CN100395338C - 一种乙烯应答元件结合蛋白及其编码基因 - Google Patents
一种乙烯应答元件结合蛋白及其编码基因 Download PDFInfo
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Abstract
本发明公开了一种乙烯应答元件结合蛋白及其编码基因。本发明的乙烯应答元件结合蛋白的氨基酸残基序列如SEQ ID NO:2所示,其编码基因是下列核苷酸序列之一:1)序列表中SEQ ID NO:1的DNA序列;2)编码序列表中SEQ ID NO:2蛋白质序列的多核苷酸。实验证明本发明的TaEREB1在干旱、高盐和病原菌的诱导下表达,并且可以特异的调控含有GCC-盒顺式元件(核心序列:GCCGCC)的基因的表达,本发明的TaEREB1为人为控制抗逆和耐逆相关基因的表达提供了基础,将在培育抗逆性和耐逆性增强的植物育种中发挥重要的作用。
Description
技术领域
本发明涉及植物中一种与胁迫相关的转录因子及其编码基因,特别涉及一种乙烯应答元件结合蛋白及其编码基因。
背景技术
干旱、高盐及低温等逆境胁迫是影响小麦生长、发育的障碍因子。因此,了解小麦对逆境条件的应答与信号传导机制,提高小麦品种的抗逆性,成为小麦遗传研究及小麦品种改良的重要任务之一。
在逆境胁迫下植物体内会产生一系列应答反应,伴随着许多生理生化及发育上的变化。明确植物对逆境的反应机制,将为抗逆基因工程研究和应用提供科学论据。目前,植物抗逆性研究已逐渐深入到细胞、分子水平,并与遗传学和遗传工程研究相结合,探索用生物技术来改进植物生长特性,其目的是提高植物对逆境的适应能力。
在干旱、高盐和低温等环境胁迫的逆境条件下,植物能够在分子、细胞和整体水平上做出相应的调整,以最大程度上减少环境所造成的伤害并得以生存。许多基因受胁迫诱导表达,这些基因的产物不仅能够直接参与植物的胁迫应答,而且能够调节其它相关基因的表达或参与信号传导途径,从而使植物避免或减少伤害,增强对胁迫环境的抗性。与胁迫相关的基因产物可以分为两大类:第一类基因编码的产物包括离子通道蛋白、水通道蛋白、渗透调节因子(蔗糖、脯氨酸和甜菜碱等)合成酶等直接参与植物胁迫应答的基因产物;第二类基因编码的产物包括参与胁迫相关的信号传递和基因表达调节的蛋白因子,如蛋白激酶、转录因子等。其中,转录因子在植物胁迫应答的基因表达调控中起着重要作用。
转录因子也称为反式作用因子,是能够与真核基因启动子区域中顺式作用元件发生特异性作用的DNA结合蛋白,通过它们之间以及与其它相关蛋白之间的相互作用,激活或抑制转录。转录因子的DNA结合区决定了它与顺式作用元件结合的特异性,而转录调控区决定了它对基因表达起激活或是抑制作用。此外,其自身活性还受到核定位及寡聚化等作用的影响。
目前已知在植物中与胁迫相关的转录因子主要有:具有AP2结构域的AP2(APETALA2)/EREBP(ethylene responsive element binding protein)转录因子家族、含有碱性区域和亮氨酸拉链的bZIP(basic region/leucine zipper motiftranscription factors)类转录因子、含有保守的WRKY氨基酸序列的WRKY转录因子家族、含有碱性螺旋-环-螺旋(bHLH)和亮氨酸拉链的MYC家族和具有色氨酸簇(Trp cluster)的MYB家族。这五个转录因子家族,除WRKY家族不参与植物的水胁迫反应外,其它四个家族均参与调节植物对干旱、高盐和低温等的逆境胁迫反应。其中,AP2/EREBP类转录因子在高等植物中广泛存在,它是植物所特有的一类转录因子,近年来,在拟南芥、烟草、玉米、水稻、大豆和油菜中均有报道,这表明AP2/EREBP类转录因子在高等植物中普遍存在并具有重要作用。
DREB(DRE-binding protein)类转录因子是AP2家族中EREBP-like亚家族中的一个成员。DREB和EREBP类转录因子在氨基酸序列上没有显著的相同性,但都含有一段非常保守的由58个左右氨基酸组成的DNA结合区域(EREBP/AP2结构域)。蛋白质三维分析表明,该区域含有3个β-折叠,对识别各类顺式作用元件起关键作用。其中位于第二个β-折叠中的第14、19位的两个氨基酸残基的差异,决定这类转录因子与不同顺式作用元件的特异结合。DREB类转录因子第14位氨基酸是缬氨酸(V14),第19位氨基酸是谷氨酸(E19),其中第19位的氨基酸并不保守,例如水稻的OsDREB1转录因子的第19位氨基酸就是缬氨酸(Dubouzet J.G.,Sakuma Y.,Ito Y.,Kasuga M.,Dubouzet E.G.,Miura S.,Seki M.,Shinozaki K.,Yamaguchi-Shinozaki K.,OsDREB genes in RICE,Oryza sativa L.,encodetranscription activators that function in drought-,high-salt-andcold-responsive gene expression.The Plant Journal,2003,33:751-763)。在DREB相关蛋白中决定DNA结合的特异性方面,V14的作用明显要比E19重要(Sakuma Y.,Liu Q.,Dubouzet J.G.,Abe H.,Shinozaki K.andYamaguchi-Shinozaki K.,DNA-Binding specificity of the ERF/AP2 domain ofArabidopsis DREBs,transcription factors involved in dehydration-andcold-Inducible gene expression.Biochemical and Biophysical ResearchCommunications,2002,290:998-1009);而ERF类转录因子第14位氨基酸是甘氨酸,第19位是天冬氨酸,因而DREB特异结合DRE/CRT顺式元件,ERF特异结合GCC-盒。AP2/EREBP结构域的C-端区还包含1个由18个氨基酸残基组成的核心序列,该序列形成双亲性的α-螺旋,该α-螺旋可能参与同其它转录因子及DNA间的相互作用。
目前,在许多植物中都发现这种含有EREBP/AP2结构域的转录因子,并分别与抗病、抗逆等信号传递有关(刘强,赵南明,Yamaguchi-Shinozaki K.,ShinozakiK.,DREB转录因子在提高植物抗逆性中的作用.科学通报,2000,第45卷1:11-16)。刘强等认为,一个DREB基因可以调控多个与植物干旱、高盐及低温耐性有关的功能基因的表达(刘强,赵南明,Yamaguchi-Shinozaki K.,Shinozaki K.,DREB转录因子在提高植物抗逆性中的作用.科学通报,2000,第45卷1:11-16)。Kasuga等的研究证实,导入到拟南芥的DREB1A基因可以同时促进与逆境胁迫耐性有关的基因rd29、rd17、kin1、cor6.6、cor15a以及erd10的表达,转基因植株的抗逆性大大增强(Kasuga M.,Liu Q.,Miura S.,Yamaguchi-Shinozaki K.,Shinozaki K.,Improving plant drought,salt,and freezing tolerance by gene transfer of asingle stress-inducible transcription factor.Nature Biotechnology,1999,17:287-292)。同样,低温耐性转录因子CBF1的转基因植株的耐低温能力有显著提高(Jaglo-Ottosen K.R.,Gilmour S.J.,Zarka D.G.,Schabenberger O.,Thomashow M.F.,Arabidopsis CBF1 overexpression induces COR genes andenhances freezing tolerance.Science,1998 280(5360):104-106)。由于植物的逆境耐性是由多基因调控的复杂性状,依靠导入单个功能性蛋白基因很难实现植物抗逆性的综合提高。因此,利用一个关键性转录因子促进多个功能基因的表达,从而增强植物的抗逆性,已经成为植物抗逆基因工程的研究热点。
根据含DNA结合区的数目,AP2/EREBP转录因子分为AP2(APETALA2)和乙烯应答元件结合蛋白EREBP(ethylene-responsive element binding protein)以及RAV三个大类型。AP2型转录因子包括拟南芥的AP2、ANT,玉米的Glossy、idsl等。这种类型的转录因子含有两个AP2/EREBP结构域,调节细胞的生长发育,在拟南芥中已发现14个AP2型转录因子基因;EREBP型转录因子仅含1个AP2/EREBP结构域,调节植物对激素(乙烯)、病原、低温、干旱及高盐等地分子应答反应。EREBP型转录因子中,已发现有烟草EREBP1-4、番茄Pti4-6、拟南芥RAV1-2、AtEBP、AtERF1-5、DREB1A-C(CBF1-3)和DREB2A-B等许多成员,分别与细胞发育、激素、抗病、低温及干旱、高盐等信号传递有关。这些EREBP型转录因子又可以再分为:EREBP(ethylene-responsive element binding protein,即ERF)亚组,包括烟草EREBP1-4、番茄Pti4-6、拟南芥AtEBP、AtERF1-5、这类转录因子与含核心序列AGCCGCC的GCC-盒特异结合,因此,它的DNA结合区又称为GCC-盒结合域(GCC-boxbinding domain,GBD),其中第2个G、第5个G、第7个C对ERF蛋白的识别有重要作用(Hao D.,Ohme-Takagi M.,Sarai A.,Unique mode of GCC box recognitionby the DNA-binding domain of ethylene responsive element-binding factor(ERFdomain)in plant.The Journal of Biological Chemistry,1998,273:26857-26861)。用核磁共振对其三维空间结构的研究表明,AtERF1的GBD通过形成3个反向的β-片层与其靶序列GCC-盒的大沟相结合;DREBP亚组,包括拟南芥DREB1A-C(CBF1-3)和DREB2A-B,这类转录因子在干旱、高盐、低温下特异结合干旱应答元件DRE/CRT,在拟南芥基因组中发现124个DREBP型转录因子基因;RAV型转录因子包括拟南芥RAV1、RAV2、含有两个不同的DNA结合区-ERF/AP2和B3,在拟南芥中已发现6个RAY型转录因子基因。还有一类特殊的转录因子AL079349,它与以上的转录因子都不同,自成一类。
最近发现EREBPs蛋白参与了干旱、高盐和低温胁迫的信号传导和基因表达调控。Mine等人从低温储藏的土豆块茎中分离到了EREBP转录因子CIP353,受低温胁迫诱导强烈表达(Mine T.,Hiyoshi T.,Kasaoka K.,Ohyama A.,2003.CIP353 Encodesan AP2/ERF-Domain Protein in Potato(Solanum tuberosum L.)and RespondsSlowly to Cold Stress.Plant Cell Physiol.,44:10-15),说明可能有EREBP蛋白参与了受低温胁迫的基因表达调控。Park等利用西红柿为材料,得到了受高盐、乙烯或茉莉酸诱导表达的EREBP转录因子Tsi基因,EMSA(Electrophoretic mobilityshift assays)试验分析发现,Tsi蛋白与GCC-box和DRE/CRT顺式元件都能结合(Park J.M.,Park C.J.,Lee S.B.,Ham B.K.,Shin R.,and Paek K.H.,Overexpression of the Tobacco Tsil gene encoding an EREBP/AP2-Type transcriptionfactor enhances resistance against pathogen attack and osmotic stress inTobacco.The Plant Cell,2001,13:1035-1046),尽管前者结合能力大于后者,但说明,某些EREBP蛋白能激活受渗透胁迫诱导表达的基因。在正常生长条件下,Tsi基因的超量表达提高了转基因植株(35S::Tsil)的耐盐性、增强了抗病性(ParkJ.M.,Park C.J.,Lee S.B.,Ham B.K.,Shin R.,and Paek K.H.,Over expressionof the Tobacco Tsil gene encoding an EREBP/AP2-Type transcription factorenhances resistance against pathogen attack and osmotic stress in Tobacco.The Plant Cell,2001,13:1035-1046),以上说明了Tsi基因可能参与了生物胁迫和非生物胁迫两条信号传导途径。由高盐胁迫激活的一条类MAPK信号传递模式(包括SIMKK和SIMK),将胁迫信号传递给EIN2(在乙烯信号传递途径的CTR1下游)(Guo H.W.and Ecker J.,The ethylene signaling pathway:new insights.CurrentOpinion in Plant Biology,2004,7:40-49),最后激活某些EREBPs转录因子,调控渗透胁迫相关基因的表达,提高植物的耐盐性。对于是否存在含GCC-box元件,且表达产物直接参与非生物胁迫响应的基因,还有待作进一步的证实。
综合目前的研究结果,植物在逆境胁迫条件下的信号传递途径至少有以下六条途径:(1)依赖于ABA的信号传递途径有三条:受干旱、高盐诱导,激活MYB、MYC类转录因子基因,调控具有MYBR或MYCR顺式作用元件的靶基因;受干旱、高盐诱导,激活ABF/AREB类转录因子基因,调控具有ABRE顺式作用元件的靶基因;受干旱、高盐诱导,激活CBF4,DREB1类转录因子基因,调控具有DRE/CRT顺式作用元件的靶基因。(2)不依赖于ABA的信号传递途径有三条:受干旱、高盐诱导,激活DREB2类转录因子基因,调控具有DRE/CRT顺式作用元件的靶基因;受低温诱导,激活CBF1-3/DREB1A-C类转录因子基因,调控具有DRE/CRT顺式作用元件的靶基因;受干旱、高盐或乙烯诱导,激活ERF类转录因子基因,调控具有DRE/CRT或GCC顺式作用元件的靶基因。
用酵母单杂交系统证明转录因子的激活特性的主要原理如图2所示,将GCC-盒顺式作用元件和突变体GCC-盒顺式作用元件分别构建到pHISi-1载体和pLacZi载体的基本启动子Pmin(minimal promoter)上游,Pmin启动子下游连接报道基因(His3、LacZ和URA3)。当连接有编码转录因子的目的基因的表达载体YEP-GAP(不含激活功能)分别转化到连有GCC-盒顺式作用元件和突变体GCC-盒顺式作用元件的酵母细胞后,如果连有突变体GCC-盒顺式作用元件的酵母细胞中的报道基因不能表达,而连有特定的GCC-盒顺式作用元件的酵母细胞中的报道基因能够表达,说明该转录因子能与GCC-盒顺式作用元件结合,且具有激活功能,激活了Pmin启动子,促使报道基因表达。从而证明了目的转录因子的体内结合特异性和激活功能。
发明创造内容
本发明的目的是提供一种乙烯应答元件结合蛋白及其编码基因。
本发明所提供的乙烯应答元件结合蛋白,名称为TaEREB1,来源于小麦属小麦(Triticum aestivum L.),是具有序列表中SEQ ID№:2氨基酸残基序列的蛋白质,或者是将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、缺失或添加且具有与SEQ ID №:2的氨基酸残基序列相同活性的由SEQ ID №:2衍生的蛋白质。
序列表中序列2的氨基酸残基序列是由355个氨基酸残基组成的蛋白质,自氨基端第27位-30位氨基酸残基序列和第106位-110位氨基酸残基序列为两个可能的核定位信号区,自氨基端第113位-170位氨基酸残基序列为保守的AP2/EREBP结构域。
乙烯应答元件结合蛋白编码基因,名称为TaEREB1,来源于小麦属小麦(Triticumaestivum L.),是下列核苷酸序列之一:
1)序列表中SEQ ID №:1的DNA序列;
2)编码序列表中SEQ ID №:2蛋白质序列的多核苷酸;
3)与序列表中SEQ ID №:1限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
序列1中的cDNA序列由1373个碱基组成,该基因的开放阅读框架为自5′端第3位-1070位碱基,共编码355个氨基酸(序列表中的序列2)。
含有本发明基因的表达载体及细胞系均属于本发明的保护范围。
扩增TaEREB1中任一片段的引物对也在本发明的保护范围之内。
实验证明本发明的TaEREB1在干旱、高盐和病原菌的诱导下表达,并且可以特异的调控含有GCC-盒顺式元件(核心序列:GCCGCC)的基因的表达,本发明的TaEREB1为人为控制抗逆和耐逆相关基因的表达提供了基础,将在培育抗逆性和耐逆性增强的植物育种中发挥重要的作用。
附图说明
图1a-1c为TaEREB1受胁迫诱导表达的Nothern杂交图谱
图2为用酵母单杂交系统证明转录因子的体内结合特异性和激活特性的原理示意图
具体实施方式
实施例1、TaEREB1的克隆
一、mRNA的分离
将水培生长10天左右的小麦三叶期幼苗按如下方法进行干旱处理2小时:将幼苗小心取出,注意不要伤根,用干净的吸水纸将叶片及根部的水分吸干,再将幼苗放在干净的吸水纸上,置于阴凉处2小时。然后用液氮速冻,-80℃保存备用。采用Quikprep Micro mRNA Purification Kit(Pharmacia)进行mRNA的分离。
二、cDNA文库的构建及滴度测定
1、cDNA文库的构建
采用TimesaverTM cDNA Synthesis Kit(Pharmacia)将步骤一中分离得到的mRNA合成cDNA双链,并加EcoRI/NotI adaptor;采用ZAPPredigestedIII Gold Cloning Kit(Stratagene)进行cDNA文库的构建,共得到500ul库液。
2、滴度的测定
(1)取1ul库液用SM Buffer稀释1000倍;
(2)分别取1ul,10ul,100ul稀释液入三个10ml离心管中,分别加100ul感受态宿主菌XL1-Blue MRF’(OD600为1.0),于37℃温浴20min;
(3)分别加入3ml顶胶(50℃)混匀,立即铺于固体NZY平板上,凝固后倒置,37℃培养过夜;
(4)根据平板噬菌斑数,求平均值,即为库容量。
计算公式:
经计算,该cDNA文库的滴度为3.0×106个噬菌斑。
三、cDNA文库的筛选
1、探针的准备
根据已克隆的DREB基因的AP2保守区序列设计引物WAPF和WAPR,以按常规方法制备的普通小麦的cDNA为模板进行PCR扩增,程序及体系如下:
引物序列:WAPF:5’ACC GCG GTG TGA GGC AGA GGA 3’
WAPR:5’TGA GAA GTT GAC ACG TGC TTT GGC 3’
反应体系(50μl):
模板(60ng/ul) 0.5μl
dNTP(10mM) 1μl
引物(25μM) 1μl
10×buffer 5μl
ddh2O 42.1μl
Taq(5U/μl) 0.4μl
扩增条件(PTC-200):
取扩增产物2ul在1.2%琼脂糖凝胶中电泳检测,溴化乙啶染色,紫外凝胶成像仪扫描观察,在180bp的位置处是否有一条亮带。
2、探针的回收
采用Agarose Gel DNA Purification Kit Ver.2.0(TaKaRa公司,Code No.:DV805A)回收纯化探针。
(1)在紫外灯下迅速切下含有目的DNA片段的带,大概在180bp的位置处,用纸巾吸尽凝胶表面液体并切碎。计算凝胶重量(提前记录1.5ml离心管重量),该重量作为一个凝胶体积(如100mg=100ul);加入3个凝胶体积的DR-I溶液,混合均匀后于75℃加热,间断混合,直至凝胶块完全熔化(约6-8min);
(2)加0.5个DR-I体积的DR-II溶液,再加入异丙醇至终浓度为20%,混合均匀;
(3)吸取步骤(2)中的混合液,转移到DNA制备管(置于2ml离心管中),3600rpm离心1min,弃滤液;
(4)将制备管置回离心管,加0.5ml RinseA溶液,3600rpm离心30s,弃滤液;
(5)将制备管置回离心管,加0.7ml RinseB溶液,3600rpm离心30s,弃滤液。以同样的方法再用0.7ml RinseB溶液洗涤一次;
(6)将制备管置于离心管中,最高速度离心1min;
(7)将制备管置于洁净的1.5ml离心管中,在DNA制备膜正中央加25ul水,室温静置1min,最高速度离心1min洗脱DNA。
(8)洗脱出的即为目的DNA,保存备用。
其中,DR-I溶液:凝胶熔化剂(含DNA保护剂,防止DNA在高温下降解)。室温密闭贮存(TaKaRa公司,Code No.:DV805A)。
DR-II溶液:高离液序列溶液(促使大于100bp的DNA片段选择性结合到DNA制备膜上)。室温密闭贮存。若出现沉淀,应于70℃温育溶解并冷却至室温后再使用(TaKaRa公司,Code No.:DV805A)。
RinseA溶液:洗涤液。室温密闭贮存(TaKaRa公司,Code No.:DV805A)。
RinseB溶液:洗涤液。室温密闭贮存(TaKaRa公司,Code No.:DV805A)。
3、转膜
(1)取1ul库液(见cDNA文库的构建部分),在直径150mm的培养皿中培养噬菌体,大概6.0×103pfu;
(2)噬菌斑培养好后需要放在4℃冷却,临用时取出置于超净台吹干,防止转膜时顶胶被膜粘起;
(3)将Hybrond-N+膜剪成圆形,比直径为150mm的培养皿稍小,用铅笔在膜上表明编号及日期(与培养皿对应);
(4)用镊子夹住膜两边,有字面朝上,中间先接触平板,慢慢松开,不要移动,不要有气泡,使膜自然摊平,完全摊平后,计时;
(5)用注射器针头在膜上扎三个不对称的孔,在培养皿背面对应的位置用记号笔做好标记;
(6)3min后,用镊子从一边开始轻轻揭起膜,不要带起顶胶;
(7)将膜迅速放入盛有变性液的培养皿中(放一层滤纸及15ml变性液)变性7min,有字面朝下,注意避免溶液到达膜的上表面;
(8)将膜转移至盛有中和液的培养皿中(放一层滤纸及15ml中和液)中和两次,每次3min;
(9)然后将膜转入漂洗液中洗30min,可轻轻摇动;
(10)取出膜,在干净的滤纸上吸干,有字面朝下;
(11)用保鲜膜将膜包好,在紫外交联仪上交联1min,4℃存放备用;
其中,
变性液:NaCl 1.5M (87.75g/1000ml)
NaOH 0.5M (20g/1000ml)
中和液:NaCl 1.5M (87.75g/1000ml)
Tris 0.5M (60.57g/1000ml)
EDTA 0.001M (2ml 0.5M贮存液/1000ml)
加入700-800ml水溶解,用HCl调pH值至8.0,最后定容至1000ml。
漂洗液:Tris-HCl(pH7.5) 0.2M(200ml 1.0M贮存液/1000ml)
2×SSC(100ml 20×SSC/1000ml)
4、预杂交和杂交反应
根据要杂交的膜的数量配制预杂交液,每10ml预杂交液的成分组成如下。
ddH2O 6.5ml
5×HSB 2ml
DenHardt’sIII 1ml
*ssDNA(鲑鱼精DNA,Sigma)(10mg/ml) 0.5ml
*ssDNA加入之前需变性:煮沸10min后立即放冰上10min。
其中,5×HSB(pH6.8) 分子量(MW) g/200ml
NaCl(3M) 58.44 35.064
PIPES(0.1M) 302.4 6.048
EDTA(20mM) 372.24 1.488
Denhardt’sIII g/100ml
2%Gelatin 2
2%Ficoll-400 2
2%PVP-360 2
10%SDS 10
5%Na4P2O·10H2O 5
将预杂交液混匀,于65℃预热至澄清后放入尼龙膜,65℃预杂交5-6h(新膜);探针标记完后,加入等体积的0.4N NaOH溶液,混匀后于室温静置10min使探针变性,然后加入到预杂交液中,65℃杂交过夜;
5、洗脱
在55℃-65℃条件下洗膜。
I洗:2×SSC/0.5%SDS,洗两次,每次15min;
II洗:0.1×SSC/0.1%SDS,洗膜过程中检测信号强度,确定洗脱时间。
用滤纸吸干洗好的膜,保鲜膜包好,压X-光片。
6、阳性克隆的二轮筛选
(1)将X光片与膜对齐,确定位置,描下膜上的三个不对称点的位置;
(2)在读片机上放上X光片及相应的培养皿,根据不对称点使培养皿定位;
(3)用去头的1ml枪头取出确定的阳性杂交斑,放在1ml SM缓冲溶液中,加入50ul氯仿;
(4)振荡30秒,室温放置1小时,离心,取上清;
(5)取10-50ul上清再次铺板,培养噬菌体进行二次筛选;
(6)二次筛选的步骤同上:转膜、预杂交和杂交反应、洗脱、压X-光片,得到单个阳性噬菌斑。
四、得到TaEREB1
1、集群内删除(Mass excision)
(1)制备XL1-Blue MRF’和XLOLR菌。用液体LB培养基培养XL1-Blue MRF’和XLOLR菌,30℃过夜,培养基中添加0.2%(w/v)麦芽糖、10mM MgSO4和抗生素,分别为12.5μg/ml四环素和50μg/ml卡那霉素;
(2)第二天,1000×g离心10min收集菌体,用10mM MgSO4重悬,使OD600达到1.0;
(2)在一个10ml的无菌离心管中加入:
1μl库液(见cDNA文库的构建部分)(约含6.0×103噬菌体颗粒)
XL1-Blue MRF’ 200μl(OD600为1.0)
ExAssist助手噬菌体 2μl(>1×1010pfu/ml)
(3)37℃温育15min;
(4)加入20ml液体NZY培养基,37℃振荡培养2.5-3h;
(5)65-70℃加热20min;
(6)1000×g离心10min,上清移至一个新管中;
(7)在一个1.5ml的离心管中混合200μl XLOLR菌和1μl上清;
(8)37℃温育15min;
(9)取10μl、100μl菌液分别涂于LB固体培养基(含氨苄50μg/ml)上,37℃培养过夜,直至长出菌落。
2、cDNA文库插入片段的检查
(1)随机挑取步骤1中集群内删除的单菌落,提取它们的质粒DNA;
(2)用限制内切酶EcoR I(Takara)消化,反应体系10μl:
10×缓冲液H 1μl
EcoR I(12U/μl) 0.5μl
质粒DNA 2μl
ddH2O 6.5μl
(3)37℃消化2h,0.8%琼脂糖凝胶电泳,发现95%以上的载体有插入片段,说明95%以上的噬菌体含有重组子,因此文库实际含有的重组子为2.85×106(cDNA文库的滴度为3.0×106)。50%以上的重组子的插入片段在800bp-4Kb之间,说明构建的文库较完整。
3、单克隆内删除(Single-clone excision)
(1)将得到单个阳性噬菌斑(见三、cDNA文库的筛选部分的步骤6二次筛选cDNA文库)从平板上抠下,放入到一个无菌的、已加有500μl SM缓冲液和20μl氯仿的离心管中,漩涡振荡10sec,4℃储存;
(2)用液体LB培养基培养XL1-Blue MRF’和XLOLR菌,30℃过夜,培养基中添加0.2%(w/v)麦芽糖、10mM MgSO4和抗生素,分别为12.5μg/ml四环素和50μg/ml卡那霉素;
(3)第二天,1000×g离心10min收集菌体,用10mM MgSO4重悬,使OD600达到1.0;
(4)在一个10ml的无菌离心管中加入:
XL1-Blue MRF’ 200μl(OD600为1.0)
噬菌体贮存液 250μl(至少含1×105噬菌体颗粒)
ExAssist助手噬菌体 1μl(>1×1010pfu/ml)
(5)37℃温育15min;
(6)加入3ml液体NZY培养基,37℃振荡培养2.5-3h;
(7)于65-70℃水浴离心管20min,然后1000×g离心15min;
(8)将上清移至一个新的离心管中,即为噬菌粒悬浮液;
(9)在一个1.5ml的离心管中加入200μl步骤(3)制备好的XLOLR菌和步骤(8)制备好的噬菌粒悬浮液100μl,再加入300μl液体NZY培养基,37℃温育45-60min;
(10)取50μl菌液涂于LB固体培养基(含氨苄50μg/ml)上,37℃培养过夜;
(11)第二天挑取阳性克隆,用液体LB培养基培养过夜,提取质粒,用EcoRI酶切,电泳检测插入片段长度。
(12)选取插入片段大于800bp的克隆进行测序,在ABI733测序仪(GenecoreBiological Company)上,采用双脱氧核苷酸链终止法测定序列,将得到的全序列与EMBL Bank以及GENEBANK核苷酸数据库比较,用DNASIS软件进行分析。发现6号克隆有一个保守的AP2/EREBP结构域,且基因结构完整。
(13)分析6号克隆的核苷酸序列及对应的氨基酸序列,结果得到具有序列表中序列1所示的核苷酸序列的TaEREB1,其开放阅读框架为自5′端第3位-1070位碱基,共编码355个氨基酸(序列表中的序列2),序列2中的自氨基端第27位-30位氨基酸残基序列和第106位-110位氨基酸残基序列为两个可能的核定位信号区,自氨基端第113位-170位氨基酸残基序列为保守的AP2/EREBP结构域。
实施例2、Northern杂交分析TaEREB1的表达特性
1、植物材料的准备
苗龄为10天的小麦幼苗,进行以下处理:
(1)干旱处理:将水培的小麦幼苗取出吸干根上的水分,置于干燥的滤纸上,干旱培养2小时、5小时、10小时后取出材料,用液氮速冻,-80℃保存备用。
(2)盐渍处理:将小麦幼苗置于2%的由NaCl和Na2SO4组成的钠盐溶液中(NaCl与Na2SO4的质量百分比为3∶2)中,光照培养2小时、5小时、10小时后分别取出材料,用液氮速冻,-80℃保存备用。
(3)ABA处理:将小麦幼苗置于200μM的ABA溶液中,光照培养2小时、5小时、10小时后分别取出并用液氮速冻,-80℃保存备用。
(4)白粉病处理:将小麦幼苗接种白粉病菌株,光照培养24及48小时后,取出并用液氮速冻,-80℃保存备用。
(5)冷害处理:将小麦幼苗置于4℃培养箱,光照培养6小时、10小时、24小时后取出并用液氮速冻,-80℃保存备用。
(6)对照的处理:直接取未经任何处理的小麦幼苗-80℃冻存作为对照。
2、转膜
(1)电泳及转膜用具的处理:0.1N NaOH/1mM EDTA浸泡1h以上,用清水冲洗干净备用;
(2)准备甲醛变性凝胶:在一个三角烧瓶中加入10ml 10×MOPs,73ml DEPC处理的水和1g琼脂糖,加热融化;当冷却到55℃左右时加入17ml甲醛,混匀,倒入RNA专用的胶槽中;
(3)在DEPC处理的0.5ml离心管中加入:
RNA+DEPC处理水 4μl(RNA约为30μg,利用Promega公司的RNAgents Total RNA Isolation System kit提取的上述幼苗的总RNA)
甲酰胺 12.5μl
甲醛 4.0μl
10×MOPs 2.5μl
溴酚兰(10×) 2.5μl
(4)样品在65℃水浴处理5min,然后点样,开始电泳,缓冲液为1×MOPs。当溴酚兰到达凝胶1/3-1/2位置时,停止电泳;
(5)用蒸馏水冲洗凝胶,用20×SSC转膜。将凝胶翻转后放在滤纸桥上,放上一张与凝胶大小相当的尼龙膜(Hybond-N+,Amarsharm),赶除气泡,在膜上放3层滤纸,与尼龙膜大小相同,赶除气泡,将凝胶周围用胶片封好,放上大小相当的吸水纸,压上约500g的重物,转膜16-24h,其间更换吸水纸。转膜完成后,用2×SSC洗膜10min,用滤纸吸干,保鲜膜包好,紫外灯照射3min,4℃存放至杂交;
3、探针的制备
在TaEREB1基因的3’设计特异引物EREB1F和EREB1R(避开AP2/EREBP区域),以克隆的TaEREB1基因为模板进行PCR扩增,程序及体系如下:
引物序列:EREB1F:5’CTG TGA TGG GTG ATG CTA ATG 3’
EREB1R:5’AGC AAT TAC AAT AAG GTA GAA ACA GG 3’
反应体系(50μl):
模板(60ng/ul) 0.5μl
dNTP(10mM) 1μl
引物(25μM) 1μl
10×buffer 5μl
ddh2O 42.1μl
Taq(5U/μl) 0.4μl
扩增条件(PTC-200):
取扩增产物2ul在1.2%琼脂糖凝胶中电泳检测,溴化乙啶染色,紫外凝胶成像仪扫描,观察在450bp左右的位置处是否有一条亮带。
4、探针的回收
采用Agarose Gel DNA Purification Kit Ver.2.0(TaKaRa公司,Code No.:DV807A)回收纯化探针(同三、cDNA文库的筛选的探针的回收)。
5、探针标记
探针标记为随机六聚体引物法,每1-2张膜加入步骤4的探针DNA,约50-100ng和,加水至14.5μl,100℃煮沸变性5min后立即置于冰浴中5min,瞬时离心,然后加入下列成分,终体积25μl,于37℃反应5h-8h。
5×Oligo缓冲液 5μl
Klenow片段(5U/μl) 0.7μl
10×缓冲液 2.5ul
BSA(10mg/ml) 1μl
α-32P-dCTP(10mCi/ml) 1.5μl
其中,1ml 5×Oligo缓冲液中含有:
160μl H2O
250μl 1M Tris-HCl
25μl 1M MgCl2
5μl 1M巯基乙醇
500μl 2M HEPES
20μl 100mM dATP
20μl 100mM dGTP
20μl 100mM dTTP
4、预杂交和杂交反应
根据要杂交的膜的数量配制预杂交液,每10ml预杂交液的成分组成如下。
ddH2O 6.5ml
5×HSB 2ml
DenHardt’sIII 1ml
*ssDNA(鲑鱼精DNA,Sigma)(10mg/ml) 0.5ml
*ssDNA加入之前需变性:煮沸10min后立即放冰上10min。
其中,5×HSB(pH6.8) 分子量(MW) g/200ml
NaCl(3M) 58.44 35.064
PIPES(0.1M) 302.4 6.048
EDTA(20mM) 372.24 1.488
Denhardt’sIII g/100ml
2%明胶 2
2%Ficoll-400 2
2%PVP-360 2
10%SDS 10
5%Na4P2O·10H2O 5
将预杂交液混匀,于65℃预热至澄清后放入尼龙膜,65℃预杂交5-6h(新膜);探针标记完后,加入等体积的0.4N NaOH溶液,混匀后于室温静置10min使探针变性,然后加入到预杂交液中,65℃杂交过夜;
5、洗脱
将杂交完的膜从杂交管中取出放入洗脱盒中,加入少量洗液I(2×SSC/0.1%SDS)漂洗除去过量杂交液,然后用洗液I于45℃洗两次,每次20min;再用洗液II(0.2×SSC/0.2%SDS)于45℃洗两次,每次15min。洗液I洗过后,检测杂交信号与背景情况,再决定是否继续洗脱。用滤纸吸干洗好的膜,保鲜膜包好(注意除去气泡);
6、放射自显影
在X-射线摄影暗盒中,将杂交膜置于增感屏上,在暗室中将X-光片放在杂交膜上,再放上另一张增感屏,压紧X-射线摄影暗盒,-70℃冰箱曝光7-15天。
Northern分析结果如图1a-1c所示,表明在干旱胁迫2小时后,以及在高盐(2%(NaCl+NaSO4)胁迫2小时后TaEREB1基因开始表达。同样,在白粉病菌诱导的条件下,TaEREB1基因表达增强,说明TaEREB1基因分别受干旱、高盐和病原菌诱导表达。但是,在冷胁迫和ABA处理的条件下,TaEREB1基因没有表达。说明该基因不受冷胁迫和ABA诱导。图1a-1c中,CK为对照。
实施例3、TaEREB1的激活特性
一、将TaEREB1基因构建到表达载体YEP-GAP上
1、获得TaEREB1基因编码氨基酸部分的全序列
根据已克隆的TaEREB1基因的序列设计引物,引物末端分别加SmaI和XhoI酶切位点,PCR扩增获得编码氨基酸部分的全序列,程序及体系如下:
引物序列:
TaEREB1-EI:5’-TTTCCCGGGATGTGCGGCGGCGC-3’
TaEREB1-XI:5’-CCCCTCGAGCCCAGCGACCTCGATTCCC-3’
反应体系(50μl):
模板(60ng/ul) 0.5μl
dNTP(10mM) 1μl
引物(25μM) 1μl
10×buffer 5μl
ddH2O 42.1μl
Taq(5U/μl) 0.4μl
扩增条件(PTC-200):
取扩增产物2ul在1.2%琼脂糖凝胶中电泳检测,溴化乙啶染色,紫外凝胶成像仪扫描,观察在1.1Kb左右的位置处是否有一条亮带。
采用Agarose Gel DNA Purification Kit Ver.2.0(TaKaRa公司,Code No.:DV807A)回收纯化PCR产物(方法同实施例1的三、cDNA文库的筛选中的步骤2、探针的回收)。
2、将TaEREB1基因构建到表达载体YEP-GAP上
将步骤1中纯化的PCR产物和表达载体YEP-GAP(Liu Q,Kasuga M,SakumaY,Abe H,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.,1998),用SmaI(Takara)和XhoI(Takara)分别酶切4-6hr,反应体系如下:
10×缓冲液H 5μl
Sma I(12U/μl) 2μl
XhoI(12U/μl) 2μl
PCR产物/YEP-GAP 20μl
ddH2O 21μl
采用Agarose Gel DNA Purification Kit Ver.2.0(TaKaRa公司,Code No.:DV807A)回收纯化酶切产物(方法同实施例1的三、cDNA文库的筛选中的步骤2、探针的回收)。
将纯化的酶切PCR产物和酶切载体YEP-GAP连接4-8hr,反应体系如下:
10×Ligase buffer 1μl
酶切PCR产物 4μl
酶切载体YEP-GAP 4μl
T4DNA Ligase 1μl
取0.5μl连接产物,电击转化JM109菌株,37℃过夜培养,挑取阳性克隆,测序分析序列是否正确,得到含有TaEREB1基因的重组表达载体YEP-GAP-TaEREB1。
二、TaEREB1的体内结合特异性和激活特性的验证
1、酵母报道子的构建
将含有4个GCC-盒元件的串联序列5’-GAATTC-GAGCCGCCAC-GAGCCGCCAC-GAGCCGCCAC-GAGCCGCCAC-GTCGAC-3’(GCC-盒的核心序列是GCCGCC)分别构建到pHis-1载体(MATCHMAKER One-Hybrid System,Clontech公司)的PminHIS3启动子和pLacZi载体(MATCHMAKER One-Hybrid System,Clontech公司)PCYCI启动子上游,分别得到重组载体pHis-1-GC和pLacZi-GC,用Xho I和Nco I内切酶分别将pHis-1-GC和pLacZi-GC切成线状。先将线状pHis-1-GC转化到酵母细胞(YM4271株系,MATCHMAKEROne-Hybrid System,Clontech公司)内,获得能在SD/His-培养基上正常生长的酵母转化子(Yeast transformant)。接着以这种酵母转化子为寄主细胞,继续转化含有4个重复GCC-盒元件的pLacZi-GC载体。这样在同时缺乏组氨酸和尿嘧啶的SD/His-/Ura-培养基上,选择获得含有pHis-1-GC和pLacZi-GC的正常双重酵母报道子;将4个GCC-盒元件的核心序列GCCGCC突变成TTTTTT(MGCC盒),即5’-GAATTC-GATTTTTTAC-GATTTTTTAC-GATTTTTTAC-GATTTTTTAC-GTCGAC-3’,按正常的双重酵母报道子构建方法,再构建一个含4个MGCC盒的突变体双重酵母报道子。
2、PEG/LiAc法转化酵母及结果分析
(1)接种酵母菌株(YM4271株系,MATCHMAKER One-Hybrid System,Clontech公司)到1ml YPD液体培养基中,剧烈震荡2分钟,分散团块后将悬浮液转至含有50ml YPD液体培养基的三角瓶中,30℃/250rpm摇过夜,测OD600=1.7-1.8(计数约4×107个/mL);
(2)取30ml步骤(1)过夜培养物接到300ml新鲜的YPD培养基中,30℃/250rpm培养,约3小时至OD600=0.5±0.1,室温1000g离心5min,收集菌体,弃上清,用1/2体积1×TE悬浮,1000g/5min离心;
(3)吸弃上清,用1.5ml新鲜配制的1×TE/LiAc溶液悬浮,振荡混匀备用;
(4)取出0.1ml酵母感受态进行转化,依次加下列溶液:0.1μg表达载体YEP-GAP-TaEREB1、0.1mg ssDNA(鲑鱼精DNA,Sigma)、0.6mlPEG/LiAc高速振荡1分钟,30℃/200rpm振荡培养30分钟;
(5)加入70ul DMSO(sigma#D8779),轻轻倒置混匀,42℃热激30分钟,其间轻轻振荡,冰浴2分钟,室温1000g离心5min;
(6)吸弃上清,加入0.5ml 1×TE buffer悬浮细胞;
(7)用接种环蘸取悬浮液,分别在含有0,15mmol/L 3-AT的SD/His-/Ura-/Trp-选择性培养基上画线培养。
(8)平板的一半培养步骤1构建的正常双重酵母报道子,另一半培养步骤1构建的突变体双重酵母报道子,以便做对照分析。
(9)颠倒放置于培养箱中,30℃培养3-4天。
(10)结果发现在0mmol/l 3-AT的SD/His-/Ura-/Trp-的培养基平板上正常的酵母报道子和突变的酵母报道子都有生长,但突变的酵母报道子的直径明显小;而在15mmol/L 3-AT的SD/His-/Ura-/Trp-的培养基平板上正常的酵母报道子能正常生长,但突变的酵母报道子被抑止没有生长。
3、半乳糖苷酶活性检测
(1)从0mmol/L 3-AT的SD/His-/Ura-/Trp-的培养基平板上分别挑取正常的酵母报道子和突变的酵母报道子菌落。转至YPD液体培养基中,于30℃振荡培养,待长至对数生长后期,取1.5ml菌液,3000rpm离心30s;
(2)弃上清,控干管中液体,将离心管置于液氮中速冻10min,取出使其自然融解,加50ul Z/X-gal溶液,30℃温育,结果发现正常的酵母报道子在6-8h内变蓝,而突变的酵母报道子在12h内没有变化,仍为白色。说明转录因子TaEREB1能与GCC-盒顺式作用元件结合,且具有激活功能,激活了Pmin启动子(包括PminHIS3启动子和PCYCI启动子),促使报道基因表达。从而证明了TaEREB1的体内结合特异性和激活功能。
三、药剂配制:
(1)YPD液体培养基
细菌培养用酵母抽提物(Bacto-Yeast Extract) 10g/L
细菌培养用胰化蛋白胨(Bacto-Peptone) 20g/L
调节pH至5.8,121℃/15min灭菌,降至60℃以后加入40%的Glucose,使其终浓度为20g/L。
(2)SD/His-/Ura-/Trp-选择性培养基
不含氨基酸的酵母氮源(Yeast nitrogen base) 6.7g/L
营养缺陷型混合物(drop-out media without Leu/Ura/Trp) 100ml
琼脂粉(Bacteriological agar) 20g/L
调节pH至5.8,121℃/15min灭菌,降至60℃后加入40%Glucose,使其终浓度为20g/L。
(3)营养缺陷型混合物(Drop-out mix): (10X)
L-Isoleucine(异亮氨酸) 300mg/L
L-Valine(缬氨酸) 1500mg/L
L-Adenine(腺嘌呤) 200mg/L
L-Arginine(精氨酸) 200mg/L
L-Histidine Hcl monohydrate(组氨酸) 200mg/L
L-Leucine(亮氨酸) 1000mg/L
L-Lysine Hcl(赖氨酸) 300mg/L
L-Methionine(甲硫氨酸) 200mg/L
L-Phenylalanine(苯丙氨酸) 500mg/L
L-Threonine(苏氨酸) 2000mg/L
L-Tyrosine(酪氨酸) 300mg/L
(4)1×PEG/LiAc:
50%(w/v)PEG3350 8ml
10×TE buffer 1ml
10×LiAc 1ml
(5)10×TE Buffer:
100mM Tris-Hcl
10mM EDTA,pH=7.5
121℃高压灭菌,室温保存。
(6)1×TE/LiAc:
10×TE buffer 1ml
10×LiAc 1ml
ddH2O 8ml
(7)Z Buffer:
Na2HPO4·7H2O 16.1g/L
NaH2PO4·H2O 5.5g/L
KCl 0.75g/L
MgSO4·7H2O 0.246g/L
调节pH至7.0,121℃/15min灭菌,4℃保存。
(8)X-gal储存液(X-gal Stock Solution):
用N,N-dimethyl-formamide(DMF)溶解X-gal,使其终浓度为20mg/ml,-20℃贮存。
(9)含有X-gal的Z buffer缓冲液100ml(Z buffer with X-gal),注意现用现配:
Z buffer 98ml
β-巯基乙醇(β-mercaptoethanol) 0.27ml
X-gal储存液(X-gal stock solution) 1.67ml
序列表
<160>2
<210>1
<211>1373
<212>DNA
<213>小麦属小麦(Triticum aestivum L.)
<400>1
ccatgtgcgg cggcgccatc ctctccgaca tcatcccgcc gccgcgccgg gccaccggcg 60
gcaacgtctg gcgggcggac aagaagagga gggccaggcc cgacgccgcc gcggggaggc 120
cccgccgcgt gcccgaggag gagttccagg aggaggaggg cgacgcggag ttcgaggccg 180
acttcgaggg gttcgtggag gcggaggagg agtccgacgg cgaggccaag cccttccccg 240
tccgcaggac cggcttctcc ggagatggac tgaaggcaac tgctgctggt gatgatgact 300
gtgcctcagg gtctgctaaa aggaagagaa agagccagtt caggggcatc cgccgccgcc 360
cttggggtaa atgggctgct gaaataagag atcctcgcaa gggtgtccgt gtctggcttg 420
gcacttacaa ctctgctgag gaagctgcca gagcctatga tgttgaagcc cgcagaattc 480
gtggcaagaa ggcaaaggtc aatttcccag aagaagctcc catggatcct cagcaacgct 540
gcgctacctc tgtgaaggtg cccgagttca acaccgaaca gaagccagta ctcaacacca 600
tgggcaacac agatgtgtat tcctgccctg ctgttgacta caccttaaat cagcaatttg 660
tgcagcctca gaacatgtcg tttgtgccta cagtgaatgc agttgaggct cctttcatga 720
atttttcctc tgaccagggg agcaactcct ttagttgctc agacttcagc tgggagaatg 780
atatcaagac ccctgacata acttctgtgc ttgcatccat tcccacctca actgaggtca 840
atgaatctgc atttctccag aacaatggca ttaattcaac ggtacctcct gtgatgggtg 900
atgctaatgt tgatcttgcc gacttggagc catacatgaa gttcctgatg gacgatggtt 960
cagatgagtc aattgacagc attctaagct gtgatgtacc gcaggacgtt gtcggcaaca 1020
tgggcctttg gacctttgat gacatgccct tgtctgctgg tttctactga gggaatcgag 1080
gtcgctgggt gcctgtatat atagacaaag gaataagtat tctggacatc aacaagtgct 1140
tgtgtctggt gcctctagaa tcgagcagta gcgacgtcag tctatggtta tgtctagctt 1200
aaatggtcag gagacctaag tcttttgcaa tagacctctg tcttgtgccc ccagactata 1260
ttatatctat atatgaaacc agtatgtgat gggaactgct tattttgtat tcctgtttct 1320
accttattgt aattgctaca agtggctgta aaccttttaa ctttgaaaaa aaa 1373
<210>2
<211>355
<212>PRT
<213>小麦属小麦(Triticum aestivum L.)
<400>2
Met Cys Gly Gly Ala Ile Leu Ser Asp Ile Ile Pro Pro Pro Arg Arg
1 5 10 15
Ala Thr Gly Gly Asn Val Trp Arg Ala Asp Lys Lys Arg Arg Ala Arg
20 25 30
Pro Asp Ala Ala Ala Gly Arg Pro Arg Arg Val Pro Glu Glu Glu Phe
35 40 45
Gln Glu Glu Glu Gly Asp Ala Glu Phe Glu Ala Asp Phe Glu Gly Phe
50 55 60
Val Glu Ala Glu Glu Glu Ser Asp Gly Glu Ala Lys Pro Phe Pro Val
65 70 75 80
Arg Arg Thr Gly Phe Ser Gly Asp Gly Leu Lys Ala Thr Ala Ala Gly
85 90 95
Asp Asp Asp Cys Ala Ser Gly Ser Ala Lys Arg Lys Arg Lys Ser Gln
100 105 110
Phe Arg Gly Ile Arg Arg Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile
115 120 125
Arg Asp Pro Arg Lys Gly Val Arg Val Trp Leu Gly Thr Tyr Asn Ser
130 135 140
Ala Glu Glu Ala Ala Arg Ala Tyr Asp Val Glu Ala Arg Arg Ile Arg
145 150 155 160
Gly Lys Lys Ala Lys Val Asn Phe Pro Glu Glu Ala Pro Met Asp Pro
165 170 175
Gln Gln Arg Cys Ala Thr Ser Val Lys Val Pro Glu Phe Asn Thr Glu
180 185 190
Gln Lys Pro Val Leu Asn Thr Met Gly Asn Thr Asp Val Tyr Ser Cys
195 200 205
Pro Ala Val Asp Tyr Thr Leu Asn Gln Gln Phe Val Gln Pro Gln Asn
210 215 220
Met Ser Phe Val Pro Thr Val Asn Ala Val Glu Ala Pro Phe Met Asn
225 230 235 240
Phe Ser Ser Asp Gln Gly Ser Asn Ser Phe Ser Cys Ser Asp Phe Ser
245 250 255
Trp Glu Asn Asp Ile Lys Thr Pro Asp Ile Thr Ser Val Leu Ala Ser
260 265 270
Ile Pro Thr Ser Thr Glu Val Asn Glu Ser Ala Phe Leu Gln Asn Asn
275 280 285
Gly Ile Asn Ser Thr Val Pro Pro Val Met Gly Asp Ala Asn Val Asp
290 295 300
Leu Ala Asp Leu Glu Pro Tyr Met Lys Phe Leu Met Asp Asp Gly Ser
305 310 315 320
Asp Glu Ser Ile Asp Ser Ile Leu Ser Cys Asp Val Pro Gln Asp Val
325 330 335
Val Gly Asn Met Gly Leu Trp Thr Phe Asp Asp Met Pro Leu Ser Ala
340 345 350
Gly Phe Tyr
355
Claims (6)
1.一种乙烯应答元件结合蛋白,其氨基酸残基序列如SEQ ID NO:2所示。
2.一种乙烯应答元件结合蛋白编码基因,是下列核苷酸序列之一:
1)序列表中SEQ ID NO:1的DNA序列;
2)编码序列表中SEQ ID NO:2蛋白质序列的多核苷酸。
3.根据权利要求2所述的基因,其特征在于:所述基因的碱基序列如SEQ ID NO:1所示。
4.含有权利要求2或3所述基因的表达载体。
5.含有权利要求2或3所述基因的细胞系。
6.扩增权利要求2或3所述基因的引物对。
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WO2002067678A1 (en) * | 2001-02-26 | 2002-09-06 | Rohm And Haas Company | A method to inhibit ethylene responses in plants |
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CN1475495A (zh) * | 2002-08-14 | 2004-02-18 | 清华大学 | 冰植物的乙烯应答结合蛋白转录因子及其编码基因与应用 |
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CN1475495A (zh) * | 2002-08-14 | 2004-02-18 | 清华大学 | 冰植物的乙烯应答结合蛋白转录因子及其编码基因与应用 |
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