CN112831504A - 三七WRKY转录因子基因PnWRKY9及其应用 - Google Patents
三七WRKY转录因子基因PnWRKY9及其应用 Download PDFInfo
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- CN112831504A CN112831504A CN202110278780.6A CN202110278780A CN112831504A CN 112831504 A CN112831504 A CN 112831504A CN 202110278780 A CN202110278780 A CN 202110278780A CN 112831504 A CN112831504 A CN 112831504A
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Abstract
本发明公开了三七WRKY转录因子基因PnWRKY9及其应用,PnWRKY9基因的核苷酸序列如SEQ ID NO:1所示,编码WRKY转录因子;本发明通过分子生物学和功能基因组学相关技术研究证实PnWRKY9基因具有提高植物抗真菌病害的功能,将本发明抗真菌基因PnWRKY9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,PnWRKY9超表达的转基因烟草对链格孢菌(Alternaria compacta)、茄腐镰刀菌(Fusarium solani)、稻黑孢霉(Nigrospora oryzae)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研究领域,特别是一种三七WRKY转录因子基因PnWRKY9及其应用。
背景技术
植物在生长发育过程中,会不断地遭遇来自外界的各种胁迫,例如病虫害,从而导致粮食作物、经济作物以及药用植物等生长发育不良,减产甚至绝收的现象。植物病害分为非侵染病害和侵染病害。侵染病害由真菌、细菌、病毒等生物因素引起,具有传染性,危害大,持续时间长。植物病原菌种类繁多,防治特别困难。传统的防治植物病害的方法主要有农药防治和培育抗病品种。化学农药在使用过程中往往危害人畜安全以及造成环境污染。抗病品种的培育耗时较长见效慢、投入巨大、抗病资源难以获得。这两种方法都不能很好的解决植物病害问题;而基因工程利用分子生物学技术克隆抗病基因并借助转基因技术可以快速培育抗病植物品种,是增强植物抗病能力的新方法。
植物通过依赖一系列复杂的信号通路调节来有效防御外部威胁,同时保持体内生长平衡。转录因子是植物生长发育和应对外界刺激反应的主要调节因子,其表达和活性受到转录、转录后以及翻译等水平的调控。植物体内转录因子家族众多,其中AP2、bHLH、bZIP、WRKY等被发现参与生物和非生物胁迫的响应。
WRKY转录因子是植物中最大的转录因子家族之一,它是一类DNA结合蛋白,可通过与靶基因的启动子中的W-box(TGACC(A/T))结合,激活或抑制下游基因的表达从而在植物应激反应中发挥作用。WRKY转录因子在结构上包含一个或两个WRKY结构域,其中包括N端高度保守的七肽WRKYGQK和C端高度保守的锌指结构基 CX4-7-CX23-28-HX1-2-(H/C),这两个基序都是WRKY蛋白与W-box (TTGACT/C)顺式元件相互作用所必需的(Cui Q, Yan X, GaoX, et al. Analysis of WRKY transcription factors and characterization of twoBotrytis cinerea-responsive LrWRKY genes from Lilium regale. PlantPhysiolBiochem. 2018, 127: 525-536)。根据WRKY结构域的数量和锌指基序的结构,可将WRKY蛋白分为三个主要类群。第Ⅰ类和第Ⅱ类都含有C2-H2 (C-X4-5-CX22-23-H-X1-H)锌指结构,且分别含有两个WRKY结构域和一个WRKY结构域。第Ⅲ类具有单个WRKY 结构域,锌指结构为C2-HC(C-X7-C-X23-H-X-C)。类群Ⅱ又可分为五个亚类群(Ⅱa、Ⅱb、Ⅱc、Ⅱd和Ⅱe)(Ansar H, Xia L, Yahong W, et al. CaWRKY22 acts as a positive regulator inpepper response to Ralstoniasolanacearum by constituting networks withCaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58. International Journal of MolecularSciences, 2018, 19(5): E1426)。编码WRKY蛋白的第一条cDNA是从甘薯(Ipomoea batatas)中克隆得到的(Laudet V, Hänni C, Coll J, et al. Evolution of thenuclear receptor gene superfamily. EMBO J. 1992, 11(3):1003-13)。目前已经从拟南芥(Arabidopsis thaliana)、野燕麦(Avena fatua)、大麦(Hordeum vulgare)、烟草(Nicotiana tabacum)等多种植物中分离了大量WRKY基因。
WRKY作为植物体内的重要转录因子,参与调节植物的生长发育及体内次生代谢物的合成。拟南芥WRKY46、WRKY54和WRKY70通过调节油菜素内酯的合成来调控拟南芥的生长(Chen J, Nolan T, Ye H,et al. Arabidopsis WRKY46, WRKY54, and WRKY70transcription factors are involved in brassinosteroid-regulated plant growthand drought responses. Plant Cell. 2017, 29(6):1425-1439)。AtWRKY75是根毛发育的负调控因子,敲除AtWRKY75的突变体植株的根毛数量和长度较野生型有所增加。丹参(Salvia miltiorrhiza)WRKY1通过与调控甲基赤藓醇磷酸(MEP)途径的1-脱氧-d-木酮糖-5-磷酸还原异构酶(SmDXR)基因,正调节丹参酮的生物合成(Wang C, Wu C, Wang Y, etal. Transcription factor OpWRKY3 is involved in the development andbiosynthesis of camptothecin and its precursors in Ophiorrhizapumila hairyroots. Int J Mol Sci. 2019, 20(16):3996)。
WRKY也是植物激素信号转导途径的重要调控因子,可通过正向或负向调控信号转导从而在植物防御反应中发挥作用。拟南芥WRKY57可直接与JA信号通路中的JAZ1和JAZ5的启动子结合,直接激活其转录,从而增强对灰霉病(Botrytis cinerea)的敏感性,WRKY57功能的丧失增强了拟南芥对灰霉病菌的抗性(Jiang Y, Yu D. The WRKY57 transcriptionfactor affects the expression of jasmonate ZIM-domain genes transcriptionallyto compromise Botrytis cinerearesistance. Plant Physiol. 2016, 171(4):2771-2782)。水稻中OsWRKY45-2和OsWRKY45的突变体,能增加JA的积累,从而增强水稻对稻瘟病(Phyricularia grisea)的抗性(Uji Y, Taniguchi S, Tamaoki D, et al.Overexpression of OsMYC2 results in the up-regulation of early JA-responsivegenes and bacterial blight resistance in rice. Plant Cell Physiol. 2016, 57(9):1814-1827)。拟南芥Atwrky18/Atwrky40和Atwrky18/Atwrky60双突变体和Atwrky18/Atwrky40/Atwrky60三突变体对半活体型细菌病原菌的抗性增强,而对死体营养型真菌病原菌灰霉病菌的敏感性更强(Liu Q, Liu Y, Tang Y, et al. Overexpression ofNtWRKY50 increases resistance to Ralstoniasolanacearum and alters salicylicacid and jasmonic acid production in tobacco. Front Plant Sci. 2017, 8:1710)。
三七(Panax notoginseng)是云南省的重要中药资源,具有“生打熟补”的功效。三七生长周期长,性喜温暖阴湿,病害严重,特别是根腐病、黑斑病、圆斑病等真菌病害,严重危害三七产量和药材的品质。因此,对于三七抗病相关基因的克隆、功能分析和应用研究尤为迫切。
发明内容
本发明目的是提供一种三七WRKY转录因子基因PnWRKY9及其应用,WRKY基因PnWRKY9来自三七,并将其应用在提高烟草对链格孢菌(Alternaria compacta)、茄腐镰刀菌(Fusarium solani)、稻黑孢霉(Nigrospora oryzae)抗性中。
本发明从三七中克隆获得的具有抗真菌活性的WRKY转录因子基因PnWRKY9的全长基因,PnWRKY9的核苷酸序列如SEQ ID NO:1所示,该基因全长为706bp,包含一个558bp的开放阅读框、52bp的5′非翻译区(untranslated region, UTR)及96bp的3′UTR,编码如SEQ IDNO:2所示氨基酸序列的蛋白质。
本发明所述WRKY转录因子基因PnWRKY9的编码区是序列表SEQ IDNO:1中第53-610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(Agrobacterium tumefaciens)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PnWRKY9。
本发明涉及分离包含PnWRKY9的DNA片段并鉴定其功能,PnWRKY9全长cDNA为706bp,包含一个558bp的开放阅读框、52bp的5′非翻译区(untranslated region, UTR)及96bp的3′UTR,其中ORF编码一个具有185个氨基酸的蛋白质,PnWRKY9编码的蛋白质序列具有WRKY家族的保守结构域,这表明其属于三七中的WRKY蛋白;超表达序列表SEQ ID NO:1所示序列可以增强烟草对链格孢菌、茄腐镰刀菌、稻黑孢霉的抗性。
上述PnWRKY9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PnWRKY9的特异引物,从接种茄腐镰刀菌后12h的三七根中提取总RNA,通过逆转录-聚合酶链式反应(reverse transcription-polymerase chainreaction,RT-PCR)扩增出PnWRKY9的编码区,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EcoRⅠ和BamHⅠ酶切pGEM-T-PnWRKY9载体和植物超表达载体pCAMBIA2300S,通过胶回收得到目的基因片段和载体大片段;再将所获得PnWRKY9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)重组载体T-DNA上具有卡那霉素抗性基因,用添加卡那霉素的分化培养基筛选转化子,并通过PCR以及RT-PCR检测得到真正的转基因植株,分析转基因植株对于植物病原真菌的抗性,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PnWRKY9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是本发明中以三七cDNA为模板,用PnWRKY9基因特异性引物经PCR扩增得到的PnWRKY9基因片段;其中Marker为DL15000 DNA Marker (大连宝生物),由15,000bp、8,000bp、5,000bp、3,000bp、2,000bp、1,500bp、1,000bp、750bp、500bp、250bp及100bp十一条DNA片段组成;1为PnWRKY9基因特异性扩增条带;
图2是本发明中部分PnWRKY9转基因烟草基因组DNA的PCR检测结果,其中Marker:DL15000 DNA Marker (大连宝生物);阳性对照:质粒pGEM-T-PnWRKY9为模板的PCR反应;WT:非转基因烟草(野生型)总DNA为模板进行的PCR反应;
图3是本发明中部分阳性PnWRKY9转基因烟草中PnWRKY9转录水平的表达分析结果图,其中Marker:DL15000 DNA Marker (大连宝生物);WT: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-PnWRKY9为模板的PCR产物;
图4是本发明中PnWRKY9转基因烟草体外抗真菌活性的抑菌效果图;其中图a、b、c中的真菌分别是稻黑孢霉、链格孢菌、茄腐镰刀菌;WT为野生型烟草的总蛋白;Buffer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:PnWRKY9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12h的根提取总RNA,用液氮将处理过的三七的根研磨成粉末,然后转入离心管中,采用TRlzol法提取总RNA;采用M-MLV逆转录酶(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50 ngoligo (dT)、2 μLdNTP Mix (2.5mM each),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4μL 5×First-standbuffer、0.5μLRNasin (200U)、1μL M-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PnWRKY9,所用上下游引物序列分别为5’ATGGAGGGTACTTATCCGATGC3’及5’TTAATTATAGGGAGGAGGGCAAATC3’’,采用AdvantageTM 2PCR Enzyme (Clontech)扩增出目的基因。PCR反应条件:95℃ 1min;94℃ 30s,58℃ 30s,72℃1min,32个循环;72℃ 5min。反应体系(20μL)为1μLcDNA、2μL 10×Advantage 2 PCRBuffer、1.8μLdNTP Mix (10mM each)、0.2μL正向引物(10μM)、0.2μL反向引物(10μM)、0.2μL Advantage 2 PCR Polymerase Mix、14.6μL PCR-Grade water;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,直接对PCR产物进行TA克隆,使用的试剂盒为pGEM-T vector kit (Promega),反应体系和操作过程为:取1.5μL PCR产物,依次加入1μLpGEM-T vector (50ng/μL)和2.5μL 2×Ligation solutionⅠ,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PnWRKY9的特异引物检测多克隆位点插入PnWRKY9的克隆。将得到的阳性克隆进行测序,最终获得的PnWRKY9全长cDNA为706bp,通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个558bp的开放读码框(见序列表)。PnWRKY9编码一个含185个氨基酸的蛋白质PnWRKY9,其分子量约为20.73KDa,等电点为9.92。
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PnWRKY9的大肠杆菌质粒pGEM-T-PnWRKY9以及植物表达载体pCAMBIA2300S质粒,取2μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性及浓度高低。用限制性内切酶EcoRⅠ和BamHⅠ分别对质粒pGEM-T-PnWRKY9和pCAMBIA2300S进行双酶切(50μL体系),反应体系和操作过程为:分别取10μLpGEM-T-PnWRKY9和pCAMBIA2300S质粒、依次加入5μL10×Hbuffer、2.5μLEcoRI、2.5μLBamHI、30μL ddH2O,混匀后短时离心,置于37℃反应3.5h。将所有酶切产物进行琼脂糖凝胶电泳,然后使用胶回收试剂盒对PnWRKY9目的片段和pCAMBIA2300S载体大片段分别进行胶回收,取2μL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PnWRKY9DNA片段和pCAMBIA2300S载体片段连接起来,反应体系(20μL)和操作过程为:取10μLPnWRKY9DNA片段依次加入2μLpCAMBIA2300S载体DNA、2μL 10×T4 DNA Ligase Buffer、1μL T4 DNA Ligase、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PnWRKY9的特异引物进行PCR,挑选出PnWRKY9与pCAMBIA2300S成功连接的克隆,在得到的阳性菌株中加入等体积甘油并置于-80℃保存备用。
采用试剂盒提取并纯化上述大肠杆菌DH5α中的pCAMBIA2300S-PnWRKY9质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PnWRKY9转入所制备的根癌农杆菌LBA4404感受态细胞中。操作步骤为:取1μgpCAMBIA2300S-PnWRKY9质粒加入含有100μL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5min,然后迅速置于37℃水浴5min,再冰浴2min,之后加入600μL LB液体培养基于28℃振荡培养4 h;将活化后的农杆菌涂于含有50mg/L Km和20mg/L利福平(Rifampin, Rif)的LB固体培养基上,28℃倒置培养;挑选单菌落摇菌,再用扩增PnWRKY9的特异性引物进行PCR反应,检测pCAMBIA2300S-PnWRKY9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(Nicotianatabacum)。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14天左右,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-PnWRKY9质粒的农杆菌LBA4404菌种,取20μL接种于5mL含有50mg/L Km和20mg/LRif的LB液体培养基中,28℃培养至培养基浑浊。吸取1mL浑浊的菌液涂布于含有50mg/L Km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(Acetosyringone,AS)的MGL液体培养基中,28℃振荡培养5-8h以活化农杆菌。
取烟草无菌苗幼嫩叶切成约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℃条件暗培养2天。烟草转化的共培养基为MS+0.02mg/L 6-BA+2.1mg/L NAA+30g/L蔗糖+6g/L琼脂。
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+0.5mg/L 6-BA+0.1mg/L NAA+30g/L蔗糖+6g/L琼脂+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)。待烟草长出芽后用含有50mg/L Km和200mg/L Cef的MS培养基(MS+30g/L蔗糖+6g/L琼脂+)继代培养。
采用CTAB法提取转基因烟草植株叶片的基因组DNA,取2μL提取的基因组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PnWRKY9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2所示,PnWRKY9转基因烟草共筛选到32株阳性转基因植株。
实施例4:转基因烟草中PnWRKY9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PnWRKY9的特异引物进行PCR,根据PCR结果分析各转基因植株中PnWRKY9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3所示,共检测到21个转基因单株中PnWRKY9在转录水平大量表达,这些单株的编号为1~21。
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)及野生型叶片放入研钵中,加入1mL蛋白提取液[1mol/LNaCl、0.1mol/L乙酸钠、1% PVP(聚乙烯吡咯烷酮),pH6.0],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0.2μg/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℃培养几天后观察真菌生长的情况,并据此来评价PnWRKY9转基因烟草的体外抗真菌活性,结果如图4所示,PnWRKY9转基因烟草蛋白对链格孢菌、茄腐镰刀菌、稻黑孢霉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WRKY转录因子基因PnWRKY9及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 706
<212> DNA
<213> 三七(Panax notoginseng)
<400> 1
tctttctttc tttcttcatt ttattctttt gtggaaccct aagttattct taatggaggg 60
tacttatccg atgctctttc ttggttcatc aactgtgccg ccttatggtt ctaacaacaa 120
caataataat attggtacta atttttacaa caaccctaat ggattctcgg gagtatcaaa 180
ctcgatggag attcgtgcct cgacatcagg aagtaaagag gttgttaata actccaacgg 240
tagtggaagc ttcttgtcgg ctgaaaatca cgaggggaaa cttgtaggta agaagaaggg 300
tgatcagaag aagattaaga aaccgcgctt tgctttccaa acaaggagcc aggttgatat 360
tcttgatgat ggatatcgat ggagaaaata tggtcaaaag gctgttaaga acaacaaatt 420
tccgagaagc tactacaagt gtacttatca agggtgcaat gtgaagaaac aagtccaacg 480
tctgtcaaag gatgagggag ttgtggtgac tacttatgaa gggatgcaca cacattccat 540
agagaagcca tctgacaatt tcgaacaaat cttaagtgag atgaagattt gccctcctcc 600
ctataattaa ttaattgctt aacctcctta tttttatgga tcataaacta gcttagctag 660
gttggtctac gtactttttg taaaggattt atgttattta atcaga 706
<210> 2
<211> 185
<212> PRT
<213> 三七(Panax notoginseng)
<400> 2
Met Glu Gly Thr Tyr Pro Met Leu Phe Leu Gly Ser Ser Thr Val Pro
1 5 10 15
Pro Tyr Gly Ser Asn Asn Asn Asn Asn Asn Ile Gly Thr Asn Phe Tyr
20 25 30
Asn Asn Pro Asn Gly Phe Ser Gly Val Ser Asn Ser Met Glu Ile Arg
35 40 45
Ala Ser Thr Ser Gly Ser Lys Glu Val Val Asn Asn Ser Asn Gly Ser
50 55 60
Gly Ser Phe Leu Ser Ala Glu Asn His Glu Gly Lys Leu Val Gly Lys
65 70 75 80
Lys Lys Gly Asp Gln Lys Lys Ile Lys Lys Pro Arg Phe Ala Phe Gln
85 90 95
Thr Arg Ser Gln Val Asp Ile Leu Asp Asp Gly Tyr Arg Trp Arg Lys
100 105 110
Tyr Gly Gln Lys Ala Val Lys Asn Asn Lys Phe Pro Arg Ser Tyr Tyr
115 120 125
Lys Cys Thr Tyr Gln Gly Cys Asn Val Lys Lys Gln Val Gln Arg Leu
130 135 140
Ser Lys Asp Glu Gly Val Val Val Thr Thr Tyr Glu Gly Met His Thr
145 150 155 160
His Ser Ile Glu Lys Pro Ser Asp Asn Phe Glu Gln Ile Leu Ser Glu
165 170 175
Met Lys Ile Cys Pro Pro Pro Tyr Asn
180 185
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 3
atggagggta cttatccgat gc 22
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial)
<400> 4
ttaattatag ggaggagggc aaatc 25
Claims (2)
1.一种三七WRKY转录因子基因PnWRKY9,其特征在于:其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的三七WRKY转录因子基因PnWRKY9在提高烟草对链格孢菌(Alternaria compacta)、茄腐镰刀菌(Fusarium solani)、稻黑孢霉(Nigrospora oryzae)抗性中的应用。
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