CN117286150A - 三七病程相关蛋白1基因PnPR1-3及其应用 - Google Patents
三七病程相关蛋白1基因PnPR1-3及其应用 Download PDFInfo
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Abstract
本发明公开了一种三七病程相关蛋白1基因PnPR1‑3及其应用，其中所述PnPR1‑3基因的核苷酸序列如SEQ ID NO：1所示，编码病程相关蛋白1，本发明通过功能基因组学相关技术研究证实PnPR1‑3基因具有提高植物抗真菌侵染的能力，将本发明PnPR1‑3基因构建到植物表达载体上并转入烟草中过量表达，PnPR1‑3转基因烟草对棒弯孢的抗性明显提高。
Description
技术领域
本发明属于分子生物学以及基因工程技术领域，特别涉及一种具有抗真菌侵染能力的三七病程相关蛋白1基因PnPR1-3及其应用。
背景技术
植物在生长过程中会不断遭受包括细菌、真菌、病毒、盐、干旱等生物及非生物因子的胁迫。与动物不同的是，植物体内缺乏可移动的防御细胞和适应性免疫系统，因此在接收外界环境刺激时，只能依靠触发启动体内的免疫系统来对环境刺激做出免疫应答反应(Nobori T,Tsuda K.The plant immune systemin heterogeneousenvironments.Current opinion in plant biology.2019,50:58-66)。植物的免疫系统包括病原体相关分子模式触发免疫(pathogen-associated molecular patterns-triggeredimmunity,PTI)和效应器触发免疫(effector-triggered immunity,ETI)(Ding LN,Li YT,Wu YZ,et al.“Plant disease resistance-related signaling pathways:recentprogress and future prospects.”International journal of molecularsciences.2022,23(24):16200)。如PTI对细胞表面上保守的病原体相关分子模式(PAMPs)作出反应，激活MAP激酶信号级联，表达病程相关蛋白(pathogenesis-related proteins,PRs)、积累胼胝质等，最终根除病原体或限制其在植物体内的传播；此外ETI与细胞内受体对病原体的识别和PR蛋白的转录激活等过程相关(Silva MS,Arraes FBM,Campos MA,etal.Review:Potential biotechnological assets related to plant immunitymodulation applicable in engineering disease-resistant crops.Plant science:aninternational journal of experimental plant biology.2018,270:72-84)。
PRs是植物免疫系统的组成部分，广泛存在于植物中，在植物防御病原体方面起着重要作用(Finkina EI,Melnikova DN,Bogdanov IV,et al.Plant pathogenesis-relatedproteins PR-10and PR-14as components of innate immunity system and ubiquitousallergens.Current medicinal chemistry.2017,24(17):1772-1787)。
PR蛋白家族成员之间的序列相似性较低，但具有一些共同的生化特征，例如低分子量，存在疏水腔，具有配体结合特性和抗菌活性。PR能够抵抗酸性pH和蛋白酶水解，从而使得它们能在液泡、细胞壁或细胞间隙中分布(Stintzi A,Heitz T,Prasad V,etal.Plant'pathogenesis-related'proteins and their role in defense againstpathogens.Biochimie.1993,75(8):687-706)。根据蛋白结构、亲缘关系和生物活性等方面的特性将PRs分为17个家族。其中，PR1家族蛋白普遍具有抗真菌活性，是病原体或水杨酸(salicylic acid,SA)诱导期间产生的最为丰富的蛋白质(Sels J,Mathys J,De ConinckBM,et al.Plant pathogenesis-related(PR)proteins:a focus on PR peptides.Plantphysiology and biochemistry.2008,46(11):941-950)。
PR1家族的第一个成员PR1-a是在烟草花叶病毒感染的烟草(Nicotiana tabacum)叶片中发现的，随后在多种植物中鉴定出了多种PR1蛋白(Chen YL,Lee CY,Cheng KT,etal.Quantitative peptidomics study reveals that a wound-induced peptide fromPR-1regulates immune signaling in tomato.The Plant cell.2014,26(10):4135-4148)。
PR1家族基因数量较多，其结构也十分保守(Van Loon L C,Van Strien E A.Thefamilies of pathogenesis-related proteins,their activities,and comparativeanalysis of PR-1type proteins.Physiological and Molecular PlantPathology.1999,55(2):85-97)。根据PR1的结构域特征，通过HMMER查找，在拟南芥(Arabidopsis thaliana)基因组中有22个PR1基因(Van Loon L C,Rep M,Pieterse C MJ.Significance of inducibledefense-related proteins in infected plants.AnnualReview of Phytopathology.2006；44(3):135-162.)，水稻(Oryza sativa)基因组中有38个PR1基因(程雨果,魏炳峥,李春霞等.水稻PR1基因响应生物胁迫的表达模式.分子植物育种.2023,1-24)，葡萄(Vitis vinifera)基因组中有10个PR1基因(Rahman F U,Khan I A,Aslam A,et al.Transcriptome analysis reveals pathogenesis-related gene1pathway against salicylic acid treatment in grapevine(Vitis vinifera L).Frontiers in Genetics.2022；13(10):1033288-1033306.)，大豆(Glycine max)基因组中有19个PR1基因(苏伟华,赵志华,齐梦楠等.病程相关蛋白基因GmPR1-6的克隆及其在大豆抵抗SMV侵染过程中的功能初探.河北农业大学学报.2023,(04):8-15)。
三七[Panax notoginseng(Burk)F.H.Chen]为五加科(Araliaceae)人参属(Panax)多年生草本植物，是我国传统名贵中药材(Ng TB.Pharmacological activity ofsanchi ginseng(Panax notoginseng).Journal of pharmacy and pharmacology.2006,58:1007-1019)。三七喜阴暗潮湿的生长环境，而且生长周期长，容易发生病害，尤其是根腐病(蒋妮,覃柳燕,叶云峰.三七病害研究进展.南方农业学报.2011,42:5)。根腐病的大面积发生不仅导致三七药材的产量大幅下降，还严重影响了药材的品质。采用农药防治三七根腐病的传统方法不能从根本上解决三七病害问题，并且会污染土壤、环境。因此，有必要进一步研究三七对根腐病的防御机制，以开发优良的三七抗病品种促进三七产业健康发展。
发明内容
本发明的目的之一是从三七中克隆获得病程相关蛋白基因PnPR1-3，PnPR1-3的核苷酸序列如SEQ ID NO:1所示，该基因ORF全长为504bp，编码如SEQ ID NO:2所示氨基酸序列的蛋白质。
本发明克隆分离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植物抵御真菌病害的能力奠定基础。
本发明另一目的是将三七病程相关蛋白基因PnPR1-3应用在提高烟草对棒弯孢(Curvularia clavata)的抗性中。
上述三七病程相关蛋白基因PnPR1-3提高烟草抗真菌特性的具体操作如下：
(1)采用扩增PnPR1-3的特异性引物，从接种棒弯孢(Curvularia clavata)后的三七根中提取总RNA，通过逆转录-聚合酶链式反应(reverse transcription-polymerasechain reaction，RT-PCR)扩增出PnPR1-3的ORF，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SmaⅠ和PstⅠ酶切pGEM-T-PnPR1-3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PnPR1-3基因片段与pCAMBIA2300s载体片段连接，构建植物超表达载体；然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检测得到阳性转基因植株，分析转基因植株对病原真菌的抗性，最后筛选出对真菌抗性明显增强的转基因植株。
本发明中来自三七的PnPR1-3基因能增强植物对棒弯孢的抗性，将该基因导入烟草中，可以获得具有真菌抗性的新品种和新材料。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，这不仅缩短了育种周期，而且操作简单，容易获得高抗材料。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，还可以帮助减少化学农药的使用，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PnPR1-3转基因烟草基因组DNA的PCR检测结果，其中Marker：DL2501 DNA Marker(上海捷瑞)，由2,000bp、1,000bp、750bp、500bp以及250bp五条DNA片段组成；阳性对照：质粒pGEM-T-PnPR1-3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PnPR1-3转基因烟草中PnPR1-3转录水平的表达分析结果图，其中Marker：DL2501 DNA Marker(上海捷瑞)；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-PnPR1-3为模板的PCR产物；
图3是本发明中PnPR1-3转基因烟草抗病性鉴定的结果图；图中是接种棒弯孢的PnPR1-3转基因烟草叶片；其中WT为野生型烟草叶片，3-1、3-3、3-6、3-13为PnPR1-3转基因烟草株系的叶片。
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：PnPR1-3全长cDNA克隆以及序列分析
用棒弯孢接种三七根部，用接种后24h的三七根部提取总RNA，用液氮将接种后的三七根研磨成粉末，转入离心管中，按照Super总RNA提取试剂盒操作说明提取总RNA。采用Go ScriptTM Reverse Transcriptase System以总RNA为模板合成cDNA第一链，反应体系和操作过程为：取5μg total RNA，依次加入1μL Oligo dT15 primer、1μL Randomprimer，用Nuclease-Free Water将反应体积补齐至10μL；混匀，70℃加热变性5min后迅速在冰上冷却5min，然后依次加入4μL 5×Reaction Buffer、4μL MgCl2(25mM)、1μL PCRNucleotide Mix、0.4μL Recombinant />Ribonuclease Inhibitor、0.4μL ReverseTranscriptase、1.2μLNuclease-Free Water，混匀并简短离心，25℃静置5min，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PnPR1-3，所用上下游引物序列分别为5’ATGGCTTCCTTTCACCAAGTAAAA3’及5’CTCAATAAGGATGGTCGCCTACG3’。PCR反应条件：95℃5min；94℃30s，59℃30s，72℃1min，30个循环；72℃5min。反应体系(50μL)为2μL cDNA、5μL10×Ex Taq Buffer(含Mg2+20mM)、4μL dNTP Mix(2.5mM each)、1μL上游引物(5μM)、1μL下游引物(5μM)、0.25μL TaKaRa Ex Taq(5U/μL)、36.75μL ddH2O。PCR结束后，取50μL进行1.2％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，使用SanPrep柱式PCR产物纯化试剂盒(上海生工)切胶回收得到PCR扩增产物，进行TA克隆，使用的试剂为pGEM-T Vector SystemⅠ(TaKaRa)，反应体系和操作过程为：取4μL PCR产物，依次加入0.7μL pGEM-T vector、0.9μLT4 DNALigase、5μL 2×Rapid Ligation Buffer，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。挑选若干个单菌落于含有Amp的LB液体培养基中，待菌液浑浊后用扩增PnPR1-3的特异性引物检测多克隆位点插入PnPR1-3的克隆。将得到的阳性克隆进行测序，通过NCBIORF finder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析最终获得的PnPR1-3全长cDNA为504bp。PnPR1-3编码一个含167个氨基酸的蛋白质PnPR1-3，其分子量约为18.49kDa，等电点为6.37。借助生物信息学软件SignalP 4.1分析PnPR1-3编码的蛋白序列，检测其是否具有N端信号肽。结果显示在PnPR1-3的N端无信号肽，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PnPR1-3的大肠杆菌质粒pGEM-T-PnPR1-3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高低。用限制性内切酶SmaⅠ(TaKaRa)和PstⅠ(TaKaRa)分别对质粒pGEM-T-PnPR1-3和pCAMBIA2300s进行双酶切(50μL体系)，反应体系和操作过程为：分别取15μL pGEM-T-PnPR1-3和pCAMBIA2300s质粒、依次加入7.5μL 10×K buffer、2.5μL SmaⅠ、2.5μL PstⅠ、17.5μL ddH2O，混匀后短时离心，置于37℃水浴锅酶切3小时。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(上海生工)对PnPR1-3 ORF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T4 DNA Ligase(TaKaRa)，将回收的PnPR1-3 ORF和pCAMBIA2300s载体片段连接起来，反应体系(20μL)和操作过程为：取10μL PnPR1-3 ORF依次加入2μL pCAMBIA2300s载体DNA、2μL 10×T4 DNA Ligase Buffer、1μL T4 DNA Ligase、5μL ddH2O，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，Kana)的固体培养基筛选阳性克隆。挑选单菌落含有50mg/LKana的LB液体培养基震荡培养，以菌液为模板用扩增PnPR1-3的特异引物进行PCR，挑选出PnPR1-3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-PnPR1-3质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300Ss-PnPR1-3转入所制备的根癌农杆菌LBA4404感受态细胞中。操作步骤为：取5μL pCAMBIA2300s-PnPR1-3质粒加入含有50μL感受态细胞的离心管中，轻轻混匀后冰浴30min，随后转入液氮中冷冻2min，然后迅速置于37℃水浴5min，再冰浴2min，之后加入500μL LB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/L Kana和25mg/L Rif的LB固体培养基上，28℃倒置培养；挑选若干个单菌落于含有50mg/L Kana和25mg/L Rif的LB液体培养基震荡培养，以菌液为模板再用扩增PnPR1-3的特异性引物进行PCR反应，检测pCAMBIA2300s-PnPR1-3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：农杆菌介导的烟草遗传转化以及转基因烟草筛选
本实验的转基因受体是烟草(Nicotiana tabacum)。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-PnPR1-3质粒的农杆菌LBA4404菌种，取20μL接种于5mL含有50mg/L Kana和25mg/L Rif的LB液体培养基中，28℃培养至菌液浑浊；吸取1mL浑浊菌液至含有50mg/L Kana和25mg/L Rif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4h以活化农杆菌。
取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℃黑暗条件下共培养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/LKana+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)。待烟草分化出芽后用含有50mg/L Kana和200mg/LCef的MS培养基继代培养。将烟草再生苗移至含有50mg/L Kana的MS培养基上使其生根，最后选用生根较好的再生幼苗进行PCR分析。
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PnPR1-3的特异性引物进行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，PnPR1-3转基因烟草共筛选到29株阳性转基因植株。
实施例4：转基因烟草中PnPR1-3的表达分析以及转基因植株抗真菌侵染的功能分析
分别取阳性转基因烟草以及非转基因烟草(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PnPR1-3的特异性引物进行PCR，根据PCR结果分析各转基因植株中PnPR1-3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。
将实验室保存的棒弯孢接种于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5天。取温室中生长良好、大小均一且完全伸展的WT烟草和PnPR1-3转基因烟草叶片，用手术剪从叶柄处剪下。用无菌塑料枪头在叶片相同位置制造大小一致的伤口，分别接种大小相等的棒弯孢菌丝块。将处理后的叶片置于铺有无菌水浸湿的滤纸的平板中，于28℃光照培养箱中培养，每天加水保湿。培养7d后收集叶片并观察各株系叶片的发病情况。结果如图3所示，接种棒弯孢后，野生型烟草的叶片形成较大的病斑，叶片出现黄化和腐烂的现象，而转基因烟草叶片的症状很轻微，形成的病斑面积也远小于野生型烟草。显然，PnPR1-3转基因烟草对棒弯孢具有很明显的抗性。
Claims (2)
1.一种三七病程相关蛋白1基因PnPR1-3，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的三七病程相关蛋白1基因PnPR1-3在提高烟草对棒弯孢(Curvularia clavata)抗性中的应用。
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| CN116555286A (zh) * | 2023-04-28 | 2023-08-08 | 昆明理工大学 | 三七富含脯氨酸蛋白基因PnPRPL1及其应用 |
| CN116555286B (zh) * | 2023-04-28 | 2024-05-14 | 昆明理工大学 | 三七富含脯氨酸蛋白基因PnPRPL1及其应用 |
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