CN108251432A - 三七类病程相关蛋白基因PnPRlike及应用 - Google Patents
三七类病程相关蛋白基因PnPRlike及应用 Download PDFInfo
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- CN108251432A CN108251432A CN201810000431.6A CN201810000431A CN108251432A CN 108251432 A CN108251432 A CN 108251432A CN 201810000431 A CN201810000431 A CN 201810000431A CN 108251432 A CN108251432 A CN 108251432A
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Abstract
本发明公开了一种三七类病程相关蛋白基因PnPRlike及其应用，PnPRlike基因的核苷酸序列如SEQ ID NO：1所示，编码类病程相关蛋白；本发明通过功能基因组学相关技术研究证实PnPRlike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PnPR like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，实验结果显示：PnPRlike超表达的转基因烟草对茄腐镰刀菌、葡萄座腔菌以及稻黑孢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类病程相关蛋白(pathogen-related protein-like isoform)基因PnPRlike及应用。
背景技术
植物在生长发育过程中会受到许多生物和非生物因子的胁迫，如干旱、寒冷、UV射线、创伤、病原物（真菌、细菌、病毒）侵染等。相应地，植物进化出一系列防御机制来抵御逆境胁迫，病程相关蛋白（pathogenesis-related proteins，PRs）的激活和积累是植物防御反应的重要组成部分（Singh A，Kirubakaran SI，Sakthivel N. Heterologousexpression of new antifungal chitinase from wheat. Protein Expr Purif，2007，56(1):100）。受到病原体攻击时，植物通过迅速改变基因表达来对病原菌和外部胁迫做出反应，诱导一些特殊蛋白的重新合成，其中大部分是病程相关蛋白（Wen YJ, He HW, HuangQS, Liang S, Bin JH. Roles of pathogenesis-relative protein 10 in plantdefense response. Plant Physiology Communications. 2008, 44: 585-592）。
PR蛋白为多基因编码，根据氨基酸序列的相似程度、血清学关系和生物学活性，目前将PR蛋白分为17类(PR1-PR17)，PRs在植物中表现为几丁质酶、类甜蛋白、蛋白酶抑制剂、内肽酶和过氧化物酶等(van Loon LC, Rep M, Pieterse CM. Significance ofinducible defense-related proteins in infected plants. Annu Rev Phytopathol.2006, 44: 135-162)。PRs基因是植物防卫反应基因，与超敏反应（hypersensitiveresponse，HR）及***获得性抗性（systematic acquire resistance，SAR）密切相关，它的诱导表达常作为SAR建立的标志，同时其基因编码产物始终是抗性研究的热点之一。PRs相对分子质量较小，一般为10～40 kD，多为酸性蛋白，半衰期为40～70 h，因此可以在胞内和胞间较好地积累；PRs有较强的稳定性，低pH值下仍可保持可溶性。采用细胞分离技术、免疫荧光显微技术、免疫金标记技术等研究表明，PRs主要分布于植物细胞间隙和液泡内，其分布与等电点及诱发菌和植物的亲和性有关。
PR3基因家族编码的几丁质酶(chitinase)在植物抗病防卫反应中备受关注。几丁质酶是一类可水解几丁质的大小分布于20-90 kD的糖基水解酶（Bhattacharya D,Nagpure A, Gupta RK. Bacterialchitinases: properties and potential. CriticalReviews in Biotechnology, 2007, 27(1): 21-28.）。其中微生物几丁质酶多为20～60kD，植物几丁质酶在20～45 kD内，昆虫几丁质酶为40～85kD。植物几丁质酶存在于植物的茎、种子、花中，它们的表达存在组织特异性。植物激素乙烯（ethylene, ETH）、茉莉酸（jasmonic acid, JA）、水杨酸（salicylic acid, SA）也诱导几丁质酶的表达。乙烯利诱导黄瓜叶片细胞间隙几丁质酶的积累，诱导的效果与黄瓜叶片对乙烯利质量浓度的感受直接相关。用茉莉酸甲酯（methyl jasmonate, MeJA）处理人参(Panax ginseng C.A. Meyer)，根系中几丁质酶活性与对照相比明显增高。外源水杨酸处理能够诱导香蕉(Musa paradisiacal)几丁质酶的活性及几丁质酶基因的上调表达。
PR5家族中的类甜蛋白（thaumatin-like proteins,TLPs）作为PRs的家族成员之一，是普遍存在于高等植物中的一类防御蛋白，在植物抵御病原体响应外界压力以适应不良环境过程中发挥作用（Datta K, Velazhahan R, Oliva N, et al. Over-expressionof the cloned rice thaumatin-like protein (PR-5) gene in transgenic riceplants enhances environmental friendly resistance to Rhizoctonia solanicausing sheath blight disease. Theoretical & Applied Genetics, 1999, 98(6-7):1138-1145.）。目前在葡萄(Vitis vinifera)、番茄(Lycopersicones culentum)、大麦（Hordeum vulgare）、小麦（Triticuma estivum）、燕麦（Avena sativa）等植物以及部分真菌中都发现了TLPs的存在。类甜蛋白氨基酸序列与非洲植物西非竹竽（Thaumatococcus daniellii）的甜蛋白(thaumatin)高度同源，故称为类甜蛋白。两种蛋白的同源性很高，但其功能完全不一样，甜蛋白具有甜味无抗真菌活性，而类甜蛋白无甜味具有抗真菌活性。
PR10编码的蛋白分子量在15～19 kD，等电点偏酸性，无信号肽序列，属胞内PR蛋白（intracellular pathogenesis-related proteins，IPR）。PR10蛋白在植物防御真菌等生物胁迫及非生物胁迫的过程中扮演着重要的角色（Liu JJ, Ekramoddoullah AKM. Thefamily 10 of plant pathogenesis-related proteins: their structure,regulation, and function in response to biotic and abiotic stresses.Physiological and Molecular Plant Pathology, 2006, 68(1): 3-13.）。PR10蛋白属于类核糖核酸酶，可以降解RNA，具有体外核酸酶活性和抗菌活性（Edreva A. Pathogenesis-related proteins: research progress in the last 15years. General and AppliedPlant Physiology, 2005, 31(1-2):105-124.）
三七为五加科(Araliaceae)人参属(Panax)植物，是我国传统名贵中药材，主产于云南文山地区。研究表明三七总皂苷具有抗肿瘤、抗病毒、降低胆固醇及提高机体免疫力等多种生物活性，是预防心脑血管疾病的重要药物。近年来三七的真菌病害日益严重，云南文山三七主产区的真菌病害主要为根腐病、黑斑病、圆斑病、疫霉病及灰霉病。根腐病常年发病率一般在5%～20%，严重的可达70%以上，甚至绝收，且生长年限越长，病害越严重（杨建忠，崔秀明，陈昱君，等. 连作地土壤处理对三七根腐病的控制效果. 特产研究，2010，32(2):37．）。病害问题目前已成为限制三七种植业发展的严重障碍。分离克隆三七的抗病功能基因，研究三七与病原物的分子互作机制，为三七的抗病遗传育种奠定基础。
发明内容
本发明的目的是提供一种从三七中克隆获得编码具有抗真菌活性的类病程相关蛋白基因PnPR like，类病程相关蛋白基因PnPRlike核苷酸序列如SEQ IDNO：1所示，该基因cDNA全长序列为976bp，包含一个717bp的开放阅读框、96bp的5’非翻译区、163bp的3’非翻译区，编码如SEQ IDNO：2所示氨基酸序列的蛋白质。
本发明中基因PnPRlike的编码区是序列表SEQ IDNO：1中第97-813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烟草中过量表达，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PnPRlike。
本发明涉及分离包含PnPRlike的DNA片段并鉴定其功能。其中所述DNA片段如序列表所示，对该基因的序列进行分析，表明PnPRlike全长cDNA为976bp，包含一个717bp的开放阅读框、96bp的5′-非翻译区(untranslated region, UTR)及163bp的3′-UTR，其中ORF编码一个具有238个氨基酸的蛋白质。BLASTp分析表明PnPRlike所编码蛋白质序列与胡萝卜（Daucus carota）的类病程相关蛋白基因（XP_017223871.1）、牵牛花（Ipomoea nil）的类病程相关蛋白基因（XP_019190454.1）、核桃（Juglans regia）的类病程相关蛋白基因（XP_018849219.1）和梅花（Prunus mume）的类病程相关蛋白基因（XP_008237645.1）同源性较高，分别为76%、68%、69%和67%，这表明其属于三七中的类病程相关蛋白。超表达序列表SEQID NO：1所示序列可以增强烟草对茄腐镰刀菌(Fusarium solani)、葡萄座腔菌(Botryosphaeria dothidea)以及稻黑孢菌(Nigrospora oryzae)的抗性。
本发明将三七类病程相关蛋白基因PnPRlike应用在提高烟草对病原真菌的抗性中，具体操作如下：
（1）采用异硫氰酸胍法提取三七根部的总RNA，以提取的RNA为模板,以oligo(dT) 18为反转录引物，通过逆转录-聚合酶链式反应(reverse transcription-polymerase chainreaction，RT-PCR)扩增出PnPRlike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EcoRI酶切pGEM-T-PnPR like，通过胶回收得到目的基因片段，用同样的内切酶酶切植物表达载体pCAMBIA2300s，胶回收获得所需载体大片段，再将所获得PnPRlike基因片段与pCAMBIA2300s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PCR以及RT-PCR检测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PnPRlike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、药材等提供方便，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PnPR like转基因烟草基因组DNA的PCR检测结果，其中Marker为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；阳性对照为质粒pGEM-T-PnPRlike为模板的PCR反应；WT为非转基因烟草(野生型，wild type)总DNA为模板进行的PCR；
图2是本发明中部分阳性PnPRlike转基因烟草中PnPRlike转录水平的表达分析结果图，其中Marker为DL2000 DNA Marker(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-PnPRlike为模板的PCR产物；
图3是本发明中PnPRlike转基因烟草体外抑制真菌生长的效果图；其中a、b、c图示中的真菌分别是茄腐镰刀菌、葡萄座腔菌以及稻黑孢菌；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：PnPRlike全长cDNA克隆以及序列分析
取三年生三七叶片提取总RNA，用液氮将三七叶片研磨成粉末，然后转入离心管中，采用异硫氰酸胍法提取总RNA，接着采用逆转录酶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），2 μLdNTP（2.5 mM each）、DEPC水至反应体积为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PnPRlike，所用上下游引物序列分别为5’CCAAATAACAATACTCTCCACAC3’及5’TTAAAGTTTGAGTTTGTTTTACATT3’。采用AdvantageTM 2PCR Enzyme（Clontech）扩增出目的基因；PCR反应条件：95℃ 1 min；94℃ 30 s，53 ℃ 30s，72℃ 1 min，30个循环；72℃ 5 min；反应体系（10 μL）为1 μL cDNA、1 μL 10×Advantage 2 PCR Buffer、0.5 μL 50×dNTP Mix （10 mM 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、6.9 μL PCR-Grade water；PCR结束后，取5 μL用于琼脂糖凝胶电泳，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（Promega），反应体系和操作过程为：取1.5 μL PCR产物，依次加入1 μLpGEM-T vector（50 ng/μL）和2.5 μL 2×Ligation solution，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。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PnPRlike的特异引物鉴定出多克隆位点***PnPRlike的克隆，并对阳性进行测序。最终获得的PnPRlike全长cDNA为976bp，通过NCBI ORF finder （http://www.ncbi.nlm.nih.gov/gorf/gorf.html）分析发现其包含一个717bp的开放读码框（见序列表），PnPRlike编码一个含238个氨基酸的蛋白质，PnPRlike其蛋白质分子质量约为26.64 kD，等电点(pI)约为5.69，表明该蛋白为酸性蛋白。其中包括32个酸性氨基酸（D，E）、32个碱性氨基酸（K、R、H）。
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***PnPRlike的大肠杆菌质粒pGEM-T-PnPRlike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及浓度高低；用限制性内切酶BamHI（TaKaRa）和EcoRI（TaKaRa）分别对质粒pGEM-T-PnPRlike和pCAMBIA2300s进行双酶切（100 μL体系），反应体系和操作过程为：取20 μL pGEM-T-PnPRlike和pCAMBIA2300s质粒、依次加入10 μL 10×Kbuffer、4 μL BamHI、6 μL EcoRI、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PnPRlike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PnPRlike 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 PnPRlike 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PnPRlike的特异引物进行PCR，挑选出PnPRlike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。
提取并纯化上述大肠杆菌中的pCAMBIA2300s-PnPRlike质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300s-PnPRlike转入根癌农杆菌LBA4404感受态细胞中。操作步骤为：取1 μg pCAMBIA2300s-PnPRlike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，之后立即冰浴2 min，加入800 μL 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℃静止培养。挑选单菌落摇菌，再用扩增PnPRlike的特异性引物进行PCR，检测pCAMBIA2300s-PnPRlike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，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6d，发芽后转至光照培养箱（25℃，16h/d光照），以后每月用1/2MS培养基继代一次。
从-80℃冰箱中取出保存的含有pCAMBIA2300s-PnPRlike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1mL浑浊的菌液至含有50mg/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+0.02 mg/L 6-BA+2.1 mg/L NAA+30 g/Lsucrose+6 g/L琼脂，22℃无光条件下共培养2天。
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），待烟草长出芽后用含有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为模板用扩增PnPRlike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，PnPRlike转基因烟草共筛选到50株阳性转基因植株。
实施例4：转基因烟草中PnPRlike的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PnPRlike的特异引物进行PCR，根据PCR结果分析各转基因单株中PnPRlike转录水平的表达，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42个转基因单株中PnPRlike在转录水平大量表达，这些单株的编号为1～41。
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2~3cm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1 g转基因烟草单株（编号分别为1、2、4）及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1 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（提取蛋白所用的buffer），28℃培养几天后观察各处理真菌生长的情况，并据此来评价PnPRlike转基因烟草的体外抗真菌活性，结果如图3所示，PnPRlike转基因烟草蛋白对茄腐镰刀菌、葡萄座腔菌、稻黑孢菌的生长具有很强的抑制作用。
序列表
<110> 昆明理工大学
<120> 三七类病程相关蛋白基因PnPRlike及应用
<160> 4
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<210> 1
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<213> 三七(Panax notoginseng)
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caaataacaa tactctccac acattgtctc acaaacatgg catctacagg atctaaggta 120
gaaaaataca ggtctttatt aggcgcagaa gatgtcaaag acgttcaatg gaggtttggt 180
gctcctccta actttgatgt tgtaaataag cttttcgaag aaggccgaac taagatatgg 240
cctgcaggtt cacttgaaga aaaagtgcag aaccttgtga aaacatggga aatggaaata 300
ttccacaaaa ctcgattaga agacttcaaa acacttgatc ccaagaagtt tactctaagc 360
cttaatggaa ggaaaggtgc gacaatggaa gaaataggaa agcttggtgg aggatacaat 420
gctatgctgc aaaccaaatt gccagaaaat tatcagtgct acaatccgac cgaggaatca 480
gctgattcgt cccatatcgt tttcaccaca gcgtttcccc gtggatttgc cctggaaatt 540
ctccaggttt attcagggcc accggtgatt gtgtacaagt ttaggcactg gggttatatg 600
gaaggccctt ttaaagggca tgctcctact ggagaaatga cagagtttta tgggattgcc 660
atttttgcgt tggatgaaga ttcgaagatt gtcaaggtgg agttctttta tgatagggga 720
gaacttctcg gaggtcttat gaagggtgga actactgata attctacaag tgaaatgtct 780
acaggctgcc ctgttttgaa aagcacagga tagctttgtc aagacattta ttagcattta 840
tcaagttcag tgttggataa atgtaaaaca aactcaaact ttaaactaat aattgcatct 900
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<213> 三七(Panax notoginseng)
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Phe Asp Val Val Asn Lys Leu Phe Glu Glu Gly Arg Thr Lys Ile Trp
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Pro Ala Gly Ser Leu Glu Glu Lys Val Gln Asn Leu Val Lys Thr Trp
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Glu Met Glu Ile Phe His Lys Thr Arg Leu Glu Asp Phe Lys Thr Leu
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Asp Pro Lys Lys Phe Thr Leu Ser Leu Asn Gly Arg Lys Gly Ala Thr
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Thr Lys Leu Pro Glu Asn Tyr Gln Cys Tyr Asn Pro Thr Glu Glu Ser
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Ala Asp Ser Ser His Ile Val Phe Thr Thr Ala Phe Pro Arg Gly Phe
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Ala Leu Glu Ile Leu Gln Val Tyr Ser Gly Pro Pro Val Ile Val Tyr
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Claims (2)
1.一种三七类病程相关蛋白基因PnPRlike，其特征在于：核苷酸序列如SEQ ID NO：1所示，编码如SEQ ID NO：2所示氨基酸序列的蛋白质。
2.权利要求1所述的三七类病程相关蛋白基因PnPRlike在提高烟草对茄腐镰刀菌(Fusarium solani)、葡萄座腔菌(Botryosphaeria dothidea)、稻黑孢菌(Nigrospora oryzae)抗性中的应用。
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| CN109234284A (zh) * | 2018-09-14 | 2019-01-18 | 昆明理工大学 | 一种三七类甜蛋白基因PnTLP5及应用 |
| CN109295068A (zh) * | 2018-09-14 | 2019-02-01 | 昆明理工大学 | 一种三七类甜蛋白基因PnTLP2及应用 |
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| CN116606361A (zh) * | 2023-04-25 | 2023-08-18 | 华中农业大学 | 一种调控植物根瘤固氮效率和/或调控植物产量的LjPRP1蛋白及其应用 |
| CN116606361B (zh) * | 2023-04-25 | 2024-04-23 | 华中农业大学 | 一种调控植物根瘤固氮效率和/或调控植物产量的LjPRP1蛋白及其应用 |
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