CN106244598A - 三七Dirigent类似蛋白基因PnDIR1及应用 - Google Patents
三七Dirigent类似蛋白基因PnDIR1及应用 Download PDFInfo
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- CN106244598A CN106244598A CN201610835012.5A CN201610835012A CN106244598A CN 106244598 A CN106244598 A CN 106244598A CN 201610835012 A CN201610835012 A CN 201610835012A CN 106244598 A CN106244598 A CN 106244598A
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Abstract
本发明公开了三七Dirigent类似蛋白基因PnDIR1及其应用，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PnDIR1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PnDIR1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，PnDIR1超表达的转基因烟草对葡萄座腔菌、茄腐镰刀菌和轮枝镰刀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研究领域，特别是具有抗真菌活性的三七Dirigent类似蛋白基因PnDIR1及应用。
背景技术
植物是能量链的最底层生物，一些植物是经济作物、粮食作物，很多植物还具有重要的药用价值。植物在生长过程中或多或少会遭受着生物或者非生物因子的胁迫，从而影响最终植物经济性状或产量性状的形成。在多种逆胁迫因子中，由细菌、真菌、病毒等引起的病害是危害植物生长发育的主要因素。培育抗性植物品种以及使用农药是目前解决植物病害问题的主要方法。传统的育种方式具有周期长，不易获得抗源、抗性品种的抗性易散失等缺点，因此不能从根本上解决植物病害问题。近年来，分子生物学和基因工程技术快速发展，已从不同的植物分离克隆了大量抗病基因，利用基因工程技术培育植物抗病品种周期短，组合不同抗病基因可获得抗谱广、抗性强的新品种，能很好弥补传统抗病育种的缺陷。
在病原体侵染植物的过程中，病原菌分泌的物质可以诱导植物的免疫系统对病原菌的侵袭做出应答。植物-病原菌的互作一直是植物病理学的研究热点。为了应对病原菌的侵袭，植物进化出抵御病原菌的多种结构和抗性蛋白质。如植物生成木脂素加固细胞壁，产生植保素(phytoalexin)、毒性酚类小分子化合物以及多种病程相关蛋白(pathogenesisrelated protein)。基因组学的发展让测序更加简单，植物的全基因组测序也更加容易。近年来，病程相关蛋白、植物凝集素、dirigent（DIR）等作为植物产生的抗病蛋白备受关注，家族中许多抗病蛋白基因都已经分离克隆并鉴定了功能。
DIR蛋白首先发现于金钟连翘(Forsythia intermedia）和红崖柏木(Thuja plicata)中 (L.B. Davin, H.B. Wang, A.L. Crowellet al., Stereoselectivebiomolecular phenoxy radical coupling by an auxiliary (Dirigent) proteinwithout an active center. Science 1997, 362-366)。Dirigent一词源于拉丁语dirigere。DIR蛋白家族参与木质素的合成，也参与了植株的抗病过程。植物细胞中细胞壁的坚固性直接影响其对病原菌的抗性。木质素是由聚合的芳香醇构成的一类物质，存在于木质组织中，主要作用是通过形成交织网来硬化细胞壁，为次生壁主要成分。DIR蛋白家族还参与合成木脂素。木脂素类成分在厚朴中分离出来的最多，如厚朴酚和和厚朴酚。木脂素还包括双环氧木脂素、单环氧木脂素及新木脂素等，是一种在植物中广泛存在的次生代谢物，参与植物抗病过程，具有抗菌活性（宋敏. 小麦JRL和DIR基因家族的鉴定与分析. 博士研究生毕业论文 2013, 12）。DIR蛋白家族基因分为六个亚家族，分别是：DIR-a、DIR-b/d、DIR-c、DIR-e、DIR-f 和DIR-g，序列分析表明这六个亚家族之间序列相似性很低，其中只有DIR-a可以指导木质素合成正确的立体结构，其它亚家族蛋白的生化功能还不清楚(L.B.Davin, H.B. Wang, A.L. Crowell, D.L. Bedgar等. Stereoselective biomolecularphenoxy radical coupling by an auxiliary (Dirigent) protein without an activecenter. Science 1997, 362-366)，因此除了DIR-a之外的亚家族也被称为DIR-like（DIR类似）基因。DIR1蛋白具有一个非常保守的由5个α螺旋和4个二硫键组成的内部空腔，在空腔的入口处有3个极性氨基酸(Gln9, Asn13, Lys16)。DIR1晶体结构的内部空腔中，在距离3.8Å的两个磷脂之间由13个疏水残基组成了一个结合口袋（Champigny MJ，Isaacs M，Carella P et al., Long distance movement of DIR1 and investigation of therole of DIR1-like during systemic acquired resistance in Arabidopsis.Frontiers in Plant Science, 2013, 4: 230）。
DIR蛋白已经在很多植物中发现，研究表明DIR蛋白影响着植物的抗病性和抗非生物胁迫的能力。利用抑制差减杂交技术获得DIR的EST为信息探针，在基因库中筛选到陆地棉(Gossypium hirsutum)的DIR蛋白基因GhDIR。实时定量荧光PCR结果显示GhDIR基因在陆地棉幼苗根部受黄萎病菌(Verticillium dahliae)诱导，可见GhDIR与陆地棉抗真菌胁迫有关（赵付安，房卫平，杨晓杰等. 陆地棉Dirigent-like基因( GhDIR) 的克隆与分析. 华北农学报, 2011, 26(5)：29-33）。此外，白菜(Brassica rapa) ‘SUN-3061’在受到尖孢镰刀菌(Fusarium oxysporum)侵染后DIR蛋白的合成量大大提高，有些DIR蛋白在白菜受到ABA和低温胁迫时含量显著提高。表明DIR基因参与白菜对生物和非生物胁迫的保护作用（Thamil Arasan SK，Park JI，Ahmed NU，Jung HJ， Hur Y， Kang KK，Lim YP，Nou IS.Characterization and expression analysis of dirigent family genes related tostresses in Brassica. Plant Physiology and Biochemistry, 2013, 67:144-53）。在甘蔗(Saccharum officinarum) ‘F39’中克隆鉴定出DIR-like基因ScDir，经H2O2、PEG和NaCl处理后的甘蔗幼苗中ScDir的转录水平明显提高，此外，将ScDir导入大肠杆菌(Escherichia coli)中表达，ScDir蛋白的表达提高了大肠杆菌对NaCl、PEG的耐受力。经H2O2、PEG和NaCl处理后的甘蔗幼苗ScDir转录水平明显提高，说明ScDir与甘蔗抗非生物胁迫能力有关（Guo JL, Xu LP, Fang JP et al., A novel dirigent protein gene withhighly stem-specific expression from sugarcane, response to drought, salt andoxidative stresses. Plant Cell Reports, 2012, 31(10):1801-1812）。
本发明中DIR-like (DIR类似)基因PnDIR1来自三七(Panax notoginseng)。三七是传统的珍贵中药，也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、圆斑病等真菌病害是危害三七产业可持续发展的主要因素。
发明内容
本发明的目的是提供三七Dirigent类似蛋白基因PnDIR1及其PnDIR1的应用，即在提高烟草对葡萄座腔菌、茄腐镰刀菌和轮枝镰刀菌抗性中的应用。
本发明从三七中克隆获得的具有抗真菌活性的Dirigent类似蛋白PnDIR1的全长基因，PnDIR1的核苷酸序列如SEQ ID NO：1所示，该基因全长为986 bp，包含一个591 bp的开放阅读框、49 bp的5′非翻译区(untranslated region, UTR)及346 bp的3′UTR，编码如SEQ ID NO：2所示氨基酸序列的蛋白质。
本发明所述Dirigent类似蛋白基因PnDIR1的编码区是序列表SEQ ID NO：1中第50-640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PnDIR1。
DIR蛋白具有体外抑菌活性，能响应生物和非生物胁迫，是植物防御系统中重要的组成部分。拟南芥DIR1是一种获得性抗性相关的脂质转移蛋白，存在于细胞外，与细胞角质层的形成等有关，从而参与细胞抗逆性过程。
本发明涉及分离包含PnDIR1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PnDIR1全长cDNA为986 bp，包含一个591 bp的开放阅读框、49 bp的5′非翻译区(untranslated region, UTR)及346 bp的3′UTR，其中ORF编码一个具有196个氨基酸的蛋白质。BLASTn分析结果显示PnDIR1的部分序列与胡萝卜(Daucus carota)抗病反应蛋白基因(XM_017378982.1)具有84%的相似性，与可可 (Theobroma cacao)的抗病反应家族蛋白基因 (XM_007040336.1)具有78%的相似性。蛋白质同源性分析表明PnDIR1编码的蛋白质序列与可可树、野草莓(Fragaria vesca)的抗病反应蛋白分别具有81%和83%的相似性。PnDIR1编码的蛋白质序列具有Dirigent超家族的保守结构域，这表明其属于三七中的Dirigent类似蛋白。超表达序列表SEQ ID NO：1所示序列可以增强烟草对葡萄座腔菌(Botryosphaeria dothidea)、茄腐镰刀菌(Fusarium solani)和轮枝镰刀菌(F. verticillioides)的抗性。
上述PnDIR1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PnDIR1的特异引物，从接种茄腐镰刀菌后12 h的三七根中提取总RNA，通过逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction，RT-PCR)扩增出PnDIR1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XbaI和EcoRI酶切pGEM-T-PnDIR1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。再将所获得PnDIR1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PnDIR1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PnDIR1转基因烟草基因组DNA的PCR检测结果，其中Marker：DL2000 DNA Marker (大连宝生物)，由2,000 bp、1,000 bp、750 bp、500 bp、250 bp以及100 bp六条DNA片段组成；阳性对照：质粒pGEM-T-PnDIR1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；
图2是本发明中部分阳性PnDIR1转基因烟草中PnDIR1转录水平的表达分析结果图，其中Marker：DL2000 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-PnDIR1为模板的PCR产物；
图3是本发明中PnDIR1转基因烟草体外抗真菌活性的抑菌效果图；其中a、b、c图示中的真菌分别是轮枝镰刀菌、茄腐镰刀菌、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：PnDIR1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12 h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，采用异硫氰酸胍法提取总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5 μg total RNA，依次加入50 ng oligo (dT)，2 μL dNTP Mix (2.5 mM each)，用DEPC水将反应体积补齐至14.5 μL；混匀后，70℃加热变性5 min后迅速在冰上冷却5 min，然后依次加入4 μL 5×First-stand buffer、0.5 μL RNasin (200 U)、1 μL M-MLV (200 U)，混匀并简短离心，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PnDIR1，所用上下游引物序列分别为5’GAAATATGGCAAGAGCAAGAAGTAC3’及5’CCTTTTGGTTGGTATACATGGCTA3’。采用AdvantageTM 2PCR Enzyme (Clontech)扩增出目的基因。PCR反应条件：95℃ 1 min；94℃ 30 s，59℃ 30s，72℃ 50 s，32个循环；72℃ 5 min。反应体系(20μL)为1 μL cDNA、2 μL 10×Advantage2 PCR Buffer、1.8 μL dNTP Mix (10mM each)、0.2 μL 正向引物(10 μM)、0.2 μL 反向引物(10 μM)、0.2 μL Advantage 2 PCR Polymerase Mix、14.6 μL PCR-Grade water。PCR结束后，取8 μL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1.5 μL PCR产物，依次加入1 μL pGEM-T vector (50 ng/μL)和2.5 μL 2×Ligation solution I，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PnDIR1的特异引物检测多克隆位点插入PnDIR1的克隆。将得到的阳性克隆进行测序，最终获得的PnDIR1全长cDNA为986 bp，通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个591 bp的开放读码框(见序列表)。PnDIR1编码一个含196个氨基酸的蛋白质PnDIR1，其分子量约为21.6 KDa，等电点为6.17。借助生物信息学软件SignalP 4.1分析PnDIR1编码的蛋白序列，检测其是否具有N端信号肽。结果显示在PnDIR1中有信号肽，因此推测PnDIR1蛋白是分泌蛋白。
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PnDIR1的大肠杆菌质粒pGEM-T-PnDIR1以及植物表达载体pCAMBIA2300S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及浓度高低。用限制性内切酶XbaI和EcoRI分别对质粒pGEM-T-PnDIR1和pCAMBIA2300S进行双酶切(100 μL体系)，反应体系和操作过程为：分别取20 μLpGEM-T-PnDIR1和pCAMBIA2300S质粒、依次加入10 μL 10×H buffer、5 μL EcoRI、5 μLXbaⅠ、60 μL ddH2O，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，然后使用试剂盒对PnDIR1片段和pCAMBIA2300s载体大片段分别进行胶回收，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PnDIR1 DNA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 PnDIR1 DNA片段依次加入2 μLpCAMBIA2300S载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μLddH2O，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PnDIR1的特异引物进行PCR，挑选出PnDIR1与pCAMBIA2300S成功连接的克隆，在得到的阳性菌株中加入甘油并置于-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-PnDIR1质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PnDIR1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-PnDIR1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，随后转入液氮中冷冻1 min，然后迅速置于37℃水浴5 min，再冰浴2 min，之后加入500μL 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50 mg/L Km的LB固体培养基上，28℃倒置培养。挑选单菌落摇菌，再用扩增PnDIR1的特异性引物进行PCR反应，检测pCAMBIA2300S-PnDIR1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30 s，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-PnDIR1质粒的农杆菌LBA4404菌种，取20 μL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浑浊的菌液至含有50 mg/L Km的LB固体培养基上，28℃培养48 h。随后将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5-8 h以活化农杆菌。
取烟草无菌苗幼嫩叶切成约1 cm2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，22℃无光条件下共培养2天。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蔗糖+6 g/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+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L蔗糖+6 g/L琼脂+50mg/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℃，16 h/d光照，8 h/d黑暗)。待烟草长出芽后用含有50 mg/L Km和200 mg/L Cef的MS培养基(MS+30 g/L蔗糖+6 g/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1 μL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PnDIR1的特异引物进行PCR反应。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，PnDIR1转基因烟草共筛选到35株阳性转基因植株。
实施例4：转基因烟草中PnDIR1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PnDIR1的特异引物进行PCR，根据PCR结果分析各转基因植株中PnDIR1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8 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29个转基因单株中PnDIR1在转录水平大量表达，这些单株的编号为1～29。
将实验室保存的几种真菌接种于PDA固体培养基(200 g/L马铃薯，15 g/L琼脂，20g/L葡萄糖)上，28℃暗培养，待菌落生长至直径约为2~3 cm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：胶孢炭疽菌(Colletorichum gloeosporioides)、核盘菌(Sclerotinia scleroterum)、葡萄座腔菌、茄腐镰刀菌和轮枝镰刀菌。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、4、10、15、17)及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，pH6.0)，充分研磨。转入1.5 mL离心管中，混匀后4℃静置过夜。4℃离心30min (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(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PnDIR1转基因烟草的体外抗真菌活性，结果如图3所示，PnDIR1转基因烟草蛋白对葡萄座腔菌、茄腐镰刀菌和轮枝镰刀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Dirigent类似蛋白基因PnDIR1及应用
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 986
<212> DNA
<213> Panax notoginseng
<220>
<221> mRNA
<222> (1)..(986)
<220>
<221> 5'UTR
<222> (1)..(49)
<220>
<221> CDS
<222> (50)..(640)
<220>
<221> 3'UTR
<222> (641)..(986)
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ttctccttcc tcggccgcat acccggcggc gacgaagaaa aagcaataca aaccatgcaa 180
acacttggtc ctattcttcc acgacattat ttacaatggt gaaaatgcag cgaatgcaac 240
ttctgccatg gtaggtgccc ctgcaggggc caacttaaca attttagcag ataaattcca 300
ttttggaaac atggtggttt ttgatgatcc aattacactg gacaacaatc ttcattcacc 360
tcccgttggt agagcacaag gaatgtatgt atatgacacc aaaaacacct acacggcgtg 420
gcttgggttt tcctttgttc taaacaccac cgattatcag ggcagcatca acttcatcgg 480
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tttcatgcac cggggagttg ccaccgttat gacggattca tttgaaggtg aagtctactt 600
taggctcagt gttgatatca acttctatga atgctggtaa tctaattact cacttaagtt 660
atagtatatg catcatgcat gcatgttgaa gaattaataa tatttattgt tcatgaattt 720
tagccatgta taccaaccaa aaggatatat gggcttcttc ttttttttgt attaatttgc 780
atgtggtcgt cggaattgag aacgttaatt actagtagta ctattgtatc aacgtttaca 840
gctacagcat gcttggcaca attaattgtg cctgcttgtt ttaattagct taattaaaat 900
gattgttaag aatgtacttc accaaatcca aatgaataag tacaagatta gttttttggt 960
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<213> Panax notoginseng
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Ser Ala Met Val Gly Ala Pro Ala Gly Ala Asn Leu Thr Ile Leu Ala
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Phe Val Leu Asn Thr Thr Asp Tyr Gln Gly Ser Ile Asn Phe Ile Gly
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Claims (2)
1.一种三七Dirigent类似蛋白基因PnDIR1，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Dirigent类似蛋白基因PnDIR1在提高烟草对葡萄座腔菌(Botryosphaeria dothidea)、茄腐镰刀菌(Fusarium solani)和轮枝镰刀菌(F. verticillioides)抗性中的应用。
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