白在盐胁迫下，双亲和性的吸收K+，不同株系的烟草由于转入AlAKT1基因的表达量不 同，因此生物量存在一定差异，推测AlAKT1可介导双亲和钾吸收且具有一定的耐盐功能， 可增强植物的耐盐性，预示其可在改良植物钾营养吸收和耐盐性中发挥有效作用。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管 参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然 可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行 等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方 案的范围。
SEQUENCE LISTING
<110> 大连理工大学
<120> 一种钾离子通道蛋白AlAKT1、其编码基因及其应用
<130> 20220516
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2293
<212> DNA
<213> 人工序列
<400> 1
atgacgtatt tgcttgaaga tgatccaaag aggatagctt ggcggtatac tactagttgg 60
tttgttcttg acgtggcctc taccgtccca acagaatttg ctcgacggat actacctcat 120
aacctcaggt cgtatggatt cttcaacatg ctgcgtcttt ggcgtcttcg tagagtcagc 180
tctctctttg ctcgattgga gaaagatagg cacttcaatt acttctgggt tcgatgtgca 240
aagctcatct gtgtcacact ttttgctgtc cactgttcgg catgcttcta ctatcttctt 300
gctgataggt atcctgaacc aacgcataca tggatcggca atgcaatacc agattttcac 360
gagagaagct tatggattcg ctatgtaacg tccatgtatt ggtcaatcac tactcttacc 420
actgtgggtt atggtgattt tcatgcggat aacacaaggg aaatgatttt caacatattt 480
tacatgctat ttaaccttgg attgactcgc ctatttgatc ggaaacatga ccaatctagt 540
tgtacatggc accagccgta ctcgaaaata tagagataca attcaagcag caaccagctt 600
tgcactaagg aatcagttac cgcatcggtt gcaagatcaa atgatctcac atcttagttt 660
gaagttcagg acagattcgg aaggtcttca acaacaagag acccttgatg cgctgcctaa 720
ggctattaga tccagcattt ctcagtatct attttttaat ctggttcaaa aggtttactt 780
gtctgaaggg gtgtcgaatg acctgatatt ccaactggtt tctgaaatga aagctgaata 840
ttttccacct agggaagatg tcattctgca gaatgaagca cccactgact tctacatcct 900
agtttctggt agcgcggagc taatagagct gcaaaatggt gcagaacagg tggctggggt 960
ggctaaatca ggagatgttg ttggtgaaat tggggttctt tgttataggc ctcaattatt 1020
cacagttcga acaaaatcct tatgccagct cctgcgtata aatcgtactg cctttctcag 1080
cattgttcaa tccaatgtgg gagatggaac tataataatg aataacctta ttcagttact 1140
aaaagagcag aaagaaaaca ctgtaatggt tggtgtcctg aaggaggttg agagcatgct 1200
agcaagaggt cgtctggatt tgccaattac cctctgtttt gcagtaaata gaggagatga 1260
ctttttgttg catcaacttc ttaagcgtgg tttggatcca aacgaatcag ataacaatgg 1320
ccatacggca ctgcatatag ctgcttctaa aggaaatgaa caatgtgtca agcatctgct 1380
agactatgat gctgatccta atgccaggga ctctgaagga aaggttccat tatgggaggc 1440
tatgtgcgaa aagcatgaca gagttgtgca gttgttagtc cagaatggtg ccgatttatc 1500
atggggggac acagccttat atgcttgtat cgctgttgaa gaaaataaca ttgagctgct 1560
taaggacatt atccgttacg gtggcgatgt aaaaagatcg ctgaaagatg gaaccactcc 1620
actgcataaa gctgtctgtg atggaaatgt tcagatggtt gagttcttgc tggaacaggg 1680
tgctgaaatt gataaactgg acaacaatgg ctggacgcca agagctctag ctgagcaaca 1740
aggccatgcc tacatacaac tcctgtttaa atcacgacga gaagcaccaa agcatcacgt 1800
tccaaataat agggtggcac cttcgttaat agggaggttt aacagtgagc cttcaatgca 1860
aaatgtagac agcgaagata ttggagtaca aaacaaagtt tttccaaaga agctccttaa 1920
aaggagggtc agttttcaga actccctttt cggtgttatt tcttcaacta atgcaagccg 1980
ggacaccggc cccctactcc caagaggtcc tgcagcaaca agtgccctaa attgcaatac 2040
caactcgctc attagggtga caatcagctg ccctgagaag gagaacaccg ctgcgaagct 2100
tgtcctccta ccacggtcaa tgcaggagtt tcttgatcta ggcgcaaaga agttcgactt 2160
caggcctacc aaggtcctga caattgaagg tgctgaggtt gatgaggttg aacttatcag 2220
agatggcgat catcttgttc tcgtcagtga tggctgggtg ccagatgatg tacaaaataa 2280
gcttcaacaa taa 2293
<210> 2
<211> 763
<212> PRT
<213> 人工序列
<400> 2
Met Thr Tyr Leu Leu Glu Asp Asp Pro Lys Arg Ile Ala Arg Arg Tyr
1 5 10 15
Thr Thr Ser Trp Phe Val Leu Asp Val Ala Ser Thr Val Pro Thr Glu
20 25 30
Phe Ala Arg Arg Ile Leu Pro His Asn Leu Arg Ser Tyr Gly Phe Phe
35 40 45
Asn Met Leu Arg Leu Trp Arg Leu Arg Arg Val Ser Ser Leu Phe Ala
50 55 60
Arg Leu Glu Lys Asp Arg His Phe Asn Tyr Phe Trp Val Arg Cys Ala
65 70 75 80
Lys Leu Ile Cys Val Thr Leu Phe Ala Val His Cys Ser Ala Cys Phe
85 90 95
Tyr Tyr Leu Leu Ala Asp Arg Tyr Pro Glu Pro Thr His Thr Trp Ile
100 105 110
Gly Asn Ala Ile Pro Asp Phe His Glu Arg Ser Leu Trp Ile Arg Tyr
115 120 125
Val Thr Ser Met Tyr Trp Ser Ile Thr Thr Leu Thr Thr Val Gly Tyr
130 135 140
Gly Asp Phe His Ala Asp Asn Thr Arg Glu Met Ile Phe Asn Ile Phe
145 150 155 160
Tyr Met Leu Phe Asn Leu Gly Leu Thr Ala Tyr Leu Ile Gly Asn Met
165 170 175
Thr Asn Leu Val Val His Gly Thr Ser Arg Thr Arg Lys Tyr Arg Asp
180 185 190
Thr Ile Gln Ala Ala Thr Ser Phe Ala Leu Arg Asn Gln Leu Pro His
195 200 205
Arg Leu Gln Asp Gln Met Ile Ser His Leu Ser Leu Lys Phe Arg Thr
210 215 220
Asp Ser Glu Gly Leu Gln Gln Gln Glu Thr Leu Asp Ala Leu Pro Lys
225 230 235 240
Ala Ile Arg Ser Ser Ile Ser Gln Tyr Leu Phe Phe Asn Leu Val Gln
245 250 255
Lys Val Tyr Leu Phe Glu Gly Val Ser Asn Asp Leu Ile Phe Gln Leu
260 265 270
Val Ser Glu Met Lys Ala Glu Tyr Phe Pro Pro Arg Glu Asp Val Ile
275 280 285
Leu Gln Asn Glu Ala Pro Thr Asp Phe Tyr Val Leu Val Ser Gly Ser
290 295 300
Ala Glu Leu Ile Glu Leu Gln Asn Gly Ala Glu Gln Val Ala Gly Val
305 310 315 320
Ala Lys Ser Gly Asp Val Val Gly Glu Ile Gly Val Leu Cys Tyr Arg
325 330 335
Pro Gln Leu Phe Thr Val Arg Thr Lys Ser Leu Cys Gln Leu Leu Arg
340 345 350
Ile Asn Arg Thr Ala Phe Leu Ser Ile Val Gln Ser Asn Val Gly Asp
355 360 365
Gly Thr Ile Ile Met Asn Asn Leu Ile Gln Leu Leu Lys Glu Gln Lys
370 375 380
Glu Asn Thr Val Met Val Gly Val Leu Lys Glu Val Glu Ser Met Leu
385 390 395 400
Ala Arg Gly Arg Leu Asp Leu Pro Ile Thr Leu Cys Phe Ala Val Asn
405 410 415
Arg Gly Asp Asp Phe Leu Leu His Gln Leu Leu Lys Arg Gly Leu Asp
420 425 430
Pro Asn Glu Ser Asp Asn Asn Gly His Thr Ala Leu His Ile Ala Ala
435 440 445
Ser Lys Gly Asn Glu Gln Cys Val Lys His Leu Leu Asp Tyr Asp Ala
450 455 460
Asp Pro Asn Ala Arg Asp Ser Glu Gly Lys Val Pro Leu Trp Glu Ala
465 470 475 480
Met Cys Glu Lys His Asp Arg Val Val Gln Leu Leu Val Gln Asn Gly
485 490 495
Ala Asp Leu Ser Trp Gly Asp Thr Ala Leu Tyr Ala Cys Ile Ala Val
500 505 510
Glu Glu Asn Asn Thr Glu Leu Leu Lys Asp Ile Ile Arg Tyr Gly Gly
515 520 525
Asp Val Lys Arg Ser Leu Lys Asp Gly Thr Thr Pro Leu His Lys Ala
530 535 540
Val Cys Asp Gly Asn Val Gln Met Val Glu Phe Leu Leu Glu Gln Gly
545 550 555 560
Ala Glu Ile Asp Lys Leu Asp Asn Asn Gly Trp Thr Pro Arg Ala Leu
565 570 575
Ala Glu Gln Gln Gly His Ala Tyr Thr Gln Leu Leu Phe Lys Ser Arg
580 585 590
Arg Glu Ala Pro Lys His His Val Pro Asn Asn Arg Val Ala Pro Ser
595 600 605
Leu Ile Gly Arg Phe Asn Ser Glu Pro Ser Met Gln Asn Val Asp Ser
610 615 620
Glu Asp Ile Gly Val Gln Asn Lys Val Phe Pro Lys Lys Leu Leu Lys
625 630 635 640
Arg Arg Val Ser Phe Gln Asn Ser Leu Phe Gly Val Ile Ser Ser Thr
645 650 655
Asn Ala Ser Arg Asp Thr Gly Pro Leu Leu Pro Arg Gly Pro Ala Ala
660 665 670
Thr Ser Ala Leu Asn Cys Asn Thr Asn Ser Leu Ile Arg Val Thr Ile
675 680 685
Ser Cys Pro Glu Lys Glu Asn Thr Ala Ala Lys Leu Val Leu Leu Pro
690 695 700
Arg Ser Met Gln Glu Phe Leu Asp Leu Gly Ala Lys Lys Phe Asp Phe
705 710 715 720
Arg Pro Thr Lys Val Leu Thr Ile Glu Gly Ala Glu Val Asp Glu Val
725 730 735
Glu Leu Ile Arg Asp Gly Asp His Leu Val Leu Val Ser Asp Gly Trp
740 745 750
Val Pro Asp Asp Val Gln Asn Lys Leu Gln Gln
755 760
Claims (9)
1.一种钾离子通道蛋白AlAKT1，其特征在于，其氨基酸序列如SEQ ID NO:2所示。
2.一种编码权利要求1所述的钾离子通道蛋白AlAKT1的基因。
3.根据权利要求2所述的基因，其特征在于，所述的基因的核苷酸序列如SEQ ID NO:1所示。
4.一种含有权利要求2或3所述基因的重组表达载体。
5.根据权利要求4所述的重组表达载体，其特征在于，所述的重组表达载体包括pYES2.0和PTF101。
6.一种含有权利要求4或5所述的重组表达载体的宿主细胞。
7.根据权利要求6所述的宿主细胞，其特征在于，所述的宿主细胞包括酿酒酵母CY162和根癌农杆菌EHA105。
8.权利要求1所述的钾离子通道蛋白AlAKT1、权利要求2-3任一项所述的基因、权利要求4-5任一项所述的重组表达载体在培育高效利用钾离子和/或耐盐转基因植物中的应用。
9.一种提高植物高效利用钾离子和耐盐的方法，其特征在于，所述方法为将权利要求2-3任一项所述的基因或权利要求4-5任一项所述的重组表达载体转入到植物中，筛选获得转基因的植物。
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