CN117568305A - 黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白及其应用 - Google Patents
黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白及其应用 Download PDFInfo
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Abstract
本发明公开了黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白及其应用,该黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白的氨基酸序列如SEQ ID NO.1所示。本发明从黄花苜蓿中克隆了钙依赖蛋白激酶基因MfCDPK14的cDNA序列,并将获得的MfCDPK14基因与植物表达载体连接,构建了重组表达载体,用构建的重组表达载体转化植物组织,然后培育成转基因植株。MfCDPK14基因受脱水刺激的诱导;本发明提供了利用MfCDPK14基因培育耐旱植物的方法,将该基因在植物过量表达后,提高了转基因植物的耐旱性。
Description
技术领域
本发明属于植物基因工程领域,具体涉及黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白及其应用。
背景技术
非生物胁迫(如干旱、盐碱、冷害和热害等)对植物的生长发育有不可忽视的影响,导致农作物产量下降,同时也影响林木、果树、花卉以及园艺观赏植物的正常生长和品质。因此,培育出耐逆性强的植物品种成为种植业的重要目标之一。为此,从耐逆性强的植物中分离出耐性基因成为必要之举。
植物体内主要存在钙调素(CaM)、类钙调素蛋白(CML)、钙调磷酸酶B类似蛋白(CBL)、钙依赖蛋白激酶(CDPK)及钙/钙调素依赖型蛋白激酶(CCaMK)等类型的钙感受器(Batistic et al.,2012;Schulz et al.,2013)。不同于其他只带有钙感受器的小分子蛋白质,CDPK和CCaMK是一类植物中特异存在的蛋白激酶。在拟南芥中,4%的基因编码了蛋白激酶(Champion et al.,2004;Zulawski et al.,2014)。其中参与解码Ca2+信号的色氨酸/苏氨酸蛋白激酶主要有三类:CDPK、CCaMK及CBL互作蛋白激酶(CIPK)(Harper et al.,2004),其中CIPK需要与CBL蛋白互作才可以参与钙信号的转导(Batistic et al.,2009)。CDPK是其中最大的家族,它与Ca2+结合后,可以直接把Ca2+信号转换成磷酸化信号,通过磷酸化信号直接激活和调控靶蛋白。这一过程并不需要其他蛋白质的参与,这使得CDPK具有双重功能:钙感受器和应答器(Harper et al.,2004)。
植物响应干旱胁迫最早的反应之一就是细胞质Ca2+浓度增加,这一变化可以通过Ca2+感受器(例如CDPK)传递至其靶蛋白,进而引起下游系列基因表达和生理生化等适应性变化,提高耐旱性。
CDPK还广泛参与植物对干旱胁迫的应答过程,主要包括磷酸化各类离子通道蛋白调控气孔的开闭、与ABA信号通路中的转录因子互作调控下游基因表达、调控ROS的清除及渗透物质的积累等。其中,气孔关闭是植物应对干旱胁迫的关键反应。当受到外界刺激后,保卫细胞内Ca2+浓度发生变化,阴离子与钾离子通道蛋白被激活从而使离子流出,渗透势上升,随之细胞收缩。拟南芥中的多个CDPK被证明参与了该过程的调控。AtCPK3/6/21/23在非洲爪蟾卵母细胞中异源表达时可激活S-型阴离子通道SLAC1和SLAH3,并且可以在体外磷酸化SLAC1(Brandt et al.,2012;Brandt et al.,2015;Geiger et al.,2011;Geiger etal.,2010;Scherzer et al.,2012),但它们间可能存在功能冗余,在cpk3、cpk6及cpk23突变体的离体枝条上只能观测到微弱的气孔表型,而在cpk3/6双突变体以及cpk5/6/11/23四突变体中ABA诱导的气孔关闭则显著减弱(Brandt et al.,2015;Geiger et al.,2010;Mori et al.,2006)。相反的,AtCPK9/33被鉴定为ABA诱导气孔关闭的负调节因子(Chen etal.,2019;Li et al.,2016)。此外,CDPK还可以通过调控K+通道调控气孔关闭,如cpk10突变体的干旱敏感表型可能是由于保卫细胞内K+通道蛋白的活性增加造成的(Zou et al.,2010)。
干旱胁迫可以引发体内大量的基因发生转录变化,其中一部分是由CDPK介导的。例如,AtCPK32与AtABF4互作并将其磷酸化对于ABA诱导ABF4的转录激活具有重要作用(Choi et al.,2005),AtCPK4可以磷酸化AtABF2并激活其转录活性(Lu et al.,2013)。此外,AtABF1/4还可以作为AtCPK4/11的底物,过表达AtCPK4/11可以增强胁迫响应基因表达来增强耐旱性,而突变体表现出相反的表型(Zhu et al.,2007)。此外,植株还可以通过调控ROS的清除及渗透物质的积累来抵御干旱胁迫。拟南芥AtCPK8通过磷酸化AtCAT3并激活AtCAT3的酶活来提高耐旱性(Zou et al.,2015),水稻OsCPK10也可以通过增加CAT的积累增强耐旱能力(Bundo et al.,2017)。另外,枳PtrCDPK10通过与PtrAPX互作并使其磷酸化,进而降低ROS的积累来参与对渗透和干旱胁迫的响应(Meng et al.,2020)。水稻OsCPK9通过调控气孔的关闭和提高渗透调节能力来增强耐旱性(Wei et al.,2014)。
CDPK存在于高等植物、绿藻、卵菌和原生生物中,但不存在于动物和真菌中,而CRK仅存在于植物中(Hrabak et al.,2003;Valmonte et al.,2014)。CDPK包含四个结构域:N端可变域、色氨酸/苏氨酸蛋白激酶域、抑制连接域和类钙调素域(CaM-LD)(Hrabak etal.,2003)。CDPK家族成员间氨基酸的差异主要集中在N端可变域内,它可能参与CDPK与靶蛋白的互作识别。
CDPK和CRK在植物中的广泛分布暗示其具有激活多种底物的潜力,目前已经发现并鉴定到了大量的底物,包括各类转录因子、离子通道蛋白、代谢酶及转运蛋白等。研究发现它们通过磷酸化特异的下游底物,在调控植物生长发育及响应各类胁迫等过程中发挥重要作用。
苜蓿是优质的豆科牧草,主要种植于我国的干旱和半干旱地区,尤其集中在西北和华北。干旱是苜蓿生产中最主要的逆境因素,严重影响了产量和品质。黄花苜蓿是一种具有强耐旱性的草种,从中克隆抗旱调控基因,并揭示其调节耐旱性的分子机制,是苜蓿育种的重要基因资源。这为农林植物的抗逆分子育种提供了坚实的基础,对于探索苜蓿的耐旱机制以及生产利用和培育耐旱新品系具有重要的指导意义。
发明内容
为克服现有技术的缺点和不足,本发明的目的是提供一种提高植物耐旱性的黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白及其编码基因和应用。
本发明的目的通过下述技术方案实现:
第一方面,本发明请求保护一种黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白,该蛋白的氨基酸序列为(a)或(b):
(a)如SEQ ID NO.1所示的氨基酸序列;
(b)由SEQ ID NO.1所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸而形成的具有同等功能的序列。
第二方面,本发明请求保护编码上述黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白的MfCDPK14基因;该MfCDPK14基因的核苷酸序列为(1)或(2):
(1)如SEQ ID NO.2所示的核苷酸序列;
(2)与(1)所述的核苷酸序列具有90%以上的同源性且具有同等功能的核苷酸序列。
第三方面,本发明请求保护包含上述MfCDPK14基因的生物材料,该生物材料为表达盒、重组载体、细胞系或重组微生物。
进一步,所述的重组载体为重组表达载体,该重组表达载体是通过将上述的MfCDPK14基因与植物表达载体连接得到的。
所述植物表达载体包括但不限于双元农杆菌载体以及用于单子叶基因枪转化的载体;所述的双元农杆菌载体包括pBI121、pCAMBIA系列载体;所述植物表达载体还可包含外源基因的3’端非翻译区域,即包含聚腺苷酸信号和任何其它效应mRNA加工或基因表达的DNA片段;
构建含有MfCDPK14基因的重组表达载体时,在其转录起始核苷酸前可使用任何一种强启动子或诱导型启动子;所述强启动子或诱导型启动子包括但不限于泛素(35Squtin)启动子和花椰菜花叶病毒(CaMV)35S启动子,它可单独使用或与其他的植物启动子结合使用;此外,构建含有MfCDPK14基因的重组表达载体时,还可使用增强子,这些增强子区域包括但不限于ATG起始密码子和邻接区域。起始密码子必须与编码序列的阅读框相同,以保证整个序列的翻译。翻译控制信号和起始密码子可以是多种不同的来源,可以是天然的,也可以是合成的。翻译起始区域可以来自转录起始区域或来自结构基因。
为了便于对转基因植株进行鉴定及筛选,可对所使用的植物表达载体进行加工,包括加入或替换植物可选择性标记。可使用的选择性标记包括编码抗除草剂的酶的基因或具有抗性的抗生素标记物;所述的除草剂包括草丁膦、草甘膦等,所述的抗生素包括硫酸卡那霉素、潮霉素、庆大霉素等。从转基因植物的安全性考虑,可以不加任何选择性标记基因,直接以逆境筛选转化植株。
本发明的具体实施方式中,所述的植物表达载体选择pCAMBIA3301。
所述重组表达载体的构建方法具体包含如下步骤:
(1)引物设计
引物①为MfCDPK14基因扩增上游引物MfCDPK14-F:
5’-ATGGGTTGTCACGGCAGC-3’;
引物②为MfCDPK14基因扩增下游引物MfCDPK14-R:
5’-TTAAAGTAATGGTCCCTGG-3’;
引物③为引入Nco I酶切位点的上游引物MfCDPK14-3301-F:
5’-CACGGGGGACTCTTGACCATGGACATGGGTTGTCACGGCAGC-3’;
引物④为引入BstEIⅠ酶切位点的下游引物MfCDPK14-3301-R:
5’-CGGGGAAATTCGAGCTGGTCACCTTAAAGTAATGGTCCCTGG-3’;
(2)MfCDPK14基因片段的获得:
以黄花苜蓿的cDNA为模板,使用引物①和引物②扩增MfCDPK14基因片段,并与Cloning Kit载体连接,构建获得pEASY-MfCDPK14载体;
(3)含有MfCDPK14基因的重组表达载体pCAMBIA3301-MfCDPK14的构建:
以pEASY-MfCDPK14载体为模板,使用引物③和引物④使MfCDPK14基因片段引入Nco I和BstEIⅠ两个酶切位点,并与植物表达载体pCAMBIA3301连接,进而构建获得含有MfCDPK14基因的重组表达载体pCAMBIA3301-MfCDPK14。
所述的黄花苜蓿优选为黄花苜蓿(Medicago falcata L.)品种海拉尔。
本发明的具体实施方式中,采用上述的重组表达载体pCAMBIA3301-MfCDPK14培育表达黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白的转基因植株,是通过将上述的含有MfCDPK14基因的重组表达载体转化植物组织,并将转化的植物组织培育成转基因植株得到的。
所述的含有MfCDPK14基因的重组表达载体转化植物组织可通过使用Ti质粒、Ri质粒、植物病毒载体、直接的DNA转化,显微注射、电穿孔等各种常规或特异的遗传转化方法导入植物细胞或组织,并将转化的植物组织培育成植株。被转化的植物宿主可以是单子叶植物或双子叶植物。所述植物以苜蓿为例。具体包含如下步骤:
(1)用电转的方法将重组表达载体pCAMBIA3301-MfCDPK14导入根瘤农杆菌EHA105中,获得含有重组表达载体pCAMBIA3301-MfCDPK14的农杆菌阳性菌落;
(2)将活化的含有重组表达载体pCAMBIA3301-MfCDPK14的农杆菌EHA105浸染苜蓿叶片,经共培养和抗生素筛选,获得转基因苜蓿。
第四方面,本发明请求保护上述的黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白,上述的MfCDPK14基因,或上述述的生物材料在提高植物抗旱性或培育耐旱性提高的转基因植物中的应用。
进一步,上述的应用为在目标植物中过表达上述的MfCDPK14基因,或提高上述MfCDPK14蛋白的表达量以提高植物的耐旱性。
进一步,在目标植物中过表达所述MfCDPK14基因的过程为:构建如上所述MfCDPK14基因的过表达载体,通过农杆菌介导法将所述的过表达载体转入到目标植物中得到耐旱性提高的转基因植株。
第五方面,本发明请求保护一种提高植物抗旱性的方法,在目标植物中过表达上述的MfCDPK14基因,或提高上述MfCDPK14蛋白的表达量以提高植物的耐旱性。
上述的植物为单子叶植物或双子叶植物,例如苜蓿,但不限于此。
第六方面,本发明请求保护一种黄花苜蓿钙依赖蛋白激酶MfCDPK14启动子,该启动子的核苷酸序列如SEQ ID NO.3所示。
上述的黄花苜蓿钙依赖蛋白激酶MfCDPK14启动子在如下(1)或(2)中的应用也属于本发明的保护范围:
(1)驱动GUS表达;
(2)调控下游基因响应逆境胁迫的表达;所述的逆境胁迫为干旱胁迫。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明从黄花苜蓿中克隆了钙依赖蛋白激酶基因MfCDPK14的cDNA序列,该MfCDPK14基因的表达受干旱诱导。
(2)本发明将获得的MfCDPK14基因与植物表达载体连接,构建了适合于双子叶植物的重组表达载体,并转化双子叶模式植物蒺藜苜蓿,转基因植株明显提高了抗旱性。
(3)本发明提供了利用MfCDPK14基因培育耐旱植物的方法。
附图说明
图1是重组表达载体pCAMBIA3301-MfCDPK14的表达框示意图。
图2是MfCDPK14基因开放阅读框序列的PCR扩增琼脂糖凝胶电泳图,其中,M是标准DNA分子;泳道1是MfCDPK14基因的扩增片段。
图3是重组表达载体pCAMBIA3301-MfCDPK14的PCR鉴定琼脂糖凝胶电泳图,其中,M是标准DNA分子;泳道1是阴性重组子;泳道2~5是阳性重组子;泳道P是阳性对照。
图4是干旱诱导MfCDPK14基因表达的结果分析柱状图,即干旱对MfCDPK14基因转录的影响,柱上方的字母a、b、c、d和e表示不同处理之间的差异显著(P≤0.05),即数据柱上方字母相同时表示没有显著差异,数据柱上方字母不同时表示差异显著。
图5是导入MfCDPK14基因的转基因苜蓿的PCR鉴定结果琼脂糖凝胶电泳图,其中,WT是野生型;编号1~11表示不同的待鉴定转MfCDPK14基因苜蓿。
图6是3个导入MfCDPK14基因的转基因苜蓿的实时定量PCR结果分析图,其中,WT表示野生型;编号OE1、2、3表示不同的转基因苜蓿;实时定量PCR检测,柱上方的字母a和b表示不同材料之间的差异显著(P≤0.05)。
图7是导入MfCDPK14基因的转基因苜蓿抗旱性检测结果分析图,其中,(A)是过表达MfCDPK14及野生型‘R108’材料在干旱胁迫下的表型,从左到右分别为:栽种示意图、干旱胁迫前、干旱胁迫后和复水10d后的表型;(B)是干旱胁迫后各个植系复水10d后存活率;(C)是干旱处理后各个植系叶片的相对电导率;柱上方的字母a、b和c表示不同材料之间的差异显著(P≤0.05)。
图8是以MfCDPK14启动子驱动GUS的转基因拟南芥纯合株系GUS染色结果,其中,(A)在幼根的成熟区、茎的韧皮部、叶脉、花中的表达GUS染色结果;(B)是qRT-PCR组织表达结果。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
黄花苜蓿(Medicago falcata L.)品种海拉尔由南京农业大学草业学院郭振飞课题组实验室提供。
根癌农杆菌(Agrobacterium tumefaciens)EHA105:为南京农业大学草业学院郭振飞课题组实验室保藏菌株。
蒺藜苜蓿(Medicago truncatula)种子:品种为R108,由南京农业大学草业学院郭振飞课题组实验室提供。
pCAMBIA3301超表达载体:由南京农业大学草业学院郭振飞课题组实验室提供
大肠杆菌DH5α:购自北京擎科生物科技股份有限公司。
上述生物材料均为现有技术以公开的生物材料。
实施例1MfCDPK14基因的克隆
一、黄花苜蓿cDNA模板制备
取少量黄花苜蓿(Medicago falcata L.)‘海拉尔’种子放入1.5mL无菌离心管内,加入1mL 70%乙醇处理30s后,用无菌水洗涤3次。加入10%次氯酸钠溶液,颠倒10min后用无菌水洗涤3-5次。然后将种子置于1/2MS培养基上,待种子发芽生根后移栽至混合培养基质(蛭石:营养土=1:1)中栽培,于温室中进行培养,温度22℃/16℃(昼/夜),光周期为16h/8h(光/暗),湿度60%,光照强度800μmol·m-2·s-1;对其进行定期修剪并施以适量的复合肥(N:P:K=15:15:15)溶液;待材料生长均匀一致后用于试验处理,取黄花苜蓿的成熟叶片,用RNA Prep Pure Plant kit试剂盒(天根公司)方法提取总RNA,用EasyScript First-Strand cDNA Synthesis SuperMix逆转录酶试剂盒(TransGen Biotech公司)反转录获得cDNA模板,-20℃保存备用。
二、扩增MfCDPK14基因引物
根据蒺藜苜蓿基因组数据(http://www.medicagohapmap.org/)MtMYB4基因的cDNA序列,进行同源性比对,在此基础上设计扩增黄花苜蓿MfCDPK14 cDNA开放阅读框(由北京擎科合成)的引物。扩增MfCDPK14的上游引物MfCDPK14-F,序列为:5’-ATGGGTTGTCACGGCAGC-3’;扩增MfCDPK14的下游引物MfCDPK14-R,序列为:5’-TTAAAGTAATGGTCCCTGG-3’;
三、扩增获得MfCDPK14基因并构建载体
用上述步骤一制备的模板和步骤二设计的引物进行PCR扩增:
PCR反应体系(50μL):KOD-Plus-DNA聚合酶(TOYOBO公司,1U·μL-1)1μL,10×buffer 5μL,dNTPs(10mmol·L-1)5μL,MgSO4(25mmol·L-1)2μL,上游引物(10μmol·L-1)1.5μL,下游引物(10μmol·L-1)1.5μL,反转录第一链cDNA 2μL,ddH2O补足至50μL;
PCR反应程序:94℃3min;94℃0.5min,55℃0.5min,72℃0.5min,35个循环;72℃10min;扩增产物以质量分数为1%的琼脂糖凝胶电泳检测(图2);
获得一条长约900bp的序列(图2),将获得的PCR产物用DNA凝胶回收试剂盒(南京诺唯赞公司)回收;
将PCR回收片段与Cloning Kit(TaKaRa)按摩尔比3:1的比例进行连接,反应体系如下:/>Cloning Vector(0.03pmol)1μL,回收的目的片段0.1pmol~0.3pmol,轻轻混合,37℃连接5min,获得连接产物;
大肠杆菌感受态细胞购于北京擎科生物科技股份有限公司,于-80℃保存备用;
连接产物热激转化大肠杆菌:将连接产物加入至100μL DH5α感受态细胞中,冰中放置5min;42℃热激45s后,再在冰中放置2min;热激后的菌体转入1mL LB液体中,于37℃恒温摇床上以200rpm振荡培养1h。取100μL菌液涂布于LB固体(含100μg/mL Kana)培养基上,于37℃培养箱内倒置培养过夜。
重组质粒pEASY-MfCDPK14的筛选、纯化及测序:挑取PCR阳性菌落,接种于2mL含100μg/mL硫酸卡那霉素的LB液体培养基中,于37℃恒温摇床上以200rpm振荡培养过夜;吸取2mL菌液,用质粒DNA纯化试剂盒(Qiagen公司)提质粒,4℃保存备用。纯化的重组质粒命名为pEASY-MfCDPK14,送交上海生工生物工程技术服务有限公司进行测序,测序结果如SEQID NO.4所示。
测序结果与文库的序列进行比对,证明所扩增出的PCR产物为MfCDPK14基因。编码所述的逆境诱导的核转录因子MfCDPK14的基因由1635个碱基组成,其中MfCDPK14的蛋白质编码区(Sequence coding for amino acids in protein,CDS)由30位~2279位的870个碱基组成,编码544个氨基酸。
实施例2重组表达载体pCAMBIA3301-MfCDPK14的构建
根据实施例1获得的MfCDPK14开放阅读框序列,设计带有Nco I和BstEⅠⅠ酶切位点的扩增引物:
引入Nco I酶切位点的上游引物MfCDPK14-3301-F:
5’-CACGGGGGACTCTTGACCATGGACATGGGTTGTCACGGCAGC-3’;
引入BstE II酶切位点的下游引物MfCDPK14-3301-R:
5’-CGGGGAAATTCGAGCTGGTCACCTTAAAGTAATGGTCCCTGG-3’;
以实施例1构建的克隆载体pEASY-MfCDPK14为模板,以MfCDPK14-3301-F和MfCDPK14-3301-R为上下游引物,进行MfCDPK14基因PCR扩增,纯化回收扩增获得的MfCDPK14基因片段。
将上述获得的MfCDPK14基因片段和pCAMBIA3301表达载体分别用限制性内切酶Nco I(NEB公司)和BstEⅠⅠ(NEB公司)双酶切,分别回收MfCDPK14基因目的片段和酶切后载体大片段,连接后,将MfCDPK14基因插入到表达质粒pCAMBIA3301 35S启动子下游多克隆位点的Nco I和BstEIⅠ之间,构建获得重组表达载体,将构建获得的重组表达载体命名为pCAMBIA3301-MfCDPK14,其中,图1是重组表达载体pCAMBIA3301-MfCDPK14的表达框示意图。
通过PCR检测,证明获得了重组子(图3)。进一步对插入片段测序,结果表明,插入片段的序列与MfCDPK14编码区的序列完全一致,并且插入片段两端的酶切位点也完全正确,从而证明成功构建了重组表达载体pCAMBIA3301-MfCDPK14,重组表达载体pCAMBIA3301-MfCDPK14的表达框示意图如图1。
实施例3MfCDPK14受逆境诱导的表达情况
一、获得逆境条件下的黄花苜蓿模板
取少量黄花苜蓿种子放入1.5mL无菌离心管内,加入1mL 70%乙醇处理30s后,用无菌水洗涤3次。加入10%次氯酸钠溶液,颠倒10min后用无菌水洗涤3-5次。然后将种子置于1/2MS培养基上,待种子发芽生根后移栽至混合培养基质(蛭石:营养土=1:1,w/w)中栽培,于温室中进行培养,温度22℃/16℃(昼/夜),光周期为16h/8h(光/暗),湿度60%,光照强度800μmol·m-2·s-1。适时施以适量的复合肥(N:P:K=15:15:15)溶液,待材料生长均匀一致后用于试验处理;
脱水处理
黄花苜蓿叶片脱水处理:取黄花苜蓿的叶片,置于超净工作台(上海锦屏仪器公司,型号:SW-CJ-2)内,变压器调至75V,室内温度为28℃,对样品进行吹风模拟干旱处理(Yang J,Guo Z.Cloning of a 9-cis-epoxycarotenoid dioxygenase gene(SgNCED1)from Stylosanthes guianensis and its expression in response to abioticstresses[J].Plant cell reports,2007,26(8):1383-1390),分别于0h、1h、2h、6h和12h后取样,取样时剪取叶片,用锡箔纸包好后投于液氮迅速冷冻,于-80℃冰箱中保存备用。
将以上存放于-80℃冰箱中保存备用的样品取出放于液氮中,加入液氮磨碎样品,用RNA Prep Pure Plant kit试剂盒(天根公司)提取叶片的总RNA,采用EasyScriptFirst-Strand cDNA Synthesis SuperMix(TransGen Biotech公司)逆转录酶试剂盒进行反转录制备获得cDNA模板,-20℃保存备用。
二、设计特异检测引物
根据MfCDPK14基因cDNA序列和黄花苜蓿β-actin的序列,用Beacon Designer 7.0软件设计出检测用定量引物:
MfCDPK14基因的上游定量引物qMfCDPK14-F:5’-GGAAGGTGAACTCGACTTTGT-3’;
MfCDPK14基因的下游定量引物qMfCDPK14-R:5’-GGGTGTTCAAGGACTTCTGTAG-3’;
β-actin基因的上游定量引物qActin-F:5’-CCCACTGGATGTCTGTAGGTT-3’;
β-actin基因的下游定量引物qActin-R:5’-AGAATTAAGTAGCAGCGCAAA-3’;
三、定量PCR检测表达差异
将步骤一制备的模板cDNA稀释50倍用于定量PCR的模板,反应体系为10μL:SYBRGreen Supermix(2×)5μL,上游和下游引物(10μM)各0.5μL,cDNA模板4μL;使用仪器为TaKaRa公司生产的Thermal Cycler Dice Real TimeSystem II,PCR反应条件设置为:95℃10s;94℃5s,59℃25s,40个循环;以不加cDNA模板作为阴性对照,每个样品设置3个重复,以持家基因β-actin作为内参基因;溶解曲线绘制:95℃变性15s,60℃退火1min,95℃变性15s,反应结束后,进行溶解曲线分析,PCR扩增效率在95%以上,利用使用2-ΔΔCT方法计算基因的相对表达量;
实时定量PCR的结果显示,MfCDPK14表达在干旱处理1h开始受到诱导,并在2h表达量最高,此后表达量降低(图4);结果表明,干旱能诱导MfCDPK14基因的表达。
实施例4转基因苜蓿的产生及分子检测
一、转基因苜蓿的产生
1、将重组表达载体pCAMBIA3301-MfCDPK14导入根癌农杆菌EHA105
EHA105感受态的制备参考J.萨姆布鲁克(黄培堂,王嘉玺,朱厚础.J萨姆布鲁克,DW拉塞尔,著[J].分子克隆实验指南,2002:27-30)的方法并加以改进:取EHA105菌种于YEP平板(含25mg/L利福平)上划线,28℃培养48h,挑取单菌落,接种于50mL液体LB中,28℃培养过夜,吸取0.5mL菌液,接种于500mL液体YEP中,28℃培养8h,至OD600为0.6,冰浴冷却10min,倒入经灭菌的200mL离心管中,平衡后4℃4000rpm离心10min,收集菌体,倒去LB,倒置于灭过菌的纸巾上,控干水分,加入50mL预冷的体积分数为10%的甘油(甘油:YEP液体培养基),于冰上摇动,悬浮菌体,4℃4000rpm离心15min,收集菌体,重复洗涤一次,加入2mL预冷的体积分数为10%的甘油(甘油:YEP液体培养基),悬浮菌体,分装,25μL/管,加液氮速冻后于-80℃冰箱保存。把感受态细胞置于冰上解冻,将0.2cm内径的电击杯置于冰上预冷,在超净工作台内,将1.5μL pCAMBIA3301-MfCDPK14质粒(20ng/μL)加至20μL解冻的感受态细胞中,轻弹管壁混匀,冰浴1min后,转移至电击杯中,置于电击仪(MicroPulser,Bio-RAD公司)的电极间,选定程序Agr,进行电击;电击结束后,在超净工作台内,迅速将1mL YEP液体培养基倒入电击杯,再用移液枪转移至摇菌管中,28℃轻柔振荡培养2h;吸取0.3mL菌液,涂布在YEP平板(含25mg/L的利福平和50mg/L的硫酸卡那霉素)上,28℃培养箱内倒置培养48h;
2、含重组表达载体pCAMBIA3301-MfCDPK14农杆菌阳性菌落的鉴定
挑取平板上的单菌落进行菌落PCR检测,并做好标记;进一步挑取PCR阳性菌落至3mL YEB液体培养基(含25mg/L的利福平和50mg/L的硫酸卡那霉素)中,28℃振荡培养40h;取2mL菌液用FastPure Plasmid Mini Kit试剂盒(诺唯赞)抽提质粒,用限制性内切酶NcoI(NEB公司)和BstEIⅠ(NEB公司)进行酶切检测,确定阳性克隆中含有植物表达载体pCAMBIA3301-MfCDPK14;取0.8mL农杆菌液,加0.2mL体积分数为80%的甘油(甘油:YEP液体培养基),混匀后保存于-80℃超低温冰箱备用。
3、转基因苜蓿植株的获得
(1)培养基配制
大量元素母液(10×):称取28.30g KNO3、4.63g(NH4)2SO4、4.00g KH2PO4、1.85gMgSO4·7H2O、1.66g CaCl2·2H2O溶解于1L去离子水;
微量元素母液(1000×):称取10g MnSO4·H2O、5g H3BO3、1g KI、1g ZnSO4·7H2O、0.2g CuSO4·5H2O、0.1g CoCl2·6H2O、0.1g Na2MoO4·2H2O溶解于1L去离子水;
SH培养基有机物母液(1000×):称取5g盐酸硫胺素、5g盐酸吡哆醇、5g烟酸溶解于1L去离子水;
SH培养基EDFS母液(50×):称取6.97g Na2FeEDTA溶解于1L去离子水;
SH3α培养基:加入100mL大量元素母液,20mL EDFS母液,1mL微量元素母液,1mL有机物母液,4mL 2,4-D贮存液(1mg/mL),0.5mL 6-BA贮存液(1mg/mL),30g蔗糖,0.1g肌醇,去离子水定容至1L,pH调至5.85,固体培养基加入8g琼脂粉,121℃高压灭菌20min;
SH9培养基:加入100mL大量元素母液,20mL EDFS母液,1mL微量元素母液,1mL有机物母液,20g蔗糖,0.1g肌醇,去离子水定容至1L,pH调至5.85,加入8g琼脂粉,121℃高压灭菌20min;
植株生根培养基:称取2.2g MS培养基干粉,15g蔗糖,溶解后加入0.5mL IBA贮存液(1mg/mL),去离子水定容至1L,pH调至5.85,加入8g琼脂粉,121℃高压灭菌20min。
1/2MS培养基:称取2.2g MS培养基干粉,15g蔗糖,去离子水定容至1L,pH调至5.85,加入8g琼脂粉,121℃高压灭菌20min。
(2)蒺藜苜蓿‘R108’的培养
选取蒺藜苜蓿‘R108’的饱满种子在砂纸上轻轻磨出缺口,之后在大培养皿中放上一层定性滤纸,用蒸馏水润湿,放至4℃春化2-3d。在24-28度黑暗放置一晚,待根长到2cm左右后,转移至营养土(营养土:蛭石=1:1)的基质中培养。生长条件:16h光照/8h黑暗,湿度60%,白天温度22℃,夜间温度16℃。生长4~6周后可用于侵染;
(3)农杆菌的活化与悬浮
取含有重组表达载体pCAMBIA3301-MfCDPK14的农杆菌EHA105贮存液,在YEB平板(含25mg/L的利福平和50mg/L的硫酸卡那霉素)上划线,28℃培养;挑分离良好的单菌落,接种于2mL液体YEB(含25mg/L的利福平和50mg/L的硫酸卡那霉素)中,28℃振荡暗培养24h;转化前1天,按1:50比例,将含阳性克隆的过夜菌液加到5-7ml含同样抗生素的YEB摇过夜;
转化当天,按1:50比例,将含阳性克隆的过夜菌液加到50ml含同样抗生素的YEB,28℃振荡培养至OD600为0.6-0.8;22℃,3000rpm,离心13min,沉淀用100ml SH3α(含100μM乙酰丁香酮)悬浮,制成侵染液。
(4)侵染、共培养及转基因苗的产生
用消毒的剪刀剪下叶片,自来水清洗表面杂质,6.25%NaClO表面消毒30min,在超净工作台内用灭菌蒸馏水清洗2-3次,除去NaClO,切掉叶柄叶缘,将叶片切成5mm×5mm小块,用于转化实验。将提前剪好的叶片和农杆菌侵染液SH3α(含100μM乙酰丁香酮且应保证农杆菌侵染液淹没愈伤组织),充分混合,抽真空,-0.1pka(>0.09pka),30min;然后在摇床上,80rpm,28℃侵染1.5h。在超净工作台内,倒出侵染液,将叶片置于多层灭菌滤纸的小平皿上干燥约10min且尽可能除去切片表面的农杆菌,然后将叶片正面向上接到有一张新灭菌滤纸的固体共培养基SH3α(含100μM乙酰丁香酮)上,封口25℃暗培养两天后,分别接入筛选培养基SH3α(含2mg/L草铵膦、200mg/L头孢霉素和200mg/L特美汀),然后封口,于25℃下暗培养2周;
抗性愈伤的分化:挑选生长状态好的抗性愈伤接入分化培养基SH9培养基(含2mg/L草铵膦、200mg/L头孢霉素和200mg/L特美汀),于25℃,光暗交替下(16h/8h)培养,每3周转一次板,有绿点的愈伤组织继续分化出抗性芽。
(5)转基因苜蓿的生长
待分化苗长至3~5cm时,将其从分化培养基中转移到100mL的组培瓶1/2MS培养基中,25℃光暗(16h/8h)交替培养4周,直到长出新的根。如果苗生长良好,新的根长得比较快,则可以提前移栽练苗。当幼苗根部长出新的长根时,将幼苗从培养基中移出,并把根部的培养基冲洗干净,分单株移栽到蛭石中。待苗成活后移栽至蛭石:营养土=2:1(V/V)的基质中,置于培养箱中进行生长。生长条件:16h光照/8h黑暗,湿度60%,白天温度22℃,夜间温度16℃。最后于温室外自然条件培养。
二转基因苜蓿的PCR检测
1.CTAB法提取植物基因组DNA
取一个复叶于2mL离心管,将其放入液氮中快速冷冻,用组织研磨仪将材料研磨成粉末,加入500μL 65℃预热的CTAB提取液,颠倒摇匀,65℃水浴30min,每隔5-10min颠倒混匀,加入等体积的氯仿/异戊醇(24/1,V/V),颠倒混匀,12,000rpm离心15min,吸取上清移至1.5mL离心管;加入350μL异丙醇,颠倒混匀,-20℃静置30min;12,000rpm离心10min,弃上清,加入1mL 75%乙醇清洗;12,000rpm离心5min,弃上清,置于超净台吹干;加入100μL的无菌水溶解DNA。
2.转基因苜蓿的PCR检测
由于侵染植物的载体pCAMBIA3301在其左右边界区内还带有bialaphos(Bar)抗性基因,因此使用能特异识别该基因的引物Bar-F(5’-ATGAGCCCAGAACGACGC-3’)和Bar-R(5’-TCAAATCTCGGTGACGGG-3’)来检测目的基因是否成功转入到植物基因组;以上一步骤微抽得到的DNA为模板进行PCR扩增,以pCAMBIA3301-MfCDPK14重组质粒为阳性对照。
反应体系(20μL):TaqDNA聚合酶0.1μL、PCR 10×buffer 2μL、dNTP(10mM)1.6μL、Bar-F(10μM)0.5μL、Bar-R(10μM)0.5μL、DNA 1μL(pCAMBIA3301-MfCDPK14质粒20ng)、ddH2O14μL;
PCR反应程序:94℃2min;94℃0.5min,57℃0.5min,72℃30s,35个循环;72℃5min。质量分数为1%的琼脂糖凝胶电泳检测PCR产物(如图5所示)。
实施例4MfCDPK14基因的组织表达模式
一、MfCDPK14基因启动子片段的获得
依据MfCDPK14基因序列在5’端设计3个反向引物,利用TAIL-PCR向上游扩增获得启动子序列。使用引物、体系和反应程序如下:
CDPK14-R1:5’-GAGGGTTTTGGATTTGAGGATGGGG-3’
CDPK14-R2:5’-GGGACGGGCAGGTGATGATCTAT-3’
CDPK14-R3:5’-CCGTGACAACCCATTATTGTGTGAT-3’
TAIL-AD1:5’-NGTCGASWGANAWGAA-3’
TAIL-AD2:5’-TGWGNAGSANCASAGA-3’
TAIL-AD3:5’-AGWGNAGWANCAWAGG-3’
TAIL-AD4:5’-SSTGGSTANATWATWCT-3’
TAIL-AD5:5’-CGSATSTCSAANAAWAT-3’
TAIL-PCR反应体系(50μL):
10×ExTaq buffer 5μL;dNTPs(10mM)4μL;Primer-R1(10μM)2μL;AD1(2、3、4、5)(20μM)6μL;ExTaq DNA聚合酶1μL;Genomic DNA 1μL;ddH2O补足至50μL;每一管DNA都要分别用AD1、AD2、AD3、AD4、AD5五个随机引物进行扩增。
第一轮PCR扩增程序:
(1)93℃,1min;95℃,1min;
(2)94℃,30s;62℃,1min;72℃,2.5min;进行5个循环;
(3)94℃,30s;25℃,3min;ramping to 72℃,over 3min;72℃,2.5min;
(4)94℃,30s;68℃,1min;72℃,2.5min;94℃,30s;68℃,1min;72℃,2.5min;94℃,30s;94℃,1min;72℃,2.5min;进行15个循环;
(5)72℃,5min。
反应完成后将产物稀释50倍,取2μL作为模板并使用R2引物进行下一步反应,反应体系同第一轮。
第二轮PCR扩增程序:
(1)94℃,30s;64℃,1min;72℃,2.5min;94℃,30s;64℃,1min;72℃,2.5min;94℃,30s;44℃,1min;72℃,2.5min;进行12个循环;
(2)72℃,5min。
反应完成后将产物稀释50倍,取2μL作为模板并使用R3引物进行下一步反应,第三轮反应体系及程序同第二轮。
反应结束后,进行电泳检测,并切胶回收,连T载体测序。
获得一条长约MfCDPK14上游1998bp的序列,将获得的PCR产物用DNA凝胶回收试剂盒(AXYGEN公司)回收。
所述的黄花苜蓿类钙依赖蛋白激酶MfCDPK14启动子序列的核苷酸序列如SEQ IDNO.3所示;
MfCDPK14基因启动子元件分析见表1。
Table 1Cis-acting elements in the MfCDPK14 promoter region
二、PMfCDPK14:GUS表达载体构建
根据步骤一获得的MfCDPK14上游的启动子序列,设计带有EcoR I和Nco I酶切位点进行同源臂扩增;同时,pCAMBIA1381表达载体分别用限制性内切酶BamH I(TaKaRa公司)和Hind III(TaKaRa公司)双酶切,将回收的P:MfCDPK14启动子同源臂扩增目的片段和酶切后载体大片段,采用Clon Express II重组反应液按摩尔比1:2的比例进行连接,反应体系如下:5×CE II Buffer 4μL,Exnase II 2Μl,回收的目的片段0.06pmol,补去离子水到20μL,37℃连接30min,置于冰上冷却后获得连接产物,重组反应液转化DH5α,挑取单克隆送测序,将测序正确的菌液和质粒保存。进一步用电击转化的方法将重组表达载pCAMBIA1381-P:MfCDPK14导入根瘤农杆菌EHA105中。
引入BamH I酶切位点的上游引物pMfCDPK14-1381-F:
5’-ATTACGAATTCCCGGGGATCCAGAGATTTAATGGTGTCTGC-3’;
引入Hind III酶切位点的下游引物pMfCDPK14-1381-R:
5’-ACGACGGCCAGTGCCAAGCTTTATTGTGTGATTTGGATTGAA-3’。
三、获得以MfCDPK14启动子驱动GUS的转基因拟南芥的遗传转化
(1)将保存的农杆菌EHA105划线到含卡那霉素和利福平抗性的YEP培养基上,
28℃培养两天;挑取单菌落,加入YEP液体培养基中,28℃,200rpm摇菌。转化前1天,按1:50比例将菌液加到50mL YEP液体培养基,28℃,200rpm,暗培养至OD600nm=0.6-0.8。
(2)4℃,5000rpm离心15min,去上清。
(3)用5%(g/100ml)的蔗糖溶液(含有0.03%silwetl-77,v/v)重悬沉淀使OD600nm=0.6-0.8,蔗糖溶液需要现用现配,无需灭菌。
(4)将生长状态良好的拟南芥花蕾浸没在重悬液中,同时轻轻旋转拟南芥植株,此步骤应尽量避免转化溶液进入基质,防止对拟南芥根系造成损伤。
(5)用保鲜膜套住植株以保持湿润状态,将植株置于暗处培养12h后恢复正常生长条件。
(6)使用草丁膦筛选和PCR鉴定获得纯合MfCDPK14启动子转基因拟南芥。
四、获得纯合MfCDPK14启动子转基因拟南芥进行GUS染色
GUS染色的配制:
(1)X-Gluc母液(20mM):X-Gluc粉末用N-N-二甲基酰胺(DMF)溶解,每管100μL,-20℃保存待用;
(2)X-Gluc基液(50mM PBS,pH 7.0):将0.78g NaH2PO4、16.5mg K3[Fe(CN)6](铁氰化钾)、1.79g Na2HPO4、100μL Triton-100、0.372g Na2EDTA、21.1mgK4[Fe(CN)6](亚铁氰化钾)和80mL加入到烧杯中混匀,用量筒加蒸馏水定容至100mL,4℃保存待用;
(3)染色液的配制:50uL X-Gluc母液+450uL X-Gluc基液(现配现用)。在GUS染色液中放入转基因拟南芥的幼苗、茎、叶、花、果荚,抽真空10min。置于37℃避光染色2-24h至颜色出现变化后倒掉染色液,用70%乙醇进行脱色3-5次,使用体式显微镜进行拍照。
以蒺藜苜蓿Actin基因作为内参,对MfCDPK14基因在蒺藜苜蓿不同组织中的表达进行定量PCR检测。与此同时,获得了以MfCDPK14启动子驱动GUS的转基因拟南芥纯合株系,GUS染色结果表明,MfCDPK14在幼根的成熟区、茎的韧皮部、叶脉、花瓣、雄蕊中有表达,但在根尖、气孔及果荚中未检测到GUS染色(图8)。qRT-PCR组织表达结果与GUS染色结果基本一致。
实施例5转基因苜蓿的抗旱性鉴定
一、抗旱性测定
测定方法为:
将转基因苜蓿及其野生型苜蓿‘R108’植株各10棵种植于同一盆中,栽种示例图见图7A,在温室正常培养生长30天后,干旱25天后取植株的第2片复叶测定相对电导率(Ionleakage)并对植株复水,复水10d后统计存活率。
结果如图7所示,干旱胁迫后过表达株系的叶片相对电导率均显著低于野生型(图7B)。复水10d后过表达株系OE-1、OE-2、OE-3的存活率分别为33.3±2.9%、40.0±5.0%、53.3±2.9%,均显著高于野生型13.3±3.3%的存活率(图7C),表明过表达MfCDPK14可以显著提高蒺藜苜蓿的耐旱性。
施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
SEQ ID NO.1
MGCHGSKEKKHAKDEFTARHHRSSPARPAAAPPSRPAGVDSNYRYINVPPQVQQSSTTVTPSSNPKPSATTAHKPVQQKVDTTILGKPYEDIKKFYTLGKELGRGQFGITYFCTENSTGLNYACKSILKRKLVSKADREDIKREIQILQHLSGQPNIVEFKGAYEDRFSVHLVMELCAGGELFDRIIAQGHYSERAAASICRDVVKVVHICHFMGVLHRDLKPENFLLSSKDDGAALKATDFGLSVFIEEGKVYRDMVGSAYYVAPEVLRRNYGKEIDIWSAGIILYILLSGVPPFWAETEKGIFNAILEGELDFVSEPWPSISESAKDLVRKMLTPDPKKRITSTEVLEHPWMREGGEASDKPIDSAVLSRMKQFRAMNKFKKLALKVMAENLSEEEIKGLKAMFANMDTDSSGMITYEELKTGLARIGSRLSEAEVKQLMEAADVDGNGSIDYLEFISATMHRHRLERDEHLYKAFQYFDKDNSGHITREELETAMTKHGMGDEATIKEIISEVDTDNDGRINYEEFCAMMRSGMPHQGPLLIVGNSSSGNGSTAPAYDFLGLKGGVWDYKGLEMK
SEQ ID NO.2
ATGGGTTGTCACGGCAGCAAGGAGAAGAAACATGCCAAAGATGAGTTTACTGCCCGTCATCATAGATCATCACCTGCCCGTCCCGCAGCTGCACCCCCTTCTCGTCCCGCCGGTGTAGATAGCAATTATCGTTACATCAATGTTCCACCTCAGGTTCAACAGTCTTCAACAACAGTTACCCCATCCTCAAATCCAAAACCCTCTGCCACCACTGCCCACAAACCAGTGCAGCAGAAGGTTGATACAACCATCTTAGGTAAACCCTATGAAGATATAAAGAAGTTTTACACTCTAGGTAAAGAATTGGGTCGAGGCCAATTTGGGATTACTTATTTTTGTACTGAGAATTCAACCGGTTTAAATTACGCGTGTAAATCTATATTAAAGAGGAAGCTTGTGTCGAAAGCGGATAGAGAAGATATTAAAAGAGAGATTCAGATTCTGCAGCATTTGTCTGGTCAACCGAATATTGTTGAGTTTAAAGGTGCTTATGAGGATAGATTCTCTGTGCATCTTGTGATGGAACTTTGTGCTGGTGGTGAGTTGTTTGACAGGATTATTGCTCAAGGTCATTATTCAGAGAGAGCTGCTGCTTCTATTTGTAGGGATGTTGTTAAAGTTGTGCACATTTGTCATTTTATGGGTGTTTTGCATCGCGATTTGAAGCCTGAGAATTTCTTGCTTTCTAGTAAGGATGATGGGGCAGCACTTAAGGCTACTGATTTTGGACTTTCTGTCTTTATTGAAGAAGGAAAAGTTTACCGTGATATGGTAGGCAGTGCTTACTATGTTGCTCCTGAGGTACTTCGTCGTAATTATGGAAAGGAAATAGACATTTGGAGTGCAGGGATCATATTGTATATTCTGCTTAGTGGTGTACCACCTTTTTGGGCTGAAACTGAAAAGGGAATATTTAATGCTATATTGGAAGGTGAACTCGACTTTGTTAGTGAACCATGGCCATCAATATCAGAAAGTGCCAAAGATCTAGTCCGGAAGATGCTGACACCAGATCCAAAGAAGAGAATCACTTCTACAGAAGTCCTTGAACACCCGTGGATGAGAGAAGGTGGAGAAGCCTCCGATAAACCGATTGATAGTGCAGTTCTATCTAGAATGAAACAATTCAGGGCAATGAACAAGTTCAAGAAACTTGCATTGAAGGTTATGGCTGAAAATTTATCAGAAGAAGAGATTAAAGGTCTCAAGGCGATGTTTGCAAACATGGACACTGATAGTAGTGGCATGATCACTTATGAAGAACTGAAGACAGGCTTGGCTCGAATTGGATCAAGACTGTCTGAGGCTGAAGTGAAACAGCTTATGGAAGCTGCTGATGTTGATGGAAATGGGTCGATTGACTATCTTGAATTCATATCGGCTACAATGCATAGGCACAGACTTGAGCGGGATGAACATCTTTACAAAGCATTCCAGTATTTTGATAAGGACAATAGTGGGCATATTACTAGAGAAGAACTGGAGACTGCCATGACAAAGCATGGAATGGGTGATGAGGCAACGATAAAGGAAATCATTTCTGAGGTGGATACAGATAATGATGGGAGAATAAATTATGAGGAATTCTGTGCAATGATGAGAAGTGGAATGCCACACCAGGGACCATTACTTTAA
SEQ ID NO.3
AGAGATTTAATGGTGTCTGCACATGTGAATATCCATACTTGCTAGTATTAAGCTGAGTAAGTTACACAAATAACCTTAATACATTTCAACTTCCTAAGCCAAAAACCTCGATAGTGTGAATGTTATTAGTTATTACACGAACTTTATTTTTATTAAATCTAACGAACTTTATTTATGTTTTTGCTGAGTCAAGGATCTTTATTTTTATTGTAATGTATGAATATATGTTTTGCTAAAAAAAAGGCATCTGATGAACTTTTGTTTTTAAATTTTAGTTAAAAAATCAACAATTTTTTTCATAAGTGTTATCCATGGATATATGTAAGGGTGGAAATGGGCTAGGCCGGGCCGGGTTTTGTAAGGCCTGAGCCTGGCCTACTTCAAAAATTATAGGCTTGAGCCAGGCCTATTACCCGTCATAGGCTTATTTTCGAGGTTTGGTCTGGCCTTTTTAAAGGTTTGATGAAGCTTGAAAGCCTATTTAAAAGCCTATTTTATGCTAAGGTCTGTCAACGCATGTAAGCCTACTCTTAAATAGGCCAAAACTTATTAAGGTCTAAAGCACATAAATATGTTTGCTACTAAATAATGAATTCTTACGAATGTCTAATCACCTTAATATATCAGCTACTAAATAACCAATCATATGAAGACTTGTGTTTATGGAAGAAAAAAAACTTCAAAAATATCAATAATGGAAGATATATTATGTATGAAAAGCTTAATAGGTCGGTCTGATGGGCTTAATAGGCTTTTTTTGCAAGCATTAGCCTGGTCTGTTTAATTAAATAGGCCTTATAAAAAGTTTGAGCCTGACCTTGTTAATAAACGAGCTGGGCCGAGCTGGGCTTTAAATAGGCTAGGTCGTAGGCCCCTGTCAGACGGCCTGGCATGTTCCCACCCCTAGATATATGTATAAACTCGTTGATAATTTAAAACTCGTAGATTATCTGCTAAACGGATATCCACACGGATATGGACAGGGTACAAATTTCATATTTATTCAATGAAGCGGACACGGATATCACACTATATGCGTGGATGGATATCCATTTACATCCCTATCAACTAGCGAAAATGTCATAATTGTTGGGTCTGTTACCGTTATTGGAGTTCAAACCCCGTCTCATCAACTTTATATCTTTGTGTCTGAATTTGAGATATGACTATTGTCATTTTGTCCATTAAAAAAAAGCTTACTTTAATAAAACATTGAATGCGTTGAATCTGCTAAATGGTAAATACTTGACAAAACATAACAATACGGAACAACACATCATAAGATATAACATCACAAACTTTTACCATATTGAACAATTTTTTGTCTTATATACGTAATGTTTAGTAGACAAAAAAACTATTTTGATATTTTAAGATATAGACAACTTGTACGTAAAACAAAAAGTTGTTGTGATTTAATGAGAAATTTAAGAACAAAAATTTCAGTTTTTATCATATCTCTTGCCTCTAATTTATGCTATCTCTACAACAATTTTACAACAAAGCCAATACAATCGAATTTATCATGTTTTATTCCATAATTCTGAACGGATCAAACACACACCTGGTAAATAATCTTTTGATCAACTTCATATCAATTCAACTAATAAAAGGAATTTGAGTAAGTGACCACCGATTTAATACCTTGTAACAGATAAAGGCGATATTCTACTTTATACTTTTTTTTTGAGGGATATTCTACTTTATATGTCGAACTCTTTGTGAAAAAACTTCCAAAAATGAATTCACCTTTATTTATATAAGGGGTGTTATTATTTTTCTTTTTTTATAAAAATAAATAAATACCAATTATGTGATTTAAACATTTATATATTTTTACACATAAATAAACAATATCAATAAACACAATGATCTGTAATCAACCAAAGCAAAGGAAATAATTTCATTTCCATTATTTCAATCCTCTTTTTCATCTTCTCTGTCTTTTCTTAGCCGCGTTCCAAATTCCAATCATCTTTCAATCCAAATCACACAATA
SEQ ID NO.4
GTAACGGCCGCCAGTCTGGAATTGCCCTTATGGGTTGTCACGGCAGCAAGGAGAAGAAACATGCCAAAG ATGAGTTTACTGCCCGTCATCATAGATCATCACCTGCCCGTCCCGCAGCTGCACCCCCTTCTCGTCCCGCCGGTGTA GATAGCAATTATCGTTACATCAATGTTCCACCTCAGGTTCAACAGTCTTCAACAACAGTTACCCCATCCTCAAATCC AAAACCCTCTGCCACCACTGCCCACAAACCAGTGCAGCAGAAGGTTGATACAACCATCTTAGGTAAACCCTATGAAG ATATAAAGAAGTTTTACACTCTAGGTAAAGAATTGGGTCGAGGCCAATTTGGGATTACTTATTTTTGTACTGAGAAT TCAACCGGTTTAAATTACGCGTGTAAATCTATATTAAAGAGGAAGCTTGTGTCGAAAGCGGATAGAGAAGATATTAA AAGAGAGATTCAGATTCTGCAGCATTTGTCTGGTCAACCGAATATTGTTGAGTTTAAAGGTGCTTATGAGGATAGAT TCTCTGTGCATCTTGTGATGGAACTTTGTGCTGGTGGTGAGTTGTTTGACAGGATTATTGCTCAAGGTCATTATTCA GAGAGAGCTGCTGCTTCTATTTGTAGGGATGTTGTTAAAGTTGTGCACATTTGTCATTTTATGGGTGTTTTGCATCG CGATTTGAAGCCTGAGAATTTCTTGCTTTCTAGTAAGGATGATGGGGCAGCACTTAAGGCTACTGATTTTGGACTTT CTGTCTTTATTGAAGAAGGAAAAGTTTACCGTGATATGGTAGGCAGTGCTTACTATGTTGCTCCTGAGGTACTTCGT CGTAATTATGGAAAGGAAATAGACATTTGGAGTGCAGGGATCATATTGTATATTCTGCTTAGTGGTGTACCACCTTT TTGGGCTGAAACTGAAAAGGGAATATTTAATGCTATATTGGAAGGTGAACTCGACTTTGTTAGTGAACCATGGCCAT CAATATCAGAAAGTGCCAAAGATCTAGTCCGGAAGATGCTGACACCAGATCCAAAGAAGAGAATCACTTCTACAGAA GTCCTTGAACACCCGTGGATGAGAGAAGGTGGAGAAGCCTCCGATAAACCGATTGATAGTGCAGTTCTATCTAGAAT GAAACAATTCAGGGCAATGAACAAGTTCAAGAAACTTGCATTGAAGGTTATGGCTGAAAATTTATCAGAAGAAGAGA TTAAAGGTCTCAAGGCGATGTTTGCAAACATGGACACTGATAGTAGTGGCATGATCACTTATGAAGAACTGAAGACA GGCTTGGCTCGAATTGGATCAAGACTGTCTGAGGCTGAAGTGAAACAGCTTATGGAAGCTGCTGATGTTGATGGAAA TGGGTCGATTGACTATCTTGAATTCATATCGGCTACAATGCATAGGCACAGACTTGAGCGGGATGAACATCTTTACA AAGCATTCCAGTATTTTGATAAGGACAATAGTGGGCATATTACTAGAGAAGAACTGGAGACTGCCATGACAAAGCAT GGAATGGGTGATGAGGCAACGATAAAGGAAATCATTTCTGAGGTGGATACAGATAATGATGGGAGAATAAATTATGA GGAATTCTGTGCAATGATGAGAAGTGGAATGCCACACCAGGGACCATTACTTTAAGGTAACAAATGGTCTTTGATAG CTGGAAGATTACCTGGAAGAACAGATAATGAGATAAAGAATTATTGGAACACTCATATAAGAAGAAAGCTTTTGAAT AGAGGAATTGACCCTGCTACTCATAGGCCTTTAAACGAAGTTTCTCATTCTCATTCTCATTCTCAATCTCAATCACA ATCTCAAACTCTTCATCTTCAAAATCAAGAAGCTGTTACTATAGCTGTAGCAGCATCTACATCAGCTCCCACTGCTA CAAAAACCCTACCAACAACTATATCTTTTGCATCATCCATCAAACAAGAACAATATCATCATCATCATCAAGAAATG AACACAAACATGGTTAAAGGGTTGGTTTTAGAACGTTGTCCTGATTTGAATCTTGAGCTAACAATTAGTCCACCACG TGTTCAAGAACATGATGAACAATTCAGAAACAGAGAGAGGAACAATCTCTGTTTTGTTTGTAGTTTGGGTTTGCAGA ATAGTAAGGATTGTACCTGTGATGAAATTGTTGGAAATTCTAGCAGTGGAAATGGTTCTACTGCACCTGCTTATGAT TTCTTGGGTTTGAAAGGTGGTGTTTGGGATTACAAAGGCTTAGAAATGAAATGAAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCAT
SEQ ID NO.5(MfCDPK14基因扩增上游引物MfCDPK14-F):
5’-ATGGGTTGTCACGGCAGC-3’;
SEQ ID NO.6(MfCDPK14基因扩增下游引物MfCDPK14-R):
5’-TTAAAGTAATGGTCCCTGG-3’;
SEQ ID NO.7(引入Nco I酶切位点的上游引物MfCDPK14-3301-F):
5’-CACGGGGGACTCTTGACCATGGACATGGGTTGTCACGGCAGC-3’;
SEQ ID NO.8(引入BstEIⅠ酶切位点的下游引物MfCDPK14-3301-R):
5’-CGGGGAAATTCGAGCTGGTCACCTTAAAGTAATGGTCCCTGG-3’。
Claims (10)
1.一种黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白,该蛋白的氨基酸序列为(a)或(b):
(a)如SEQ ID NO.1所示的氨基酸序列;
(b)由SEQ ID NO.1所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸而形成的具有同等功能的序列。
2.编码权利要求1所述黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白的MfCDPK14基因;该MfCDPK14基因的核苷酸序列为(1)或(2):
(1)如SEQ ID NO.2所示的核苷酸序列;
(2)与(1)所述的核苷酸序列具有90%以上的同源性且具有同等功能的核苷酸序列。
3.包含权利要求2所述MfCDPK14基因的生物材料,其特征在于,该生物材料为表达盒、重组载体或重组微生物。
4.根据权利要求3所述的生物材料,其特征在于,所述的重组载体为重组表达载体,该重组表达载体是通过将权利要求2所述的MfCDPK14基因与植物表达载体连接得到的。
5.根据权利要求4所述的生物材料,其特征在于,所述重组表达载体的构建方法具体包含如下步骤:
(1)引物设计
引物①为MfCDPK14基因扩增上游引物MfCDPK14-F:
5’-ATGGGTTGTCACGGCAGC-3’;
引物②为MfCDPK14基因扩增下游引物MfCDPK14-R:
5’-TTAAAGTAATGGTCCCTGG-3’;
引物③为引入Nco I酶切位点的上游引物MfCDPK14-3301-F:
5’-CACGGGGGACTCTTGACCATGGACATGGGTTGTCACGGCAGC-3’;
引物④为引入BstEIⅠ酶切位点的下游引物MfCDPK14-3301-R:
5’-CGGGGAAATTCGAGCTGGTCACCTTAAAGTAATGGTCCCTGG-3’;
(2)MfCDPK14基因片段的获得:
以黄花苜蓿的cDNA为模板,使用引物①和引物②扩增MfCDPK14基因片段,并与-Blunt Cloning Kit载体连接,构建获得pEASY-MfCDPK14载体;
(3)含有MfCDPK14基因的重组表达载体pCAMBIA3301-MfCDPK14的构建:
以pEASY-MfCDPK14载体为模板,使用引物③和引物④使MfCDPK14基因片段引入Nco I和BstEIⅠ两个酶切位点,并与植物表达载体pCAMBIA3301连接,进而构建获得含有MfCDPK14基因的重组表达载体pCAMBIA3301-MfCDPK14。
6.权利要求1中所述的黄花苜蓿钙依赖蛋白激酶MfCDPK14蛋白,权利要求2中所述的MfCDPK14基因,或权利要求3~5中任一所述的生物材料在提高植物抗旱性或培育耐旱性提高的转基因植物中的应用。
7.根据权利要求6所述的应用,其特征在于:在目标植物中过表达权利要求2中所述的MfCDPK14基因,或提高权利要求1中所述MfCDPK14蛋白的表达量以提高植物的耐旱性。
8.一种提高植物抗旱性的方法,其特征在于,在目标植物中过表达权利要求2中所述的MfCDPK14基因,或提高权利要求1中所述MfCDPK14蛋白的表达量以提高植物的耐旱性。
9.权利要求6或7中所述的应用,或权利要求8中所述的方法,其特征在于,所述的植物为苜蓿。
10.一种黄花苜蓿钙依赖蛋白激酶MfCDPK14启动子,该启动子的核苷酸序列如SEQ IDNO.3所示。
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