CN105755020A - 三七丝裂原活化蛋白激酶激酶基因PnMAPKK1及其应用 - Google Patents
三七丝裂原活化蛋白激酶激酶基因PnMAPKK1及其应用 Download PDFInfo
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- CN105755020A CN105755020A CN201610246112.4A CN201610246112A CN105755020A CN 105755020 A CN105755020 A CN 105755020A CN 201610246112 A CN201610246112 A CN 201610246112A CN 105755020 A CN105755020 A CN 105755020A
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Abstract
本发明公开了一种三七丝裂原活化蛋白激酶激酶基因<i>PnMAPKK1</i>，<i>PnMAPKK1</i>基因的核苷酸序列如SEQ ID NO：1所示，编码丝裂原活化蛋白激酶激酶，本发明通过功能基因组学相关技术研究证实<i>PnMAPKK1</i>基因具有提高植物对病原真菌抗性的功能，将本发明抗真菌基因<i>PnMAPKK1</i>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；<i>PnMAPKK1</i>超表达的转基因烟草对茄腐镰刀菌、胶孢炭疽菌、轮枝镰刀菌和葡萄座腔菌的生长均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领域相关技术，特别是具有抗真菌活性的三七丝裂原活化蛋白激酶激酶基因PnMAPKK1及应用。
背景技术
在全球范围内，由细菌、病毒和真菌等病原菌引起的粮食减产现象一直是一个重要的问题。由于全球人口的不断增加，在未来的40年里，对粮食的需求还会继续增加，到2050年，粮食产量需要再增加70%才能满足人们的需求。由于病原菌的感染，玉米、大麦、大米和大豆平均减产约为12%，花生和马铃薯平均减产约为24%，小麦和棉花的减产量分别约为50%和80%(OerkeEC.Croplossestopests.JAgricSci,2006,144(1):31–43)。仅仅在美国，每年由于病原菌感染造成的经济损失就高达400亿美元。尽管传统育种在提高作物抗真菌病害能力方面做出了显著贡献，但其固有的缺点如育种周期长、抗性品种不足以对抗新的毒性生理小种的不断进化，使其不能彻底解决作物生产过程中的真菌病害问题。此外，针对许多重要的植物病原真菌，育种家无法获得自然的抗性植物资源。使用化学农药对病原菌的防治有一定效果，但是病原菌对农药的快速适应迫使农药投入逐年增长。另一方面，化学农药对环境和人类健康产生的负面影响受到日益增强的关注，迫切需要采用有效、环保、可持续发展的方法用于植物保护。随着分子生物学和基因工程技术的迅速发展，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，从根本上解决真菌病害问题开辟了新途径。在生物技术快速发展的推动下，利用基因工程技术来提高植物的抗病能力是一种有效可行的方法。
促分裂原活化蛋白激酶(mitogen-activatedproteinkinase，MAPK)级联途径在植物的生长发育和病原物侵染、机械损伤、低温等多种生物和非生物胁迫反应中起到重要的调节作用(陈娅斐,冯斌,赵小明等.MAPK级联途径在植物信号转导中的研究进展.植物学通报,2005,22(3):357–365.)。MAPK信号级联系统至少由3种激酶组成，即MAPK，MAPKK，MAPKKK(ZhangT,LiuY,YangT,etal.DiversesignalsconvergeatMAPKcascadesinplant.PlantPhysiolBioch,2006,44(5–6):274–283.)，这3种激酶通过逐级磷酸化MAPKKK→MAPKK→MAPK，使信号逐级放大并传递到下游。MAPKKKs通过使S/TXXXXXS/T基序(X代表任意氨基酸)中丝氨酸或苏氨酸残基磷酸化进而激活MAPKKs，随后，MAPKKs使Thr-X-Tyr(T-X-Y)基序中的保守苏氨酸和酪氨酸磷酸化从而激活MAPKs(TenaG,AsaiT,ChiuWL,etal.Plantmitogen-activatedproteinkinasesignalingcascades.CurrOpinPlantBio,2001,4(5):392–400.)，MAPKs使下游调控蛋白磷酸化从而激活或抑制信号通路。MAPKKK是级联反应的第一部分，对不同的环境刺激产生反应，可被小G蛋白家族Ras、Rho等信号活化，进而实现MAPKK的磷酸化。MAPKK又称MEK(MAPK／extracelluar-regulatedkinasekinase)，位于级联系统的中心，在信号交叉网络中占据核心位置(杨洪强,接玉玲.植物MAPK及其在病原信号传递中的作用.植物病理学报,2003,33(1):8–13.)，对其下游的MAPK磷酸化使信号逐级传递。
目前针对MAPK级联系统的研究多集中在最后的MAPK，已经分离鉴定了为数不少的MAPK基因，但是对MAPK级联途径的上游，即MAPKK及MAPKKK的研究还很少。植物MAPKK被分为四大类，即A、B、C和D四大类，大部分A和C型成员在多种非生物胁迫、病原物侵染或化学刺激下显示出活性(韩静.水稻MAPKK家族基因克隆及转基因研究.上海海洋大学硕士学位论文,2009)。紫花苜蓿(Medicagosativa)中的SIMKK(stress-inducedMAPkinasekinase)，在盐胁迫下活化SIMK(KiegerlS,CardinaleF,SiliganC,eta1.SIMKK,amitogen-activatedproteinkinase(MAPK)kinase,isaspecificactivatorofthesaltstress-inducedMAPK,SIMK.PlantCell,2000,12(11):2247–2258.)，苜蓿中另一类MAPKK激酶SAMK(stress-activatedMAPkinase)与病原菌介导的抗病反应信号途径有关(CardinaleF,JonakC,LigterinkW,eta1.DifferentialactivationoffourspecificMAPKpathwaysbydistinctelicitors.JBiolChem,2000,275(47):36734–36740.)。
MAPKK基因的表达具有组织特异性。在番茄[(Solanum.lycopersicum)cv.Micro-Tom]中已经鉴定出5个MAPKKs基因，分别为SlMAPKK1、SlMAPKK2、SlMAPKK3、SlMAPKK4、SlMAPKK5。SlMAPKK1在根中的表达量最高，SlMAPKK2主要在花中表达，SlMAPKK3主要在茎和叶中表达，SlMAPKK4在茎中的表达量最高，SlMAPKK5除了在果实中的表达量较低外，在其他组织中的表达量均较高(WuJ,WangJ,PanC,etal.Genome-wideidentificationofMAPKKandMAPKKKgenefamiliesintomatoandtranscriptionalprofilinganalysisduringdevelopmentandstressresponse.PLoSOne,2014,9(7):e103032.)。Wang等对黄瓜(Cucumissativus)MAPKs信号级联系统中的基因进行全基因组鉴定，发现在黄瓜中有6个MAPKKs，即CsMKK2-1、CsMKK2-2、CsMKK3、CsMKK4、CsMKK6、CsMKK9，并且它们的表达具有特异性。其中CsMKK3，CsMKK6在根、茎、叶、花和果实中的表达量都很低，而CsMKK2-1在上述组织中均有相当高的表达量，CsMKK2-2主要在根、茎和果实中表达，CsMKK4主要在茎、花和果实中表达，CsMKK9主要在根、茎、花和果实中表达(WangJ,PanC,WangY,etal.Genome-wideidentificationofMAPK,MAPKK,andMAPKKKgenefamiliesandtranscriptionalprofilinganalysisduringdevelopmentandstressresponseincucumber.BMCGenomics,2015,16:386.)。
植物在受到病原菌侵染后，会激活MAPK信号级联系统，传递信号并引起植物的防卫反应。拟南芥(Arabidopsisthaliana)AtMKK7基因编码MAPKK7，AtMKK7正调控植株的基础抗性及系统获得抗性。丁香假单胞菌Pseudomonassyringaepv.maculicola和霜霉菌Hyaloperonosporaparasitica侵染会诱导拟南芥AtMKK7的表达，在bud1突变体中，AtMKK7基因的表达量增加，进而促进水杨酸的积累并组成性表达病程相关蛋白基因，从而增强了对丁香假单胞菌和霜霉菌的抗性，而通过反义RNA来抑制AtMKK7mRNA的表达不仅降低了基本抗性，也阻断了系统获得抗性的建立(ZhangX,DaiY,XiongY,eta1.OverexpressionofArabidopsisMAPkinasekinase7leadstoactivationofplantbasalandsystemicacquiredresistance.PlantJ,2007,52(6):1066–1079.)。番茄SlMAPKK4的表达受病原菌P.syringes强烈诱导(WuJ,WangJ,PanC,etal.Genome-wideidentificationofMAPKKandMAPKKKgenefamiliesintomatoandtranscriptionalprofilinganalysisduringdevelopmentandstressresponse.PLoSOne,2014,9(7):e103032.)。与对照组相比，在SlMAPKK2和SlMAPK2同时沉默的番茄植株中，Xanthomonascampestrispv.Vesicatoria病原菌的数量显著增加(Melech-BonfilS,SessaG.TheSlMKK2andSlMPK2genesplayaroleintomatodiseaseresistancetoXanthomonascampestrispv.vesicatoria.PlantSignalBehav,2011,6(1):154–156.)。
本发明中丝裂原活化蛋白激酶激酶基因PnMAPKK1来自三七[Panaxnotoginseng(Burk)F.H.Chen]。三七是我国的传统名贵中药材，其种植区域主要分布于我国云南省，由于三七性喜温暖阴湿，对光敏感，因而要求在遮阳网下栽培，其独特的生长环境易诱发病虫害的发生，尤其是真菌病害，其中主要由茄腐镰刀菌引起的根腐病是三七的主要病害，严重影响了三七原药材的产量和品质。
发明内容
本发明的目的是提供一种从三七中克隆获得具有抗真菌活性的丝裂原活化蛋白激酶激酶的全长基因PnMAPKK1，PnMAPKK1的核苷酸序列如SEQIDNO：1所示，该基因全长为1230bp，包含一个960bp的开放阅读框、66bp的5′非翻译区(untranslatedregions，UTR)及204bp的3′UTR，编码如SEQIDNO：2所示氨基酸序列的蛋白质。
本发明所述丝裂原活化蛋白激酶激酶基因PnMAPKK1的编码区是序列表SEQIDNO：1中第67-1026位所示的核苷酸序列。
本发明分离克隆三七的一个抗真菌相关基因的完整cDNA片段，通过根癌农杆菌(Agrobacteriumtumefaciens)介导将目的基因转入受体植物中过量表达，并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PnMAPKK1。
MAPK信号级联系统是病原相关分子模式(pathogen-associatedmolecularpatterns，PAMP)触发的免疫反应(PAMP-triggeredimmunity,PTI)和病原菌效应子触发的免疫反应(effector-triggeredimmunity,ETI)早期信号传递的一部分，在植物防卫反应中处于中心地位。当遭受病原菌侵染时，植物通过其细胞膜上的模式识别受体(patternrecognitionreceptors,PRRs)识别病原菌/微生物相关的分子模式(pathogen/microbe-associatedmolecularpatterns,PAMPs/MAMPs)和损伤相关分子模式(damage-associatedmolecularpatterns,DAMPs)，PAMPs/MAMPs和DAMPs均可引起PTI；另一方面，植物细胞内的抗病蛋白识别抑制植物免疫反应的病原菌效应子，这些效应子可引起ETI。此外，细胞感知外界刺激后，MAPK信号级联途径的MAPKKKs—MAPKKs—MAPKs及其下游的转录因子和酶被逐级磷酸化，将信号逐级放大并传递到细胞中，使细胞表面受体和胞内严格调控的靶位点连接起来，从而调控防卫激素的合成及信号传递，激活防卫基因，促进气孔关闭和类似超敏反应的细胞死亡，推动代谢流向抗菌代谢物生成的方向流动，以增强植物对病原菌的抗性。
本发明涉及分离包含PnMAPKK1的DNA片段并鉴定其功能。其中所述DNA片段如序列表所示，对该基因进行分析，表明PnMAPKK1全长cDNA为1230bp，包含一个960bp的开放阅读框、66bp的5′UTR及204bp的3′UTR，其中ORF编码一个具有319个氨基酸的蛋白质。PnMAPKK1编码蛋白具有丝裂原活化蛋白激酶激酶的保守结构域，与来自白梨(Pyrusxbretschneideri)、苹果(Malusdomestica)、梅花(Prunusmume)等物种的丝裂原活化蛋白激酶激酶具有较高的相似性，表明其属于三七的丝裂原活化蛋白激酶激酶。超表达序列表SEQIDNO：1所示序列可以增强烟草对茄腐镰刀菌(Fusariumsolani)、胶孢炭疽菌(Colletotrichumgloeosporioides)、轮枝镰刀菌(Fusariumverticillioides)和葡萄座腔菌(Botrosphaeriadothidea)的抗性。
上述PnMAPKK1基因可以应用于提高烟草的抗真菌特性，具体操作如下：
(1)采用扩增PnMAPKK1的特异引物，从接种茄腐镰刀菌后的三七根中提取总RNA，通过两步法逆转录-聚合酶链式反应(reversetranscription-polymerasechainreaction，RT-PCR)扩增出PnMAPKK1的全长编码区，然后将其连接到pGEM-T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BamHI和Pst酶切pGEM-T-PnMAPKK1载体和植物表达载体pCAMBIA2300S，通过胶回收得到目的基因片段和载体大片段，再将PnMAPKK1基因片段与pCAMBIA2300S载体片段连接，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)以表达载体T-DNA上具有的抗性标记筛选转化子，并通过PCR以及RT-PCR检测得到阳性转基因植株，分析转基因植株对于几种病原真菌的抗性，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可以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。本发明中来自三七的PnMAPKK1基因能增强植物对几种病原真菌的抗性，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。利用基因工程技术培育抗性植物品种和材料具有明显的优势和不可取代的重要性。它不仅可以为大规模生产作物、花卉、药用植物等提供方便，减少化学农药的使用，还可以为农业生产节约成本、减少环境污染，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PnMAPKK1转基因烟草基因组DNA的PCR检测结果，其中Marker为DL2000DNAMarker(大连宝生物)，由2,000bp、1,000bp、750bp、500bp、250bp以及100bp六条DNA片段组成；阳性对照为质粒pGEM-T-PnMAPKK1为模板的PCR反应；WT为非转基因烟草(野生型，wildtype)总DNA为模板进行的PCR；
图2是本发明中部分阳性PnMAPKK1转基因烟草中PnMAPKK1转录水平的表达分析结果图，其中Marker为DL2000DNAMarker(大连宝生物)；WT为非转基因烟草总RNA逆转录cDNA为模板的PCR产物；阳性对照：质粒pGEM-T-PnMAPKK1为模板的PCR产物；
图3是本发明中PnMAPKK1转基因烟草体外抗真菌活性的抑菌效果图；其中a、b、c、d图示中的真菌分别是茄腐镰刀菌、胶孢炭疽菌、轮枝镰刀菌和葡萄座腔菌；WT为野生型烟草的总蛋白；CK为空白对照，即无蛋白对照(用于提取蛋白的缓冲液)。
具体实施方式
下面通过附图和实施例对本发明进一步说明，但本发明保护范围不局限于所述内容，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：PnMAPKK1全长cDNA克隆以及序列分析
用茄腐镰刀菌接种三七的根，用接种后8h的根提取总RNA，用液氮将处理过的三七根研磨成粉末，然后转入离心管中，采用异硫氰酸胍法提取总RNA。采用逆转录酶M-MLV(promega)以总RNA为模板合成cDNA第一链，反应体系和操作过程为：取5μg总RNA，依次加入50ngoligo(dT)，2μLdNTPMix(2.5mMeach)，用DEPC水将反应体积补齐至14.5μL；混匀后，70℃加热变性5min后迅速在冰上冷却5min，然后依次加入4μL5×First-standbuffer、0.5μLRNasin(200U)、1μLM-MLV(200U)，混匀并简短离心，42℃温浴1.5h，取出后70℃加热10min，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PnMAPKK1，所用上下游引物序列分别为5’CCTTGTTTCCAACTTCAACCCA3’及5’GATTCCGTCACGGGTAACAAATTG3’。采用AdvantageTM2PCREnzyme(Clontech)扩增出目的基因。PCR反应条件：94℃2min；94℃30s，61℃30s，72℃70s，32个循环；72℃5min。反应体系(20μL)为1μLcDNA、2μL10×Advantage2PCRBuffer、1.8μLdNTPMix(10mMeach)、0.2μL正向引物(10μM)、0.2μL反向引物(10μM)、0.2μLAdvantage2PCRPolymeraseMix、14.6μLPCR-Grade水。PCR结束后，取8μL进行琼脂糖凝胶电泳，用以检测扩增产物的特异性以及大小。
所得到PCR产物只有一条DNA带，因此直接对PCR产物进行TA克隆，使用的试剂盒为pGEM-Tvectorsystem(Promega)，反应体系和操作过程为：取1.5μLPCR产物，依次加入1μLpGEM-Tvector(50ng/μL)和2.5μL2×LigationsolutionI，混匀后置于16℃过夜反应。通过热激转化法将连接产物转入大肠杆菌DH5α感受态中。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PnMAPKK1的特异引物检测多克隆位点插入PnMAPKK1的克隆。将得到的阳性克隆进行测序，最终获得的PnMAPKK1全长cDNA为1230bp，通过NCBIORFfinder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个960bp的开放读码框(见序列表)。PnMAPKK1编码一个含319个氨基酸的蛋白质PnMAPKK1，其分子量约为35.88KDa，等电点为6.86。借助生物信息学软件SignalP4.1分析PnMAPKK1编码的蛋白序列，检测其是否具有N端信号肽。结果显示在PnMAPKK1的N端没有信号肽，因此推测该蛋白不是分泌蛋白。
实施例2：植物超表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取插入PnMAPKK1的大肠杆菌质粒pGEM-T-PnMAPKK1以及植物表达载体pCAMBIA2300S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及浓度。用限制性内切酶BamHI(TaKaRa)和Pst(TaKaRa)分别对质粒pGEM-T-PnMAPKK1和pCAMBIA2300S进行双酶切(100μL体系)，反应体系和操作过程为：分别取20μLpGEM-T-PnMAPKK1和pCAMBIA2300S质粒、依次加入10μL10×Kbuffer、4.5μLBamHI、5.5μLPst、60μLddH2O，混匀后短时离心，置于37℃过夜反应。将所有酶切产物进行琼脂糖凝胶电泳，然后使用SanPrep柱式DNA胶回收试剂盒对PnMAPKK1片段和pCAMBIA2300s载体大片段分别进行胶回收，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，置于-20℃保存备用。
利用T4DNALigase(TaKaRa)，将回收的PnMAPKK1DNA片段和pCAMBIA2300S载体片段连接起来，反应体系(20μL)和操作过程为：取10μLPnMAPKK1DNA片段依次加入2μLpCAMBIA2300S载体DNA、2μL10×T4DNALigaseBuffer、1μLT4DNALigase、5μLddH2O，混匀后短时离心，然后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50mg/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PnMAPKK1的特异引物进行PCR，挑选出PnMAPKK1与pCAMBIA2300S成功连接的克隆，并向检测得到的阳性菌株中加入甘油并置于-80℃保存备用。
提取并纯化上述大肠杆菌DH5α中的pCAMBIA2300S-PnMAPKK1质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PnMAPKK1转入所制备的根癌农杆菌LBA4404感受态细胞中。操作步骤为：取100ngpCAMBIA2300S-PnMAPKK1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，随后转入液氮中冷冻1min，然后迅速置于37℃水浴5min，再冰浴2min，之后加入500μLLB液体培养基于28℃振荡培养4h。将活化后的农杆菌涂于含有50mg/LKm的LB固体培养基上，28℃倒置培养。挑选单菌落摇菌，再用扩增PnMAPKK1的特异性引物进行PCR反应，检测pCAMBIA2300S-PnMAPKK1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(NicotianatabacumL.)。将烟草种子用75%的酒精浸泡30s，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-PnMAPKK1质粒的农杆菌LBA4404菌种，取20μL接种于5mL含有50mg/LKm和20mg/L利福平的LB液体培养基中，28℃培养至培养基浑浊。吸取1mL浑浊的菌液至含有50mg/LKm的LB固体培养基上，28℃培养48h。随后将LB固体培养基上的农杆菌刮下适量接种于附加有20mg/L的乙酰丁香酮的MGL液体培养基中，28℃振荡培养5-8h以活化农杆菌。
取烟草无菌烟草幼嫩叶片切成约1cm2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，25℃浸染15min。用无菌滤纸吸干叶盘表面的菌液，将叶盘置于共培养基上，22℃无光条件下共培养2天。烟草转化的共培养基为MS+0.02mg/L6-BA+2.1mg/LNAA+30g/L蔗糖+6g/L琼脂。
将共培养后的叶盘转到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/L6-BA+0.1mg/LNAA+30g/L蔗糖+6g/L琼脂+50mg/LKm+200mg/L头孢霉素(cefotaximesodiumsalt，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)。待烟草长出芽后用含有50mg/LKm和200mg/LCef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/LKm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，取1μL所得基因组DNA进行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PnMAPKK1的特异引物进行PCR反应。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，PnMAPKK1转基因烟草共筛选到25株阳性转基因植株。
实施例4：转基因烟草中PnMAPKK1的表达分析以及转基因植株抗真菌活性分析
分别取阳性转基因植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PnMAPKK1的特异引物进行PCR，根据PCR结果分析各转基因植株中PnMAPKK1转录水平的表达量。总RNA提取以及RT-PCR的方法与实施例1中相同。PCR结束之后，取8μL用于琼脂糖凝胶电泳，部分单株的检测结果如图2所示。
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℃暗培养，待菌落生长至直径约为2~3cm时添加蛋白，分析转基因植株体外抗真菌活性。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转基因烟草单株(编号分别为1、6、8、13)及野生型叶片放入研钵中，加入1mL蛋白提取液(1MNaCl，0.1M乙酸钠，1%PVP，pH6.0)，充分研磨。转入1.5mL离心管中，混匀后4℃静置过夜。4℃离心30min(12,000g)，取上清于新的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5μg/μL，然后分别取20μL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(蛋白提取液)。28℃培养几天后观察平板中真菌生长的情况，并据此来评价PnMAPKK1转基因烟草的体外抗真菌活性。结果如图3所示，PnMAPKK1转基因烟草蛋白对茄腐镰刀菌、胶孢炭疽菌、轮枝镰刀菌和葡萄座腔菌的生长具有明显的抑制作用。
序列表
<110>昆明理工大学
<120>三七丝裂原活化蛋白激酶激酶基因PnMAPKK1及其应用
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<170>PatentInversion3.3
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<221>mRNA
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Claims (2)
1.一种三七丝裂原活化蛋白激酶激酶基因PnMAPKK1，其特征在于：其核苷酸序列如SEQIDNO：1所示。
2.权利要求1所述的三七丝裂原活化蛋白激酶激酶基因PnMAPKK1在提高烟草对茄腐镰刀菌、胶孢炭疽菌、轮枝镰刀菌、葡萄座腔菌抗性中的应用。
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| CN109706132A (zh) * | 2018-12-10 | 2019-05-03 | 中国农业科学院棉花研究所 | 棉花抗黄萎病相关蛋白GhMAPK13及其编码基因和应用 |
| CN110128515A (zh) * | 2019-03-29 | 2019-08-16 | 广西壮族自治区农业科学院 | 一种水稻粒型生长发育相关编码基因及其应用 |
| CN111560389A (zh) * | 2020-06-11 | 2020-08-21 | 云南中烟工业有限责任公司 | 烟草丝裂原活化蛋白激酶基因NtMAPK8及其应用 |
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| CN106244598A (zh) * | 2016-09-21 | 2016-12-21 | 昆明理工大学 | 三七Dirigent类似蛋白基因PnDIR1及应用 |
| CN106244598B (zh) * | 2016-09-21 | 2019-07-05 | 昆明理工大学 | 三七Dirigent类似蛋白基因PnDIR1及应用 |
| CN109706132A (zh) * | 2018-12-10 | 2019-05-03 | 中国农业科学院棉花研究所 | 棉花抗黄萎病相关蛋白GhMAPK13及其编码基因和应用 |
| CN109706132B (zh) * | 2018-12-10 | 2022-07-05 | 中国农业科学院棉花研究所 | 棉花抗黄萎病相关蛋白GhMAPK13及其编码基因和应用 |
| CN110128515A (zh) * | 2019-03-29 | 2019-08-16 | 广西壮族自治区农业科学院 | 一种水稻粒型生长发育相关编码基因及其应用 |
| CN111560389A (zh) * | 2020-06-11 | 2020-08-21 | 云南中烟工业有限责任公司 | 烟草丝裂原活化蛋白激酶基因NtMAPK8及其应用 |
| CN111560389B (zh) * | 2020-06-11 | 2022-07-01 | 云南中烟工业有限责任公司 | 烟草丝裂原活化蛋白激酶基因NtMAPK8及其应用 |
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