CN101736015A - 火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP及应用 - Google Patents
火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP及应用 Download PDFInfo
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Abstract
本发明涉及一种来源于火把梨的真菌抗性基因多聚半乳糖醛酸酶抑制蛋白基因PpPGIP及应用。基因PpPGIP具有序列表SEQ ID所述的碱基序列,PpPGIP基因长1032bp,其中990bp为开放阅读框,编码含330个氨基酸的多聚半乳糖醛酸酶抑制蛋白。本发明通过功能基因组学相关技术证实PpPGIP基因具有提高植物抗真菌的功能。本发明将抗真菌PpPGIP基因构建到植物表达载体上并转入烟草中过量表达,表达PpPGIP的转基因烟草具有很强的体外抗真菌活性,对黑曲霉、拟茎点霉属真菌、交链孢菌、青霉菌等多种真菌具有明显的抑制作用。
Description
技术领域
本发明涉及分子生物学以及基因工程技术领域,特别是一种火把梨的具有抗真菌活性的多聚半乳糖醛酸酶抑制蛋白基因PpPGIP及应用。
背景技术
在农业生产中,植物病害是一个非常棘手的问题,尤其是真菌病害,由其引起的病害占植物总病害的80%以上,且一种作物上可发现几种甚至几十种真菌病害。在近一个世纪以来,育种学家和植物病理学家为控制植物真菌病害付出了很大的努力。到目前为止,控制植物真菌病害方法主要有三种。一是采用几种不同的栽培管理方式,如作物轮作以避免使用含有病原真菌的土壤;二是通过有性杂交利用作物本身或近缘种中抗性基因选育抗性品种;三是施用化学农药。以上三种方法各自都具有一定的弊端和局限性,不能从根本上解决真菌病害问题。
随着分子生物学和基因工程的迅速发展,通过DNA重组技术导入抗真菌基因,最终培育出抗真菌植物新品种,为从根本上解决真菌病害问题开辟了新途径。因此,获得抗真菌病害基因并研究其抗性机理无疑是至关重要的。近年来,对植物-病原菌相互识别机制、信号传导途径、防卫反应的调控以及抗病和致病基因本质的研究不断深入,一系列专化抗性和广谱抗性的抗真菌基因陆续被克隆,利用转基因技术改良植物的真菌病害抗性已取得初步成效。
植物抵抗真菌侵染的一个重要屏障就是细胞壁,病原真菌为了渗透植物细胞壁,会分泌一系列降解植物细胞壁的水解酶。多聚半乳糖醛酸酶(polygalacturonases,PGs)是病原真菌侵染植物时分泌的第一个水解酶,因此它是真菌重要的致病因子之一。多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting proteins,PGIPs)是与植物自身免疫相关的多功能蛋白,属于植物胞外富含亮氨酸重复序列(extracellular leucine-richrepeat,eLRR)蛋白超家族,能特异性结合真菌分泌的PGs,降低PGs水解活性的同时在植物体内累积大量能激活各种防御反应的长链寡聚半乳糖醛酸(oligogalacturonides,OGs),最终抑制真菌对植物的侵染(De Lorenzo G,D’Ovidio R,Cervone F.The role ofpolygacturonase inhibiting proteins(PGIPs)in defense against pathogenic fungi.Annual Review of Phytopathology,2001,39:313-335.)。自1971年首次在蚕豆和番茄的细胞壁中发现PGIP蛋白以来,国内外研究人员已在苹果、梨、番茄、草莓、桃等二十多种植物中证实有PGIP的存在,并克隆得到了相应基因序列。
菜豆PGIPs家族有4个成员PvPGIP1-4,位于第10染色体共50Kb的区域上,这些基因的核苷酸序列相似度在80-99%之间,它们所编码的PGIPs都能抑制灰霉菌和炭疽病菌的PGs,然而只有PvPGIP1和PvPGIP2能抑制黄曲霉的PGs,串珠镰刀菌的PGs只有PvPGIP2能抑制(D’Ovidio R,Raiola A,Capodicasa C,et al.Characterization of the complexlocus of bean encoding polygalacturonase-inhibiting proteins revealssubfunctionalization for defense against fungi and insects.Plant Physiology,2004,135:2424-2435.)。在拟南芥中有两个相似度达80%的基因AtPGIP1和AtPGIP2,都存在于第5染色体上。与菜豆PGIPs相比,拟南芥PGIPs不能抑制黄曲霉和串珠镰刀菌的PGs,但能抑制炭疽病菌、玉米穗腐病菌和灰霉菌的PGs(Ferrari S,Vairo D,AusubelFM,et al.Tandemly dupl icated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transductionpathways in response to fungal infection.The Plant Cell,2003,15:93-106.)。对于单子叶植物而言,目前在小麦中发现一个基因编码PGIP,其对小麦根腐病菌有很强的抑制作用(Kemp G,Bergmann CW,Clay R,et al.Isolation of a polygalacturonase-inhibiting protein(PGIP)from wheat.Molecular plant-microbe interactions,2003,16(11):955-961.)。
将葡萄Vvpgip1转入烟草并高效表达,转基因烟草对灰霉菌和黑曲霉的抗性都有不同程度的增加(Joubert DA,Slaughter AR,Kemp G,et al.The grapevinepolygalacturonase-inhibiting protein(VvPGIP1)reduces Botrytis cinereasusceptibility in transgenic tobacco and differentially inhibits fungalpolygalacturonases.Transgenic Res,2006,15(6):687-702.)。苹果PGIPs能有效抑制羽扇豆炭疽菌和黑曲霉的PGs,将MdPGIP1导入烟草,转基因烟草可抑制羽扇豆炭疽菌和苹果真菌Botryosphaeria obtusa和Diaporthe ambigua,而对黑曲霉却无抑制作用。究其原因,PGIPs在原位或离体时有抗菌作用,一旦转入其他植物后,抗菌性可能降低甚至消失,也有可能是因为MdPGIP1仅仅具有苹果PGIPs的部分活性(Oelofse D,Dubery IA,Meyer R,et al.Apple polygalacturonase inhibiting protein1 expressed intransgenic tobacco inhibits polygalacturonases from fungal pathogens of apple andthe anthracnose pathogen of lupins.Phytochemi stry,2006,67(3):255-263.)。将树莓PGIP转入豌豆后,利用琼脂糖扩散实验检测转基因植株和野生型植株中PGIPs抑制羽扇豆炭疽菌和玉米穗腐病菌PGs活性的强度,结果表明23株转基因植株中有8株的抑制活性高达80%以上(Richter A,Jacobsen HJ,de Kathen A,et al.Transgenic peas(Pisum sativum)expressing polygalacturonase inhibiting protein from raspberry(Rubus idaeus)and stilbene synthase from grape(Vitis vinifera).Plant cellreports,2006,25(11):1166-1173.)。
本发明的PGIP基因来自云南火把梨。火把梨是云南本地的沙梨品种,具有稳定遗传的红色果皮表型。火把梨对土壤适应性强,抗黑星病、腐烂病,对梨木虱有较强的抗性,并且抗晚霜,耐低温能力也很强。
发明内容
本发明的目的是提供一种具有抗真菌活性的火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP及应用。
本发明分离克隆云南火把梨中携带的一个抗真菌相关基因的完整cDNA片段,利用农杆菌介导的遗传转化使目的基因在转基因受体植物中过量表达,通过进一步试验验证该基因是否具有抗真菌活性,为后期利用该基因改良烟草以及其他植物抵御真菌病害的能力奠定基础。本发明将这个基因命名为PpPGIP。
PGIPs能与PGs特异性结合形成一个双分子复合物,从而降低PGs的活性,有助于维护植物细胞壁的完整性,使其不易被病原真菌分泌的其他细胞壁降解酶所渗透,从而减慢了真菌对细胞壁的降解。OGs能激活植物的自身防御系统,其激活能力与其具有的半乳糖醛酸个数有关。真菌侵染植物时,PGs降解果胶能产生OGs。PGIPs能抑制PGs的部分酶活性,从而改变具有不同激活能力OGs间的平衡,使具有激活能力的OGs在植物体内的稳定期相对延长,更有效地激活植物体内的防御系统,调控防御基因的表达,以抵抗病原真菌的入侵。
本发明涉及分离包含PpPGIP的DNA片段并鉴定其功能,具有该基因片段的植物在一定程度上具有抵抗特定真菌浸染的表型。其中所述DNA片段如序列表SEQ ID所示,或者基本相当于SEQ ID所示的DNA序列,或者其功能相当于SEQ ID所示序列的部分片段。对该基因进行序列分析,通过同源法克隆获得的PpPGIPcDNA长1032bp,具有990bp的开放阅读框(ORF),编码含有330个氨基酸的蛋白质。PpPGIP编码的蛋白具有植物eLRR蛋白结构的典型特征,即右手超螺旋折叠结构,与来自西洋梨(Pyrus communis)、苹果(Malus×domestica)以及其他物种的PGIPs蛋白高度相似,表明其属于火把梨中的PGIP。超量表达序列表所示序列可以增强烟草对拟茎点霉属真菌、交链孢菌、派伦霉属真菌、黑曲霉、青霉等真菌的抗性。
上述PpPGIP基因可以应用于提高植物的真菌抗性,具体操作如下:
(1)将基因与适宜的植物超表达载体如pCAMBIA1301、pK2GW7、pCAMBIA2300s等连接,构建植物超表达载体。
(2)将构建的重组载体通过农杆菌介导转入目标植物中。
(3)以重组载体T-DNA上具有的抗性标记筛选转化子,并通过聚合酶链式反应(Polymerase Chain Reaction,PCR)获得真正的转基因植株,接种特定真菌或是分析转基因植物蛋白对真菌生长的抑制活性,最后筛选出对真菌抗性明显增强的转基因植株。
本发明为提高植物对真菌病害的抗性提供了一种新的方法,通过基因工程手段培育抗病植物能克服传统育种的不足,不仅育种周期短,而且操作简单,容易获得高抗材料。本发明来自火把梨的PpPGIP基因能增强植物对真菌的抗性,将该基因导入烟草、康乃馨、洋桔梗等植物中,可以产生具有真菌抗性的种质和品种。利用基因工程技术防治病害具有明显的优势和不可取代的重要性。它可以为作物、花卉等大规模生产提供方便,大量减少抗病剂的使用,为农业生产节约成本、减小环境污染且提高管理水平,因此本发明具有广阔的市场应用前景。
附图说明
图1是部分转基因烟草基因组DNA的PCR凝胶电泳图谱。
Marker:DL2000 DNA Marker(大连宝生物),由2,000bp、1,000bp、750bp、500bp、250bp以及100bp六条DNA片段组成。正对照:以质粒pMD-18T-PpPGIP为模板的PCR产物;WT:以非转基因烟草总DNA为模板的PCR产物;空白对照:PCR体系中不包含模板DNA的反应产物。
图2是部分阳性转基因烟草中PpPGIP转录水平表达分析的凝胶电泳图谱。
Marker:DL2000DNA Marker(大连宝生物);1-24分别为不同的阳性转基因烟草单株;WT:非转基因烟草总RNA逆转cDNA为模板的PCR产物;空白对照:PCR体系中不包含模板DNA的反应产物;正对照:质粒pMD-18T-PpPGIP为模板的PCR产物。
图3是PpPGIP转基因烟草体外抗真菌活性分析抑菌效果图。
A、B、C、D、E图示中的真菌分别是拟茎点霉属真菌、交链孢菌、派伦霉属真菌、黑曲霉和青霉菌;WT为野生型烟草的总蛋白;空白对照为无蛋白对照(用于提取蛋白的缓冲液)。
具体实施方式
实施例:
1、PpPGIP全长基因克隆以及序列分析
用液氮将火把梨的红色果皮研磨成粉末以后,转入离心管中,采用异硫氰酸胍法提取总RNA。采用逆转录酶M-MLV(天根)以总RNA为模板合成cDNA第一链,反应体系和操作过程为:取5μg Total RNA,依次加入50ng oligo(dT)15、2μL dNTP(2.5mM each)、ddH2O(RNase-free)至反应体积为13.5μL;混匀后,70℃加热变性5min后迅速在冰上冷却5min,然后依次加入4μL 5×First-stand buffer、0.5μL RNasin(200U)、1μLM-MLV(200U)、1μL DTT(0.1M),混匀并短时离心,42℃温浴1.5h,取出后95℃加热5min,终止反应。cDNA第一链合成后置于-20℃保存备用。
以合成的第一链cDNA为模板,扩增目的基因PpPGIP,所用引物序列分别为5’-ATTGTCGACCCAAAACAATGGAACTC-3’和5’-ATTGCGGCCGCAGTTGTGGCCTT-3’。采用大连宝生物的高保真DNA聚合酶ex taq扩增出目的基因。PCR反应条件:94℃ 3min;94℃ 30s,60℃ 30s,72℃ 1.5min,32个循环;72℃ 10min。反应体系(20μL)为1μL cDNA、2μL10×Long taq Buffer、0.4μL dNTP(10mM each)、0.1μL正向(20μM)、0.1μL反向引物(20μM)、0.2μL ex Taq DNA polymerase(5U/μL)、16.2μL ddH2O。PCR结束后,取5μL用于琼脂糖凝胶电泳,检测扩增产物的特异性以及大小。
由于PCR产物只有一条DNA带,故直接对PCR产物进行TA克隆,使用的试剂盒为pMD18-T vector kit(大连宝生物),反应体系和操作过程为:取1.5μL PCR产物,依次加入1μL pMD18-T vector(50ng/μL)和2.5μL 2×Ligation solution I,混匀后置于16℃过夜反应。采用热激转化法将连接产物转入大肠杆菌DH5α中。再涂于含有X-Gal、IPTG、氨苄青霉素(ampicillin,Amp)的LB固体培养基筛选阳性克隆。挑选若干个白色菌落,摇菌后用扩增PpPGIP的特异引物进行PCR从而鉴定出多克隆位点插入PpPGIP的克隆。将鉴定的克隆进行测序,最终获得的PpPGIPcDNA为1032bp,通过NCBI ORFfinder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个990bp的开放读码框(见序列表)。PpPGIP编码一个含330个氨基酸的蛋白质,PpPGIP的分子量为36.5KDa,等电点6.64。就整个蛋白质的氨基酸组成而言,疏水氨基酸亮氨酸的含量最高,约为15%。所有中性氨基酸含量为71%,酸性氨基酸含量为20%,碱性氨基酸含量为9%。还含有7个半胱氨酸(C),分别位于第27、57、58、62、65、298、320位。借助生物信息学软件SignalP 3.0分析PpPGIP编码的蛋白序列,检测它是否具有N端信号肽。结果表明该蛋白的第1-24位氨基酸残基是一段信号肽,说明PpPGIP是一种分泌蛋白。
2、植物表达载体构建
采用碱裂解法提取插入PpPGIP的大肠杆菌质粒pMD-18T-PpPGIP以及植物表达载体pCAMBIA2300S的质粒,取1μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性和浓度高低。用PstI(Fermentas)和BamHI(Fermentas)分别对质粒pMD-18T-PpPGIP和pCAMBIA2300S进行双酶切(100μL体系),反应体系和操作过程为:取10μLpMD-18T-PpPGIP或pCAMBIA2300S质粒、依次加入20μL 2×Tango buffer、2μL PstI、2μL BamHI、66μL ddH2O,混匀后短时离心,置于37℃过夜反应。将所有酶切产物点于琼脂糖凝胶孔中进行电泳,然后对PpPGIP片段和pCAMBIA2300S大片段分别进行胶回收,整个过程使用UNIQ-10柱式DNA胶回收试剂盒(上海生工)。具体操作流程为:切下含有目的DNA条带的琼脂糖凝胶块置于1.5mL离心管中,按400μL/100mg琼脂糖凝胶的比例加入Binding Buffer II,置于60℃水浴10min,使胶彻底融化;将融化的凝胶溶液转移至2mL收集管内的UNIQ-10柱中,室温放置2min后离心1min(6,000g/min);取下UNIQ-10柱,倒掉收集管中的废液,再将UNIQ-10柱放入收集管中,加入500μL Wash Solution,室温离心1min(8,000g/min),再重复此步骤一次;取下UNIQ-10柱,倒掉收集管中的废液,再将UNIQ-10柱放入收集管中,室温离心2min(12,000g/min);取下UNIQ-10柱放入新的1.5mL离心管中,在膜中央加入预热的20μL Elution Buffer,室温放置2min,室温离心1min(12,000g/min),离心管中收集液体即为回收的DNA片段。取1μL回收产物通过琼脂糖凝胶电泳检测回收片段的大小以及浓度,-20℃保存备用。
利用T4 DNA Ligase(大连宝生物)将回收的PpPGIP DNA片段和pCAMBIA2300S载体片段连接起来,反应体系(20μL)和操作过程为:取10μL PpPGIPDNA片段依次加入2μLpCAMBIA2300S载体DNA、2μL 10×T4DNA Ligase Buffer、1μL T4DNA Ligase、5μLddH2O,混匀后短时离心,置于16℃金属浴过夜反应。接着采用热激转化法将连接产物转入大肠杆菌DH5α中,在含有50mg/L卡那霉素(kanamycin,Km)的固体培养基上筛选阳性克隆。挑选单菌落摇菌,以菌液为模板,用扩增PpPGIP的特异引物进行PCR,挑选出PpPGIP与pCAMBIA2300S成功连接的克隆,阳性菌株中加入20%甘油混匀,置于-80℃保存备用。
用碱裂解法提取并纯化上述大肠杆菌中的pCAMBIA2300S-PpPGIP质粒。制备农杆菌LBA4404菌株的感受态细胞,操作过程为:将实验室保存的LBA4404菌株在LB固体培养基(含利福平20mg/L)上划线,28℃培养至长出单菌落后,挑取一个单克隆于含20mg/L利福平的2mL LB液体培养基,28℃振荡培养至混浊;取5mL菌液转接于含20mg/L利福平的100mL LB液体培养基中,28℃振荡培养至OD600为0.5;4℃离心5min(5,000g/min)收集菌体,弃上清,加入10mL预冷的0.1M CaCl2,轻轻充分悬浮菌体,冰浴20min;接着4℃离心5min(5,000g/min)收集菌体,弃上清,加入4mL预冷的含15%甘油的0.1M CaCl2溶液,轻轻悬浮后分装于1.5mL离心管中,每管200μL,液氮速冻后置于-80℃保存备用。
采用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PpPGIP转入所制备的农杆菌LBA4404感受态细胞中。操作步骤为:取2μg质粒加入含有200μL感受态细胞的离心管中,轻轻混匀后冰浴5min,接着转入液氮中冷冻1min,然后迅速置于37℃水浴5min,之后立即冰浴2min,加入800μL LB液体培养基,28℃振荡培养4h。将活化后的农杆菌涂于含有50mg/L Km的LB固体培养基上,28℃倒置培养。挑选单菌落摇菌,用扩增PpPGIP的特异引物进行PCR,检测pCAMBIA2300S-PpPGIP是否转入农杆菌中。对于阳性克隆,加入20%甘油混匀后置于-80℃保存备用。
3、农杆菌介导的植物遗传转化以及转基因植物筛选
本实验的转基因受体是烟草(Nicotiana tabacum L.)。将烟草的种子用75%的酒精浸泡30s,用无菌水洗涤后用0.1%的HgCl2浸泡8min,然后再用无菌水洗涤若干次,播种于1/2MS培养基上,28℃暗培养5-8d,发芽后转至光照培养箱(25℃,16h/d光照),以后每月用MS培养基继代一次。
从-80℃冰箱中取出保存的含有pCAMBIA2300S-PpPGIP质粒的农杆菌LBA4404菌种,接种于5mL含有50mg/L Km和20mg/L利福平的LB液体培养基中,28℃培养至浑浊。吸取1mL浑浊的菌液至含有50mg/L Km的LB固体培养基上,28℃培养48h。将LB固体培养基上的农杆菌刮下适量接种于MGL液体培养基中,附加一定量的乙酰丁香酮,28℃振荡培养2-3h以活化农杆菌。
取烟草的无菌苗叶子切成1cm2左右的叶盘,完全浸泡于上述含有活化农杆菌的MGL液体培养基中15min。用无菌滤纸吸干叶片表面的菌液,将叶盘置于共培养基上进行室温培养。烟草转化的共培养基为MS+0.02mg/L 6-BA+2mg/L NAA+30g/L sucrose,培养时间为2d。将共培养后的叶盘转到加有抗生素的筛选培养基中分化成苗,同时筛选转基因植株。烟草筛选培养基为MS+0.5mg/L 6-BA+0.1mg/L NAA+30g/L sucrose+50mg/L Km+200mg/L头孢霉素(cefotaxime sodium salt,Cef)。筛选培养时将培养瓶转移至光照培养箱培养(25℃,16h/d光照,8h/d黑暗)。烟草出芽后,用含有50mg/L Km和200mg/L Cef的MS培养基继代培养。因烟草愈伤分化率较高,故需要对再生植株进行进一步筛选。将烟草再生苗移至含有50mg/L Km和200mg/L Cef的MS培养基上使其生根,最后选用生根较好的再生苗做分子水平的检测。采用CTAB法提取转基因烟草植株叶片的基因组DNA,将提取的基因组DNA取1μL通过琼脂糖凝胶电泳检测其完整性和浓度。以转基因植株的基因组DNA为模板、扩增PpPGIP的特异引物进行PCR。PCR结束后,取8μL产物用于琼脂糖凝胶电泳以检测阳性转基因植株。部分烟草转基因植株的扩增结果如图1所示。PpPGIP转基因烟草共筛选到40株阳性植株,分别编号为1~40。
4、PpPGIP表达分析以及转基因植株抗真菌活性分析
取阳性转基因单株以及非转基因烟草(野生型)的嫩叶提取总RNA,逆转录生成cDNA第一链,并以此为模板、以扩增PpPGIP的特异引物进行PCR,根据PCR结果分析各转基因单株中PpPGIP转录水平的表达。总RNA提取以及RT-PCR的方法与步骤与1中相同。PCR结束后,取5μL用于琼脂糖凝胶电泳,部分单株的结果如图2所示。共检测到11株转基因烟草中PpPGIP在转录水平有表达,这些单株的编号为3、5、6、9、13、15、20、21、22、23、24。
将实验室保存的几种真菌接种于PDA固体培养基(200g/L马铃薯,15g/L琼脂,20g/L葡萄糖)上,28℃倒置培养,待菌落生长至直径约为3cm添加蛋白,分析转基因植株体外抗真菌活性。供试真菌共有5种:青霉菌(Penicillium sp.)、黑曲霉(Aspergillus niger)、拟茎点霉属(Phomopsis sp.)真菌、派伦霉属(Peyroneuaea)真菌和交链孢菌(Alternariasp.)。
为了防止其它杂菌污染所提取的蛋白,整个植物蛋白提取过程均是无菌操作。首先取1g左右转基因烟草单株(编号分别为3、5、6、9、13)或野生型叶片放入研钵中,加入1mL蛋白提取液(1M NaCl,0.1M乙酸钠,1%PVP,pH6),充分研磨。转入2mL离心管中,混匀后4℃静置过夜。4℃离心30min(12,000g/min),取上清液于新的1.5mL离心管中,并取适量用紫外分光光度仪测定总蛋白浓度。将转基因和野生型植株的总蛋白浓度调整至4mg/mL,然后分别取20μL滴于各真菌培养基的滤纸上。在每个真菌的平板上除了添加不同转基因烟草植株的总蛋白,同时平行添加野生型烟草的总蛋白和空白对照(提取蛋白所用的溶液)。28℃培养几天后观察各处理抑菌真菌生长的情况,并据此评价PpPGIP转基因烟草的体外抗真菌活性。结果如图3所示,PpPGIP转基因烟草蛋白对拟茎点霉属真菌、派伦霉属真菌、交链孢菌、黑曲霉和青霉菌具有明显的抑制作用。
序列表(SEQ ID)
<110>昆明理工大学
<120>火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP及应用
<130>
<160>2
<170>PatentIn version 3.5
<210>1
<211>1032
<212>DNA
<213>Pyrus pyrifolia Nakai
<220>
<221>mRNA
<222>(1)..(1032)
<220>
<221>5’UTR
<222>(1)..(17)
<220>
<221>CDS
<222>(18)..(1007)
<220>
<221>3’UTR
<222>(1008)..(1032)
<400>1
attgtcgacc caaaaca atg gaa ctc aag ttc tca acc ttc ctc tcc cta 50
Met Glu Leu Lys Phe Ser Thr Phe Leu Ser Leu
1 5 10
acc cta ctc ttc tcc tcc gtc cta aac ccc gct ctc tcc gat ctc tgc 98
Thr Leu Leu Phe Ser Ser Val Leu Asn Pro Ala Leu Ser Asp Leu Cys
15 20 25
aac ccc gac gac aaa aaa gtc ctc cta caa atc aag aaa gcc ttc ggc 146
Asn Pro Asp Asp Lys Lys Val Leu Leu Gln Ile Lys Lys Ala Phe Gly
30 35 40
gac ccc tac gtc ttg gcc tca tgg aaa tca gac act gac tgc tgc gat 194
Asp Pro Tyr Val Leu Ala Ser Trp Lys Ser Asp Thr Asp Cys Cys Asp
45 50 55
tgg tac tgc gtc acc tgt gac tcc acc aca aac cgc atc aac tcc ctc 242
Trp Tyr Cys Val Thr Cys Asp Ser Thr Thr Asn Arg Ile Asn Ser Leu
60 65 70 75
acc atc ttc gcc ggc cag gta tca ggc caa atc ccc gcc cta gta gga 290
Thr Ile Phe Ala Gly Gln Val Ser Gly Gln Ile Pro Ala Leu Val Gly
80 85 90
gac ttg cca tac ctt gaa acc ctt gaa ttc cac aag caa ccc aat ctc 338
Asp Leu Pro Tyr Leu Glu Thr Leu Glu Phe His Lys Gln Pro Asn Leu
95 100 105
act ggc cca atc caa ccc gcc att gcc aag ctc aaa gga ctc aag tct 386
Thr Gly Pro Ile Gln Pro Ala Ile Ala Lys Leu Lys Gly Leu Lys Ser
110 115 120
ctc agg ctc agc tgg acc aac ctc tca ggc tct gtc cct gac ttc ctc 434
Leu Arg Leu Ser Trp Thr Asn Leu Ser Gly Ser Val Pro Asp Phe Leu
125 130 135
agc caa ctc aag aac ctc aca ttc ctc gac ctc tcc ttc aac aac ctc 482
Ser Gln Leu Lys Asn Leu Thr Phe Leu Asp Leu Ser Phe Asn Asn Leu
140 145 150 155
acc ggt gcc atc ccc agc tcg ctt tct gag ctc cca aac ctc ggc gct 530
Thr Gly Ala Ile Pro Ser Ser Leu Ser Glu Leu Pro Asn Leu Gly Ala
160 165 170
ctt cgt cta gac cgc aat aag ctc aca ggt cat att ccg ata tcg ttt 578
Leu Arg Leu Asp Arg Asn Lys Leu Thr Gly His Ile Pro Ile Ser Phe
175 180 185
ggg cag ttc att ggc aac gtt cca gac ctg tat ctc tcc cac aac cag 626
Gly Gln Phe Ile Gly Asn Val Pro Asp Leu Tyr Leu Ser His Asn Gln
190 195 200
ctt tct ggt aac att cca acc tca ttc gct cag atg gac ttc acc agc 674
Leu Ser Gly Asn Ile Pro Thr Ser Phe Ala Gln Met Asp Phe Thr Ser
205 210 215
ata gac tta tca cgg aac aag ctc gaa ggt gac gca tcc gtg ata ttt 722
Ile Asp Leu Ser Arg Asn Lys Leu Glu Gly Asp Ala Ser Val Ile Phe
220 225 230 235
ggg ctg aac aag aca acc cag att gtg gac ctg tcc agg aac ttg ctg 770
Gly Leu Asn Lys Thr Thr Gln Ile Val Asp Leu Ser Arg Asn Leu Leu
240 245 250
gaa ttt aat ctg tca aag gtg gag ttt ccg aca agc tcg acc tcg ctg 818
Glu Phe Asn Leu Ser Lys Val Glu Phe Pro Thr Ser Ser Thr Ser Leu
255 260 265
gat atc aac cac aat aag atc tac ggg agt atc cca gtg gag ttt acg 866
Asp Ile Asn His Asn Lys Ile Tyr Gly Ser Ile Pro Val Glu Phe Thr
270 275 280
caa ctg aat ttc cag ttc ctg aac gtg agc tac aac agg ctg tgt ggt 914
Gln Leu Asn Phe Gln Phe Leu Asn Val Ser Tyr Asn Arg Leu Cys Gly
285 290 295
cag att cct gtg ggt gga aag ttg cag agc ttc gac gag tat tct tat 962
Gln Ile Pro Val Gly Gly Lys Leu Gln Ser Phe Asp Glu Tyr Ser Tyr
300 305 310 315
ttc cat aac cga tgc ttg tgc ggt gct cca ctc cca agc tgc aag 1007
Phe His Asn Arg Cys Leu Cys Gly Ala Pro Leu Pro Ser Cys Lys
320 325 330
taaaggccac aactgcggcc gcaat 1032
<210>2
<211>330
<212>PRT
<213>Pyrus pyrifolia Nakai
<400>2
Met Glu Leu Lys Phe Ser Thr Phe Leu Ser Leu Thr Leu Leu Phe Ser
1 5 10 15
Ser Val Leu Asn Pro Ala Leu Ser Asp Leu Cys Asn Pro Asp Asp Lys
20 25 30
Lys Val Leu Leu Gln Ile Lys Lys Ala Phe Gly Asp Pro Tyr Val Leu
35 40 45
Ala Ser Trp Lys Ser Asp Thr Asp Cys Cys Asp Trp Tyr Cys Val Thr
50 55 60
Cys Asp Ser Thr Thr Asn Arg Ile Asn Ser Leu Thr Ile Phe Ala Gly
65 70 75 80
Gln Val Ser Gly Gln Ile Pro Ala Leu Val Gly Asp Leu Pro Tyr Leu
85 90 95
Glu Thr Leu Glu Phe His Lys Gln Pro Asn Leu Thr Gly Pro Ile Gln
100 105 110
Pro Ala Ile Ala Lys Leu Lys Gly Leu Lys Ser Leu Arg Leu Ser Trp
115 120 125
Thr Asn Leu Ser Gly Ser Val Pro Asp Phe Leu Ser Gln Leu Lys Asn
130 135 140
Leu Thr Phe Leu Asp Leu Ser Phe Asn Asn Leu Thr Gly Ala Ile Pro
145 150 155 160
Ser Ser Leu Ser Glu Leu Pro Asn Leu Gly Ala Leu Arg Leu Asp Arg
165 170 175
Asn Lys Leu Thr Gly His Ile Pro Ile Ser Phe Gly Gln Phe Ile Gly
180 185 190
Asn Val Pro Asp Leu Tyr Leu Ser His Asn Gln Leu Ser Gly Asn Ile
195 200 205
Pro Thr Ser Phe Ala Gln Met Asp Phe Thr Ser Ile Asp Leu Ser Arg
210 215 220
Asn Lys Leu Glu Gly Asp Ala Ser Val Ile Phe Gly Leu Asn Lys Thr
225 230 235 240
Thr Gln Ile Val Asp Leu Ser Arg Asn Leu Leu Glu Phe Asn Leu Ser
245 250 255
Lys Val Glu Phe Pro Thr Ser Ser Thr Ser Leu Asp Ile Asn His Asn
260 265 270
Lys Ile Tyr Gly Ser Ile Pro Val Glu Phe Thr Gln Leu Asn Phe Gln
275 280 285
Phe Leu Asn Val Ser Tyr Asn Arg Leu Cys Gly Gln Ile Pro Val Gly
290 295 300
Gly Lys Leu Gln Ser Phe Asp Glu Tyr Ser Tyr Phe His Asn Arg Cys
305 310 315 320
Leu Cys Gly Ala Pro Leu Pro Ser Cys Lys
325 330
Claims (6)
1.一种火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP,其特征在于它具有序列表SEQID所述的碱基序列。
2.根据权利要求1所述的火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP,其特征在于PpPGIP基因长1032bp,其中990bp为开放阅读框,编码含330个氨基酸的多聚半乳糖醛酸酶抑制蛋白质。
3.根据权利要求1或2所述的火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP,其特征在于编码区是序列表第18-1007位所示的核苷酸序列,或是编码蛋白质与SEQID编码蛋白质相同的其它DNA序列。
4.根据权利要求1所述的火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP,其特征在于编码蛋白PpPGIP分子量为36.5KDa,等电点6.64。
5.权利要求1所述的火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP在植物抗真菌中的应用,对黑曲霉、拟茎点霉属真菌、交链孢菌、青霉菌多种真菌具有明显的抑制作用,以提高植物的抗真菌活性。
6.权利要求5所述的火把梨多聚半乳糖醛酸酶抑制蛋白基因PpPGIP在植物抗真菌中的应用方法,其特征在于提高植物的真菌抗性具体操作如下:
(1)将基因PpPGIP与适宜的植物超表达载体pCAMBIA1301或pK2GW7或pCAMBIA2300s连接,构建植物超表达载体;
(2)将上述构建的重组载体通过农杆菌介导转入目标植物中;
(3)以重组载体T-DNA上具有的抗性标记筛选转化子,并通过聚合酶链式反应获得真正的转基因植株,接种特定真菌或是分析转基因植物蛋白对真菌生长的抑制活性,最后筛选出对真菌抗性明显增强的转基因植株。
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