CN103194456B - 岷江百合抗真菌基因Lr14-3-3及其应用 - Google Patents
岷江百合抗真菌基因Lr14-3-3及其应用 Download PDFInfo
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Images
Abstract
本发明公开了一种具有抗真菌活性的岷江百合基因Lr14-3-3，该Lr14-3-3基因核苷酸序列如SEQIDNO：1所示，编码14-3-3蛋白，本发明通过功能基因组学相关技术证实Lr14-3-3基因具有提高植物抗真菌的功能，将本发明抗真菌Lr14-3-3基因构建到植物表达载体上并转入烟草中过量表达，转基因烟草植株具有很强的体外抗真菌活性，表达Lr14-3-3的转基因烟草对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌的生长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相关技术研究领域，特别是一种岷江百合的具有抗真菌活性的14-3-3蛋白基因Lr14-3-3及应用。
背景技术
植物病害是农业生产中一个非常棘手的问题，特别是真菌病害，由其引起的病害约占植物总病害的80％，不仅造成作物产量的巨大损失，还严重影响粮食及其他食品的品质。控制植物真菌病害传播的方法主要是依靠培育抗性品种和化学农药，或者是采取轮作等耕作制度。虽然也取得了一定成效，但是也存在严重的弊端，如使用起来费时费力、周期长、化学农药残留高、危害人畜健康且易对环境造成污染等，所以传统病害防治方法不能从根本上解决病害问题。随着重组DNA技术的创立和发展，利用转基因技术培育植物新品种来应对真菌病害已取得初步成效，是一种可以彻底解决真菌病害的育种新方法。
当植物受到病原菌攻击或处于其他逆境胁迫时，植物会产生复杂的生物化学和生理学上的响应，如植物感受逆境信号后通过信号转导过程随后调节细胞内抗逆相关蛋白基因的表达。作为真核生物中普遍存在的一类信号调控因子，14-3-3与植物的防卫反应密切相关。14-3-3是一类序列高度保守的蛋白，在大多数物种中由一个基因家族编码。现已确定植物中14-3-3的靶蛋白超过300种，暗示其参与多种信号传导途径和植物的生理过程。14-3-3广泛参与细胞生长分化、细胞周期和凋亡的调控、信号转导和迁移等生理生化过程。此外，14-3-3与能量代谢的关键酶结合，从而调控其代谢过程；14-3-3还通过结合质膜H+-ATPase来控制物质的跨膜运输过程。
14-3-3蛋白的氨基酸序列在物种种间和种内都高度保守，每个14-3-3蛋白均由3部分组成：特定的氨基末端，高度保守的核心区域和特定的羧基末端。根据同工型核心区域高度保守的氨基酸序列将植物14-3-3s分为ε和非ε两大类。14-3-3蛋白结合靶蛋白的保守结构域有2种：RSXpSXP（mode I）和RXY/FXpSXP（mode II），pS为磷酸丝氨酸（Ottmann C，Marco S，Jaspert N，Marcon C，Schauer N，Weyand M，Vandermeeren C，Duby G，Boutry M，Wittinghofer A，et al. Structure of a
14-3-3 Coordinated Hexamer of the Plant Plasma Membrane H+-ATPase by
Combining X-Ray Crystallography and Electron Cryomicroscopy. Molecular Cell，2007，25: 427–440）。14-3-3蛋白可以通过与蛋白相互作用，或者结合靶蛋白的磷酸丝氨酸/磷酸苏氨酸来实现其调控功能（Li XY，Dhaubhadel S. Soybean
14-3-3 gene family: identification and molecular Characterization. Planta，2011，233: 569–582）。
近年来的研究显示14-3-3能参与植物对病害的防御。如14-3-3 λ表达下调的拟南芥植株对白粉病菌感染的抗性减弱，而14-3-3 λ的超表达能够增加其抗性，并引起植物超敏反应，14-3-3通过与水杨酸信号通路中的RPW8.2蛋白（一种R基因产物）相互作用增强对白粉病真菌的抗性。（Yang X，Wang W，Coleman M，Orgil U，Feng J，Ma X，Ferl R，Turner JG，Xiao S. Arabidopsis 14-3-3
lambda is a positive regulator of RPW8-mediated disease resistance. Plant J，2009，60: 539–550）。分布于胞外空间的14-3-3也参与对病害的防御，如在雨生红球藻（Haematococcus pluvialis）的细胞壁和小麦（Triticum aestivum）幼苗根的细胞壁中都能检测到14-3-3（Wang SB，Hu Q，Sommerfeld M，Chen F. Cell wall
proteomics of the green alga Haematococcus pluvialis （Chlorophyceae）. Proteomics，2004，4: 692–708；Kong FJ，Oyanagi A，Komatsu S. Cell wall
proteome of wheat roots under flooding stress using gel-based and LC
MS/MS-based proteomics approaches. Biochim Biophys Acta，2010，1804: 124–136）。将豌豆（Pisum sativum Linn）根尖与R18肽（能与14-3-3受体特异结合，从而去除14-3-3受体）同时处理，根腐病的发生增强，降低了根对病原真菌血红丛赤壳（Nectra haematococca）的抗性（Wen F，VanEtten HD，Tsaprailis G，Hawes MC. Extracellular
proteins in pea root tip and border cell exudates. Plant Physiol，2007，143:773–783）。在豌豆和玉米（Zea mays）中利用免疫荧光方法（Immunofluorescence，IF）将制备的抗体与拟南芥14-3-3相互作用，在根边缘细胞检测到胞外荧光，暗示14-3-3可协助细胞外的蛋白质参与植物应对病原菌的入侵。
14-3-3还能响应番茄溃疡性病菌（Pseudomonas syringae pv tomato）的效应毒性蛋白，从而参与番茄中PTO（一种R基因的产物）介导的程序化细胞凋亡（programmed cell death，PCD）（Konagaya K，Kasahara YMM，Nyunoya H. Members of
14-3-3 protein isoforms interacting with the resistance gene product N and the
elicitor of Tobacco mosaic virus. J Gen Plant Pathol，2004，70: 221–231）。促分裂素原活化蛋白激酶（mitogen-activated protein
kinases，MAPK）途径参与植物对生物胁迫的防御反应，且能正调节程序性细胞凋亡以应对P. syringae。感染P. syringae的番茄细胞中14-3-3（TFT7）与番茄MAPKKK蛋白激酶和其下游激酶MAPKK存在相互作用（Oh CS，Pedley KF，Martin GB. Tomato 14-3-3
protein 7 positively regulates immunity-associated programmed cell death by
enhancing protein abundance and signaling ability of MAPKKK {alpha}. Plant Cell，2010，22: 260–272）。14-3-3蛋白在稳定MAPKKK蛋白的过程中起作用，因而它可以激活下游MAPK级联导致PCD。下游激酶MAPKK的突变体与14-3-3结合能力降低却仍然能够有效诱导PCD，可见PCD的诱导不依赖于14-3-3与MAPKK的结合，而是14-3-3在胞内可以把MAPKKKα和MAPKK联系在一起。
本发明的14-3-3蛋白基因Lr14-3-3来自岷江百合（Lilium regale Wilson）。岷江百合又名王百合，多年生草本植物，是我国的百合特有种。仅分布于四川西岷江流域海拔800～2700m的河谷到山腰的岩石缝中，具有极强的抗真菌、抗病毒等特性，是百合抗病育种的优良种质资源。
发明内容
本发明的目的是提供一种从岷江百合中克隆获得具有抗真菌活性的14-3-3蛋白的全长基因Lr14-3-3，抗真菌基因Lr14-3-3的核苷酸序列如SEQ ID NO：1所示，该基因全长1067bp，包含一个780bp的开放读码框，54bp的5’非翻译区及233bp的3’非翻译区，编码如SEQ ID NO：2所示氨基酸序列的蛋白质。
本发明中抗真菌基因Lr14-3-3的编码区是序列表SEQ ID NO：1中第55-834位所示的核苷酸序列。
本发明分离克隆岷江百合的一个抗真菌相关基因的完整cDNA片段，利用根癌农杆菌介导法将目的基因转入受体植物中并过量表达，通过进一步实验验证该基因是否具有抗真菌的活性，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，发明人将这个基因命名为Lr14-3-3。
14-3-3是真核生物中的一类信号调控因子，在植物抗病防卫反应中起重要作用，植物14-3-3通过调节关键的生理过程以应对不断变化的外部环境，14-3-3广泛参与细胞生长分化、细胞周期和凋亡的调控、信号转导和迁移等生理生化过程；此外，14-3-3与参与能量代谢的关键酶结合，从而调控其代谢过程，如14-3-3通过调节硝酸还原酶、蔗糖磷酸合酶、海藻糖-6磷酸合酶、甘油三磷酸脱氢酶、谷氨酰胺合成酶等碳、氮代谢关键酶的活性参与植物碳、氮代谢途径等，采取蛋白与蛋白的直接作用方式调节靶蛋白参与的一系列生理活动，在植物抵抗病原真菌入侵及其它氧化胁迫过程中起重要作用。
本发明涉及分离包含Lr14-3-3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侵染的表型，其中所述DNA片段如序列表SEQ ID：1所示。对该基因进行序列分析，表明Lr14-3-3全长序列为1067bp，包含一个780bp的开放阅读框（Open reading frame，ORF），54bp的5’非翻译区（untranslated region，UTR）以及233bp的3’UTR，编码259个氨基酸。Lr14-3-3编码蛋白具有14-3-3蛋白的保守结构域，Lr14-3-3与麝香百合（Lilium longiflorum）14-3-3（AAF05737）的同源性为99%，与来自拟南芥（Arabidopsis thaliana）、棉花（Gossypium hirsutum）、水稻（Oryza sativa）、烟草（Nicotiana attenuate）、油菜（Brassica campestris）、番茄（Lycopersicon esculentum）以及其它物种的14-3-3蛋白高度相似，表明其属于岷江百合中的14-3-3，超量表达序列表SEQ ID：2所示序列可以增强烟草对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌的抗性。
本发明另一目的是将岷江百合抗真菌基因Lr14-3-3应用在提高烟草对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌抗性中，具体操作如下：
（1）采用扩增Lr14-3-3的特异引物，从接种尖孢镰刀菌后的岷江百合根中提取总RNA，通过RT-PCR扩增出Lr14-3-3的全长编码区，然后将其连接到pMD-18T载体上，经测序获得具有目的基因的克隆；
（2） 用限制性内切酶PstⅠ和EcoRI酶切pMD-18T-Lr14-3-3载体，通过胶回收得到目的基因片段，用同样的内切酶对植物表达载体pCAMBIA2300s进行酶切，胶回收获得所需载体大片段；将所获得基因片段与pCAMBIA2300s载体大片段连接，构建植物超表达载体，之后将所构建的重组载体通过根癌农杆菌介导转入烟草中表达；
（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）以及RT-PCR（Reverse Transcription-PCR）方法得到真正的转基因植株，分析转基因植物蛋白对真菌生长的抑制作用，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，不仅育种周期缩短，而且操作简单，容易获得高抗材料。本发明来自岷江百合的Lr14-3-3基因能增强植物对真菌的抗性，将该基因导入烟草中，可以产生具有真菌抗性的新品种和新材料；利用基因工程技术减少真菌带来的病害具有明显的优势和不可取代的重要性；它可以为大规模生产作物、花卉等提供方便，大量减少化学农药的使用，还可以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是本发明中部分转基因烟草基因组DNA的PCR检测结果示意图，其中：Marker是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；正对照是质粒pMD-18T-Lr14-3-3为模板的PCR反应；WT是非转基因烟草（野生型）总DNA为模板进行的PCR；
图2是本发明中部分阳性转基因烟草中Lr14-3-3转录水平的表达分析结果示意图，其中：Marker是DL2000 DNA Marker（大连宝生物）；WT是非转基因烟草总RNA逆转录cDNA为模板的PCR产物；正对照是质粒pMD-18T-Lr14-3-3为模板的PCR产物；
图3是本发明中Lr14-3-3转基因烟草体外抗真菌活性的抑菌效果示意图，其中：a、b、c、d图示中的真菌分别是葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌；WT为野生型烟草的总蛋白；CK为空白对照，即用于提取蛋白的缓冲液。
具体实施方式
下面通过实施例对本发明作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明的均按常规方法操作，所用试剂如无特殊说明的采用常规试剂或按常规方法配置的试剂。
实施例1：Lr14-3-3全长基因克隆以及序列分析
用尖孢镰刀菌接种岷江百合，取接种24 h后的根提取总RNA，用液氮将处理过的岷江百合的根研磨成粉末，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（promega）以总RNA为模板合成cDNA 第一链，反应体系和操作过程为：取5 μg Total RNA，依次加入50 ng oligo（dT）、DEPC水至反应体积为12.5 μL；混匀后，70℃加热变性5min后迅速在冰上冷却5min，然后依次加入4 μL 5×First-stand buffer、2 μL dNTP（2.5mM each）、0.5 μL RNasin（200 U）、1 μL M-MLV（200 U），混匀并短时离心，42 ℃温浴1.5 h，取出后70 ℃加热10 min，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Lr14-3-3，所用上下游引物序列分别为5’CTGCAGTCCTCGCTCCTATCTAGGTTTCACC3’、5’GAATTCAGCCACACAATAGGTTT GCTGAGC3’；采用AdvantageTM 2
PCR Enzyme（Clontech）扩增出目的基因，PCR反应条件：95℃ 1min；95℃ 30s，64 ℃ 30s，72℃ 70s，30个循环；72℃ 2min。反应体系（20μL）为2 μL cDNA、2 μL 10×Advantage 2 PCR Buffer、0.5 μL 50×dNTP Mix（10mM each）、0.2 μL 正向引物（10μM）、0.2μL 反向引物（10μM）、0.2 μL Advantage 2 PCR
Polymerase Mix、14.9 μL PCR-Grade water。PCR结束之后取5 μL进行琼脂糖凝胶电泳，用于检测扩增产物的特异性以及大小。
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1.5 μL PCR产物，依次加入1 μL pMD18-T vector （50 ng/μL） 和2.5 μL 2×Ligation solution I，混匀置于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。使用含有氨苄青霉素（ampicillin，Amp）的LB固体培养基筛选阳性克隆。挑选若干个单菌落，摇菌后用扩增Lr14-3-3的特异引物鉴定出多克隆位点插入Lr14-3-3的克隆。将所鉴定的克隆进行测序，最终获得的Lr14-3-3全长cDNA为1067bp，通过NCBI ORF finder （http://www.ncbi.nlm.nih.gov/gorf/gorf.html）分析发现其包含一个780bp的开放读码框（见序列表），Lr14-3-3编码一个含259个氨基酸的蛋白质Lr14-3-3，其分子量约为29.03KDa，等电点为4.79，含有2个半胱氨酸残基（C），位于第103和198位，因此单体蛋白可能形成一个Cys103-Cys198二硫键，借助生物信息学软件SignalP 4.1分析Lr14-3-3编码的蛋白序列，检测其是否具有N端信号肽。结果表明在该蛋白中没有检测到信号肽的存在。
实施例2：植物表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒（上海生工）从大肠杆菌中提取插入Lr14-3-3的质粒pMD-18T-Lr14-3-3以及植物表达载体pCAMBIA2300s的质粒，取1 μL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。用限制性内切酶EcoRI（TaKaRa）和PstⅠ（TaKaRa）分别对质粒pMD-18T-Lr14-3-3和pCAMBIA2300s进行双酶切（100 μL体系），反应体系和操作过程为：取20 μL pMD-18T-Lr14-3-3和pCAMBIA2300s质粒、依次加入10 μL 10×H buffer、5 μL EcoRI、5 μL PstⅠ、60 μL 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中进行电泳，然后对Lr14-3-3片段和pCAMBIA2300s大片段分别进行胶回收，整个过程使用SanPrep柱式DNA胶回收试剂盒（上海生工）。取1 μL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，置于-20℃保存备用。
利用T4 DNA Ligase（TaKaRa），将回收的Lr14-3-3DNA片段和pCAMBIA2300s载体片段连接起来，反应体系（20 μL）和操作过程为：取10 μL Lr14-3-3DNA片段依次加入2 μL pCAMBIA2300s载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μL ddH2O，混匀后短时离心，然后置于16℃水浴过夜反应。接着采用热激转化法将连接产物转入大肠杆菌DH5α中，用含有50 mg/L卡那霉素（kanamycin，Km）的固体培养基筛选阳性克隆。挑选单菌落摇菌，以菌液为模板用扩增Lr14-3-3的特异引物进行PCR，挑选出Lr14-3-3与pCAMBIA2300s成功连接的克隆，所检测的菌株若为阳性，加入甘油并置于-80℃保存备用。
采用SanPrep柱式质粒抽提试剂盒（上海生工）提取并纯化上述大肠杆菌中的pCAMBIA2300s-Lr14-3-3质粒，随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300s-Lr14-3-3转入所制备的根癌农杆菌LBA4404感受态细胞中，操作步骤为：取0.2 μg pCAMBIA2300s-Lr14-3-3质粒加入含有200 μL感受态细胞的离心管中，轻轻混匀后冰浴5 min，接着转入液氮中冷冻1 min，然后迅速置于37℃水浴5 min，之后立即冰浴2 min，加入800 μL LB液体培养基，28℃振荡培养4 h。将活化后的农杆菌涂于含有50 mg/L Km的LB固体培养基上，28℃静止培养。挑选单菌落摇菌，用扩增Lr14-3-3的特异性引物进行PCR，检测pCAMBIA2300s-Lr14-3-3是否转入农杆菌中。对于阳性克隆，加入甘油后置于-80℃保存备用。
实施例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.），将烟草种子用75%的酒精浸泡30s，用无菌水洗涤后用0.1%的HgCl2浸泡8 min，然后再用无菌水洗涤若干次，播种于1/2 MS培养基上，28℃暗培养5-8 d，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-Lr14-3-3质粒的农杆菌LBA4404菌种，接种于5 mL含有50 mg/L Km和20 mg/L利福平的LB液体培养基中，28℃培养至培养基浑浊。吸取1 mL浑浊的菌液涂布于含有50 mg/L Km的LB固体培养基上，28℃培养48 h。将LB固体培养基上的农杆菌刮下适量接种于附加有20 mg/L的乙酰丁香酮的MGL液体培养基中，28℃振荡培养2-3 h以活化农杆菌。
取烟草无菌苗叶子切成1 cm2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，浸染时间为15 min，用无菌滤纸吸干叶盘表面的菌液，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+0.02 mg/L 6-BA+2.1 mg/L
NAA+30 g/L sucrose+6 g/L琼脂，22℃无光条件下共培养2 d。
将共培养后的叶盘转到加有抗生素的MS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+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L
sucrose+6 g/L琼脂+50 mg/L Km+200 mg/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），出芽后用含有50 mg/L Km和200 mg/L Cef的MS培养基继代培养，将烟草再生苗移至含有50 mg/L Km的MS培养基上使其生根，最后选用生根较好的再生苗做进一步的检测。
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1 μL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为模板用扩增Lr14-3-3的特异引物进行PCR，PCR结束后，取8 μL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，Lr14-3-3转基因烟草共筛选到25株阳性转基因植株。
实施例4：Lr14-3-3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，并以此为模板用扩增Lr14-3-3的特异引物进行PCR，根据PCR结果分析各转基因单株中Lr14-3-3转录水平的表达。总RNA提取以及RT-PCR的方法与步骤与实施例1中相同，PCR结束后，取5 μL用于琼脂糖凝胶电泳，部分单株的检测结果如图2所示，共检测到17株转基因单株中Lr14-3-3在转录水平有表达，这些单株的编号为1～17。
将实验室保存的几种病原真菌接种于PDA固体培养基（200 g/L 马铃薯，15 g/L 琼脂，20 g/L葡萄糖）上，28℃暗培养，待菌落生长至直径为2～3cm时添加蛋白，分析转基因植株体外抗真菌活性，供试真菌共有5种：葡萄座腔菌（Botrosphaeria dothidea），尖孢镰刀菌（Fusarium oxysporum），拟茎点霉属真菌（Phomopsis sp.），交链孢菌（Alternaria sp.），灰葡萄孢菌（Botrytis cinerea）。
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1 g转基因烟草单株（编号分别为1、2、5、7、10）或野生型叶片放入研钵中，加入1 mL蛋白提取液（1 M NaCl，0.1 M 乙酸钠，1% PVP，pH6），充分研磨。转入1.5 mL离心管中，混匀后4℃静置过夜。4℃离心30 min （12,000 g/min），取上清于新的1.5 mL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0.2 μg/μL，然后分别取20 μL滴于各真菌培养基的无菌滤纸上。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（CK，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，并据此来评价Lr14-3-3转基因烟草的体外抗真菌活性，结果如图3所示，Lr14-3-3转基因烟草蛋白对葡萄座腔菌、拟茎点霉属真菌的生长具有很强的抑制作用，对交链孢菌、尖孢镰刀菌也有明显的抑制活性。
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Claims (2)
1.一种岷江百合抗真菌基因Lr14-3-3在提高烟草对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌抗性中的应用，其中所述岷江百合抗真菌基因Lr14-3-3的核苷酸序列如SEQ ID NO：1所示。
2.根据权利要求1所述的岷江百合抗真菌基因Lr14-3-3的应用，其特征在于提高烟草的真菌抗性的具体操作如下：
（1） 将上述抗真菌基因与植物表达载体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根癌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，分析转基因植物蛋白对真菌生长的抑制作用，最后筛选出对真菌抗性明显增强的转基因植株。
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