CN104211791B - 一个白粉病抗性相关蛋白mil16及其编码基因和其应用 - Google Patents
一个白粉病抗性相关蛋白mil16及其编码基因和其应用 Download PDFInfo
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Abstract
本发明公开了一种白粉病抗性相关蛋白MIL16及其编码基因与应用。该蛋白MIL16，是具有下述氨基酸残基序列之一的蛋白质：1)序列表中的SEQ ID №.2的氨基酸残基序列；2)将序列表中的SEQ ID №.2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物白粉病抗性相关的由1)衍生的蛋白质。降低植物中本发明蛋白或其编码基因的表达，可实现植物对病原菌抗性的提高和品种的改良，对农业生产具有重要的理论和现实意义。
Description
技术领域
本发明属于生物技术领域，涉及一种白粉病抗性相关蛋白及其编码基因与应用。
背景技术
在自然环境中，植物不断受到病原菌的侵染，包括细菌、真菌以及病毒等。为了抵御病原菌的入侵，植物进化出复杂的防御系统，使植物能够有效阻止绝大多数病原菌的感染。
植物的抗性反应包括PAMP(pathogen-associated molecular pattern)引发的免疫反应PTI(PAMP-triggered immunity)，和效应蛋白引发的免疫反应ETI(effector-triggered_immunity)。寄主能识别病原菌相关的分子模式PAMP，比如细菌的鞭毛蛋白，通过激活有丝分裂原活化蛋白激酶(MAPK)信号传导途径及WRKY转录因子的介导，使植物产生基础抗性，即PTI。在寄主与病原菌的共同进化过程中，病原菌向植物细胞释放效应蛋白，以抑制植物的PTI免疫反应，病原菌得以在植物细胞中繁殖，使植株感病。而植物细胞中存在着抗病(R)蛋白，能直接或间接地识别病原菌的效应蛋白，启动效应蛋白引发的免疫反应ETI(effector-triggered immunity)，产生过敏反应等，植物表现抗病。还有一些证据表明，囊泡运输与植物对病原菌的免疫反应有关，是基于植物细胞自主免疫的抗病机制。
研究表明，一些信号分子，如水杨酸、茉莉酸和乙烯等在植物抗病反应中起着非常重要的作用。其中水杨酸是植物抗病反应中的一个中心信号分子。外部使用水杨酸能激活防御相关基因PR1基因的表达，并诱导对多种病原菌的抗性。
目前植物抗病基因的克隆方法主要有转座子标签技术(transposon tagging)和图位克隆技术(map-based cloning)等。利用克隆的植物抗病基因可以提高植物对病原菌的抗性。通过转座子标签法克隆的玉米抗圆斑病基因Hm1，使玉米获得抗性。携带簇毛麦抗白粉病基因Pm21的6VS·6AL易位系引入小麦，赋予小麦持久、广谱的白粉病抗性。Xa21是水稻中第一个被克隆的抗白叶枯病基因，具有广谱抗性。
对于模式植物拟南芥，图位克隆分离基因成为最主要的方法之一。尤其是全基因组测序计划的完成和各种分子标记的发现，以及DNA提取方法的日趋简化，使克隆基因花费时间大幅缩短。利用模式植物研究植物抗病相关基因的目的，就是为了寻找重要作物中具有类似功能的同源基因，通过植物基因工程技术，提高植物对病原菌的抗性。例如，EDR1，RPW8和NPR1是在拟南芥中发现的调控抗病反应的重要基因，在水稻中，通过抑制OsEDR1基因的表达可以改良水稻对白叶枯病的抗性；在烟草中表达RPW8基因可提高对白粉病菌的抗性；此外，在水稻中过表达拟南芥基因NPR1能提高水稻对细菌的抗性。
经典的杂交育种技术可以成功地培育出新抗病作物品种。研究植物与病原菌的相互作用机制，利用抗性相关基因，通过基因工程技术，可以快速和高效地培育持久抗病的作物品种，达到控制病害发生、保障农业安全生产的目的。
发明内容
本发明的目的是提供一种白粉病抗性相关蛋白MIL16及其编码基因与应用。MIL16蛋白来源于拟南芥(Arabidopsis thaliaha)。
本发明的一个目的是提供一种蛋白，是如下1)或2)的蛋白：
1)序列表中的SEQ ID №.2所示的氨基酸序列组成的蛋白质；
2)将序列表中的SEQ ID №.2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物白粉病抗性相关的由1)衍生的蛋白质。
本发明的另一个目的是提供所述蛋白的编码基因。
所述编码基因具有下述核苷酸序列之一：
1)序列表中SEQ ID №：1所示的核苷酸序列；
2)编码序列表中SEQ ID №：2蛋白质序列的多核苷酸序列；
3)在高严谨条件下可与序列表中SEQ ID №：1限定的DNA序列杂交的核苷酸序列；
4)与1)或2)或3)限定的DNA序列具有90％以上同源性。
上述高严谨条件可为用6×SSC，0.5％SDS的溶液，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
本发明的另一个目的是提供包含所述编码基因的表达盒，所述表达盒具有SEQ ID№：3所示的核苷酸序列。
本发明的又一个目的是提供所述蛋白、所述编码基因或所述表达盒在使植物的白粉病抗性增强中的应用。
本发明的再一个目的是提供所述蛋白、所述编码基因或所述表达盒在培育白粉病抗性增强植物中的应用。
具体的，所述植物为双子叶植物或单子叶植物；所述双子叶植物具体为拟南芥。
本发明通过图位克隆的方法在拟南芥中分离得到一个与白粉病抗性相关的基因。该基因编码框全长1872bp，其核苷酸序列如SEQ ID No.1所示。所述基因编码的蛋白共有624个氨基酸组成，其氨基酸序列如SEQ ID No.2所示。
所述基因丧失功能后导致拟南芥对白粉病抗性的增强。而用所述基因的野生型基因组DNA转化突变体能够实现突变体回复野生型表型。
拟南芥中MIL16基因突变后，白粉菌抗性增强和表现白粉菌诱导的细胞死亡。因此，本发明基因在培育具有抗病性状的作物品种中具有重要应用价值。
附图说明
图1为野生型Col-0和突变体植株在生长4-6周后，大量接种白粉菌(G.cichoracearum)8天后的表型；其中图a为Col-0和突变体接种白粉菌的幼苗表型；图b为Tyrpan blue染色结果；图c为DAB染色结果；图d为Aniline blue染色结果。
图2为野生型Col-0和突变体植株在生长4-6周后，小量接种白粉菌(G.cichoracearum)8天后的单克隆的分生孢子梗数，其中*代表野生型与突变体的差异达显著水平。
图3为野生型Col-0和突变体植株SA含量的检测结果图。
图4为野生型Col-0和突变体植株PR1基因的表达检测结果图。
图5为野生型Col-0和突变体植株MPK蛋白激酶的活性检测结果图。
图6为PCR验证T1代转基因阳性植株结果图。
图7为回复突变实验结果图。
图8为插入突变实验的RT-PCR结果图。
图9为插入突变实验结果图。
具体实施方式
下述实施例中所使用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材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、白粉病抗性相关基因MIL16的制备
(一)、白粉病抗性增强突变体拟南芥的筛选
诱变野生型拟南芥Col-0，从诱变群体中筛选白粉病抗性增强的突变体。具体筛选方法为：把野生型Col-0和诱变后的突变体种子播种到MS培养基，4℃春化2-3天后，转移到22℃，9小时光照/15小时黑暗光照条件下的植物生长室。7-10天后，把幼苗移至土中。幼苗生长4-6周后，用大量(100clones/mm2)和小量(1clone/mm2)两种方法接种白粉病菌(G.cichoracearum UCSC1)(Wang Y，Nishimura MT，Zhao T，Tang D(2011)ATG2，PlantJ68：74-87)。先用保鲜膜覆盖保湿1天，后移去保鲜膜，植物继续生长8天后，进行抗病表型鉴定。小量接种白粉菌只用于白粉定量实验；大量接种白粉菌用于其它抗病表型的分析。将具有典型表型的叶片剪下，照相并进行台盼蓝(Trypan-Blue)染色。DAB染色和Anilineblue染色在接白粉菌2天后进行叶片离体染色。实验结果见图1和图2。
图1结果表明在大量接种白粉菌8天后，野生型的叶片上布满白粉，而突变体的叶片上基本上无可见的白粉，并且突变体表现出白粉菌诱导的细胞死亡(见图1a)。Trypanblue染色也证明了上述结果：野生型的叶片布满了菌丝，而突变体中死亡的叶肉细胞被染成蓝色(见图1b)。DAB染色结果显示，在接种白粉菌2天后，野生型的叶片中，只是被孢子侵染的位点产生过氧化氢，而突变体的叶片中，被孢子侵染的整个细胞由于过氧化氢的产生被染成黄色(见图1c)。Aniline blue染色的结果显示，野生型的叶片，在菌丝可能侵染的部位出现清晰的胼胝质的积累；而突变体的叶片上，在菌丝侵染的整个细胞都出现了胼胝质的积累(附图1d)。
图2结果显示在小量接种白粉菌8天后，突变体叶片上单克隆的分生孢子梗数显著低于野生型。
白粉病菌定性和定量结果表明，筛选出的突变体较野生型拟南芥具有更强的白粉菌抗性。
(二)突变体和野生型拟南芥中病程相关分子的检测
1.SA含量的检测
(1)材料和方法
拟南芥野生型和突变体的幼苗在22℃，9小时光照/15小时黑暗光照条件下正常生长4-6周后，每个材料取0.2克，液氮中速冻，然后在-70℃储存，提SA备用。
用文献Li，X.，Zhang，Y.，Clarke，J.D.，Li，Y.，and Dong，X.(1999).Cell98，329-339.中所述方法提取SA，用HPLC测定SA的含量
(2)结果与分析
结果见图3。图3所示结果显示突变体中游离SA和总SA的平均含量分别是野生型的4.5倍和3.7倍。
2.PR1基因的表达
为了验证MIL16基因是否参与植物的抗病反应，我们用实时定量PCR的方法检测野生型和突变体植株在接种白粉菌后PR1基因的表达模式。所用仪器为德国EppendorfMastercycler ep realplex4S。
(1)材料和方法
拟南芥在22℃，9小时光照/15小时黑暗光照条件下生长4-6周后，大量接种白粉病菌。分别在接种前、接种后1天和3天选取叶片，液氮中速冻，然后在-70℃储存，提RNA备用。
用Trizol(Invitrogen)试剂提取总RNA；后用DNase I(Promega)把污染的DNA消化半小时；用反转录酶M-MLV(Invitrogen)，在20μl反应体系反转2μg总RNA获得第一链cDNA；稀释5倍留作PCR模板备用。
基因表达的分析的反应体系包括：SYBR Green supermix reagent(Takara)：10μl；cDNA模板：2μl；10μM引物：1μl；用重蒸水把体系补至20μl。PCR反应程序：95℃预变性2min，进入循环后，95℃变性15s，55℃退火15s，72℃延伸20s，运行40个循环，每个样品重复3次。基因特异引物序列：PR1F：5′-CGGAGCTACGCAGAACAACT-3′；R：5′-CTCGCTAACCCACATGTTCA-3′；ACT2作为内标，用其表达量衡量并均一化样品RNA的含量。特异引物序列为ACT2F：5′-TCTCCCGCTATGTATGTCGCC-3′，ACT2R：5′-GTCACGTCCAGCAAGGTCAAGA-3′。
(2)结果与分析
结果见图4。图4所示结果显示在未接白粉菌之前，在野生型和突变体中都检测不到PR1基因的表达。接菌后1天和3天，突变体中PR1基因的转录产物显著地高于野生型。表明突变体中发生突变的基因可能作为负调控因子参与植株对白粉病菌的抗性反应。
4.MPK蛋白激酶的活性检测
(1)材料和方法
拟南芥在22℃，9小时光照/15小时黑暗光照条件下生长4-6周后，大量接种白粉病菌。分别在接种前、接种后3天取突变体和野生型的叶片，在液氮中速冻，然后在-70℃储存，提蛋白备用。
在液氮预冷的研钵中，把叶片磨碎。0.2克样品加入400μl新鲜配制的总蛋白提取缓冲液：50mM的Tris-HCl，150mM的NaCl，1mM的EDTA，10％的甘油，1％Triton X-100，用前加1mM DTT和蛋白酶抑制剂Cocktail。冰上放置30分钟，16000g离心30分钟，取上清。跑12％的SDS-PAGE凝胶电泳。通过湿转把蛋白转移到PVDF膜。室温下，5％的TBS-milk封闭2小时。一抗Anti-pETpY(cell signalling)用5％的BSA，1％的TBST按1∶2000稀释，在4℃孵育过夜。二抗用兔源HRP耦联的IgG(国产)抗体按1∶10000稀释，在常温下孵育1小时。用HRP化学发光底物(Millipore)检测，最后显影、定影。
(2)结果与分析
结果见图5。图5所示结果显示在未接白粉菌之前，在野生型和突变体中都检测不到MPK激酶的活性。在接白粉菌3天后，突变体中MPK激酶被大大激活，远高于野生型。MPK3蛋白的表达在各个处理间都有很强的表达。Rubi sco亚基作为对照。
(三)、白粉病抗性相关基因MIL16的扩增
以野生型拟南芥Col-0基因组cDNA作为模板，PCR扩增MIL16基因片段，引物序列为：
上游引物：5’-ATGGCGGAGAATGGTGAAGAG-3’
下游引物：5’-TCATTTTCTTCCCGTGGTAGTCC-3’
PCR反应体系为25μl，其中，cDNA模板：2μl，10xPCR buffer：2.5μl，2.5mM dNTP：2μl，10μM引物1：0.5μl，10μM引物2：0.5μl，rTaq(Takara)：0.2μl，ddH2O：17.3μl。
PCR反应程序：95℃预变性5分钟，循环周期95℃变性30秒，55℃退火30秒，72℃延伸2分钟，30个循环，最后72℃延伸5分钟。
将PCR扩增产物进行测序。测序结果表明，上述PCR产物共1875bp，具有序列表中SEQ ID №：1所示的核苷酸序列，其中第1-1872位核苷酸为编码框，编码序列表中SEQ ID№：2所示的氨基酸序列，共624个氨基酸残基。将该具有序列表中SEQ ID №：1所述的核苷酸序列的片段命名为MIL16。
实施例2、白粉病抗性相关基因MIL16的功能验证
(一)、回复突变实验
以野生型Col-0的DNA为模板，用下述含有EcoR1和Sal1酶切位点(带有下划线的部分)的引物序列进行PCR扩增：
上游引物：5’-TGCTGAATTCTAGAGGCTGCATCATCGAAG-3’，
下游引物：5’-CATCGTCGACTCAAGTTTGATGTTGGCAAT-3’；
经测序，上述PCR产物共4241bp，具有序列表中SEQ ID №：3所示的核苷酸序列，其中SEQ ID №：3为表达盒序列，包含启动子序列、外显子序列和终止子序列；SEQ ID №：3的第1532到第3403位核苷酸为编码框，共1872bp，编码序列表中SEQ ID №：2所示的氨基酸序列，共624个氨基酸残基。扩增的片段经过回收纯化与pCAMBIA1300同时用EcoR1和Sal1双酶切，酶切回收的载体pCAMBIA1300与PCR扩增片段经T4DNA连接酶(NEB)连接，转化大肠杆菌DH-5α后，获得的阳性克隆进行插入片段的测序，测序正确的质粒转化农杆菌GV3101。正确的质粒是在pCAMBIA1300载体的EcoR1和Sal1之间插入了上述PCR产物。用花浸染的方法转化农杆菌GV3101至实施例1筛选出的突变体mil16，4周后获得T1代种子。对种子消毒、清洗，然后播种到添加潮霉素(80μg/ml)的MS培养基中，7-10天后，筛选出T1代转基因阳性植株。用PCR的方法鉴定T1代转基因阳性植株的真实性，即是否为突变体背景的转基因阳性植株。
提取T1代转基因和野生型植株Col-0的DNA作为模板进行PCR检测，所用引物序列为：
RP：5′-AAGATCGTAGTCAGTGAGTGGC-3′，
LB：5′-GGGTGAGATTCCTTGAAGTTGAG-3′；
RP：5′-AAGATCGTAGTCAGTGAGTGGC-3′，
LP：5′-CCTGGTAAGTAACAACCAGTTCGT-3′；
PCR检测结果见图6，图6结果表明T1转基因株系为转基因阳性植株。
在22℃，9小时光照/15小时黑暗条件下，当T1代转基因阳性植株幼苗长到4-6周，接种白粉菌，8天后观察表型。结果见图7，在接种白粉菌8天后，把MIL16基因转入突变体mil16所获得的转基因阳性植株mil16/gMIL16完全恢复成野生型Col-0的表型，叶片表面布满白粉菌的菌丝，而突变体mil16出现白粉菌诱导的细胞死亡。上述结果表明，突变体mil16表现的白粉菌的抗病表型正是由于基因MIL16突变所造成。
(二)、MIL16基因插入突变实验
自ABRC订购MIL16基因的2个T-DNA插入SALK突变体N328818和N717829，T-DNA插入位点分别距起始密码子的1405bp和1540bp。这2个突变体，MIL16基因均因T-DNA插入而功能丧失，检测不到转录产物。
1.N328818和N717829突变体转录水平的检测
(1)材料和方法
取野生型Col-0、实施例1筛选出的mil16突变体和2个salk突变体N328818和N717829的叶片，提取RNA，再反转录成cDNA，再以cDNA为模板，进行MIL16基因的转录水平的检测，ACT2内参基因的表达作为对照。
(2)结果与分析
RT-PCR结果见图8，图8显示，只有对照野生型Col-0能检测MIL16基因的表达，而mil16和2个T-DNA salk突变体N328818和N717829都检测不到。这说明3个突变体都是MIL16功能丧失突变体。
2.N328818和N717829突变体白粉病抗性实验
(1)材料和方法
野生型Col-0、mil16和2个salk突变体N328818和N717829在22℃，9小时光照/15小时黑暗条件下生长4-6周后，大量(100clones/mm2)接种白粉病菌。7天后，进行抗病表型鉴定。
(2)结果与分析
在大量接种白粉菌7天后观察表型，结果见图9，N328818和N717829突变体表现出与实施例1筛选出的突变体mil16同样的表型即白粉菌诱导的细胞死亡。Trypan blue染色的结果也表明3个突变体中死亡的叶肉细胞被染成蓝色。实验结果表明，在接种白粉菌的条件下，与野生型相比，三个突变体都出现了植物叶片细胞死亡的表型。突变体表现出白粉病抗性增强的表型。由于实施例1筛选出的突变体mil16与2个T-DNA插入突变体都是MIL16基因的等位突变体，并且接种白粉菌后表型类似，进一步证明突变体表现出白粉病抗性增强的表型是由于MIL16基因功能丧失产生的。
Claims (4)
1.一种蛋白或其编码基因在使拟南芥的白粉病抗性增强中的应用；
所述蛋白由序列表中的SEQ ID NO.2所示的氨基酸序列组成；
所述白粉病是由白粉菌G.cichoracearum UCSC1侵染引起的。
2.根据权利要求1所述的应用，其特征在于：所述编码基因的核苷酸序列为序列表中SEQ ID NO.1所示的核苷酸序列。
3.一种蛋白或其编码基因在培育白粉病抗性增强的拟南芥中的应用；
所述蛋白为由序列表中的SEQ ID NO.2所示的氨基酸序列组成；
所述白粉病是由白粉菌G.cichoracearum UCSC1侵染引起的。
4.根据权利要求3所述的应用，其特征在于：所述编码基因的核苷酸序列为序列表中SEQ ID NO.1所示的核苷酸序列。
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