CN116555286A - 三七富含脯氨酸蛋白基因PnPRPL1及其应用 - Google Patents
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Abstract
本发明公开了一种三七富含脯氨酸蛋白基因PnPRPL1及应用，PnPRPL1基因的核苷酸序列如SEQ ID NO：1所示，编码富含脯氨酸蛋白，本发明通过功能基因组学相关技术研究证实PnPRPL1基因具有提高植物抗真菌侵染的功能，将本发明抗真菌PnPRPL1基因构建到植物表达载体上并转入烟草中过量表达，PnPRPL1转基因烟草对弯孢霉的抗性明显提高。
Description
技术领域
本发明属于分子生物学以及基因工程相关技术领域，特别是具有抗真菌效果的三七富含脯氨酸蛋白基因PnPRPL1及应用。
背景技术
病原菌是引起植物产量和品质损失的重要影响因素，由病原菌引起的植物病害，可使作物减产高达50％(王玉芬.植物内生菌的研究进展.现代园艺,2019(11):23-24)；其中真菌病害是植物病害中数量最大的一类，约占病害总数的70％-80％(高峰,郭惠珊.植物与土传病原真菌的攻防战及干预.自然杂志,2020,42(06):499-504)；传统控制植物病害的方法主要是使用化学农药、改进栽培管理措施和培育抗性新品种，这些方法虽然取得了一定成效，但传统育种周期长，栽培措施效果差，农药易导致环境安全和食品安全等问题。随着生物技术的快速发展，利用基因工程的方法来培育抗病新品种不仅能克服上述防治方法的诸多弊端，还可最大限度地降低对土壤中有益微生物的伤害，实现农业的可持续发展。
植物细胞壁是植物抵御病原体侵染极其有效的物理屏障，细胞壁中结构蛋白的类型及其活性氧介导的交联与植物抗病性密切相关。细胞壁中的结构蛋白主要有5类：伸展蛋白(extensin)、富含羟脯氨酸糖蛋白(hydroxyproline-rich glycoproteins,HRGPs)、植物凝集素(solanaceous,SL)、阿拉伯半乳聚糖蛋白(arabinogalactan proteins,AGPs)、富含脯氨酸蛋白(proline-protein,PRP)和富含甘氨酸蛋白(glycine-rich proteins,GRPs)，这些蛋白对于细胞壁结构形成具有关键作用(JosèM,Puigdomènech P.Structure andexpression of genes coding for structural proteins of the plant cell wall.NewPhytologist,1993,125(2):259-282)。
PRPs是植物细胞壁结构蛋白的重要组成成分。大部分的PRPs基因中内含子较少或没有内含子，其氨基酸大小一般为122～761aa，具有重复的Pro-Pro-X-Y-Lys五肽序列，X、Y代表缬氨酸、酪氨酸、组氨酸或谷氨酸(Rajasheker G,Nagaraju M,Varghese RP,etal.Identification and analysis of proline-rich proteins and hybrid proline-rich proteins super family genes from Sorghum bicolor and their expressionpatterns to abiotic stress and zinc stimuli.Frontiers in Plant Science,2022,13:952732)。植物PRPs可根据结构域的差异及N端有无信号肽分为3类：第一类是具有2个结构域的PRPs，含有N端信号肽和富含Pro重复区域；第二类是具有3个结构域的PRPs，含有N端信号肽、富含Pro重复区域和C端富含半胱氨酸区；第三类是N端不具有信号肽、C端含有若干个类似的PPVXYK重复序列的PRPs(Zhang XL,Gong XQ,Li DY,et al.Genome-wideidentification of PRP genes in apple genome and the role of MdPRP6 inresponse to heat stress.International Journal Molecular Science,2021,22(11):5942)。
鹰嘴豆(Cicer arietinum)中CanPRP的表达具有发育阶段特性，在未成熟的上胚轴中几乎检测不到该基因的表达；随着上胚轴的生长发育，该基因的表达也逐渐增强；当上胚轴停止生长、细胞壁开始增厚时，该基因的表达达到高峰(FJ,Dopico B,LabradorE.A cDNA encoding a proline-rich protein from Cicer arietinum.Changes inexpression during development and abiotic stresses.Physiologia Plantarum,1998,102(4):582-590)。Kauss等在黄瓜(Cucumis sativus)中发现了一个PRP基因，系统获得性抗性(systemic acquired resistance,SAR)可诱导该PRP蛋白积累，PRR蛋白能够将原硅酸合成不溶性二氧化硅，从而加固植物细胞壁，阻止真菌的侵染(Kauss H,Seehaus K,Franke R,et al.Silica deposition by a strongly cationic proline-rich proteinfrom systemically resistant cucumber plants.Plant Journal,2003,33(1):87-95)。大豆(Glycine max)中的SbPRP作为一种防御基因，参与了抗大豆花叶病毒(soybeanmosaic virus,SMV)的防御反应(He CY,Wu XL,Yang DF,et al.Isolation andcharacterization of a new defense gene from soybean.Science In China.SeriesC,Life Science,2001,44,409–420)。吕高强等人通过RAPD、半定量RT-PCR以及qRT-PCR发现芝麻(Sesamum indicum)中SiPRP基因受青枯雷尔氏菌(Ralstonia solanacearum)诱导后在茎中的表达显著下降(吕高强,吴向阳,王心宇.芝麻中一个富含脯氨酸新基因的克隆与特征分析.作物学报,2015,41(12):1810-1818)。拟南芥(Arabidopsis thaliana)中PRP4、PRP11和PRP16响应线虫的侵染表达上调，而假单胞杆菌(Pseudomonas syringae)侵染可诱导PRP9和PRP10的表达(Fowler TJ,Bernhardt C,Tierney ML.Characterizationand expression of four proline-rich cell wall protein genes in Arabidopsisencoding two distinct subsets of multiple domain proteins.American Society ofPlant Biologists,1999,(4):1081-1092)。
三七[Panax notoginseng(Burk)F.H.Chen]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文山州。由于三七性喜温暖阴湿，对光敏感，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，尤其是根腐病、黑斑病等真菌病害，严重影响了三七原药材的产量和品质，迫切需要加强三七与根腐病等病害的分子互作机制研究，并且通过转录组测序，发掘和克隆响应病原菌入侵的防卫相关基因，为三七的抗病育种提供理论基础。
发明内容
本发明提供了一种从三七中克隆获得的富含脯氨酸蛋白基因PnPRPL1，PnPRPL1的核苷酸序列如SEQ ID NO:1所示，该基因全长为766bp，包含一个444bp的开放阅读框、编码45bp的5’非翻译区、277bp的3’非翻译区，编码SEQ ID NO:2所示氨基酸序列的蛋白质。
本发明所述三七富含脯氨酸蛋白基因PnPRPL1的编码区是序列表SEQ ID NO:1中第46-489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富含脯氨酸蛋白基因PnPRPL1应用在提高烟草对弯孢霉的抗性中。
上述三七富含脯氨酸蛋白基因PnPRPL1提高烟草抗真菌特性的具体操作如下：
(1)采用扩增PnPRPL1的特异引物，从接种茄腐镰刀菌(Fusarium solani)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PnPRPL1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EcoRⅠ和BamHⅠ酶切pGEM-T-PnPRPL1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PnPRPL1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，分析转基因植株对于病原真菌的抗性，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，不仅缩短了育种周期，而且操作简单，容易获得高抗材料。本发明中来自三七的PnPRPL1基因能增强植物对弯孢霉的抗性，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PnPRPL1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-PnPRPL1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PnPRPL1转基因烟草中PnPRPL1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-PnPRPL1为模板的PCR产物；
图3是本发明中PnPRPL1转基因烟草抗病性鉴定的结果图；图中是接种弯孢霉的PnPRPL1转基因烟草叶片；其中WT为野生型烟草叶片，2、10、11、15为PnPRPL1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：PnPRPL1全长cDNA克隆以及序列分析
用茄腐镰刀菌接种三七根，用接种后24h的根提取总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 Random primer，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 PCR NucleotideMix、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PnPRPL1，所用上下游引物序列分别为5’ATGGCCTATTCAAAGGCTTTC3’及5’TTAGTTACTACATTGGAAGCCAG3’。采用TAKARA Ex扩增出目的基因。PCR反应条件：95℃5min；95℃30s，59℃30s，72℃1min，30个循环；72℃5min。反应体系(50μL)为2μL cDNA、5μL 10×Ex Taq Buffer(含Mg2+20mM)、4μL dNTP Mix(2.5mMeach)、1μL上游引物(5μM)、1μL下游引物(5μM)、0.25μL TaKaRa Ex Taq(5U/μL)、36.75μLddH2O。PCR结束后，取50μL进行1％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、0.9μL T4 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PnPRPL1的特异引物检测多克隆位点插入PnPRPL1的克隆。将得到的阳性克隆进行测序，最终获得的PnPRPL1全长cDNA为766bp，通过NCBI ORF finder(http:// www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个444bp的开放读码框。PnPRPL1编码一个含147个氨基酸的蛋白质PnPRPL1，其分子量约为15.486KDa，等电点为8.53。借助生物信息学软件SignalP 4.1分析PnPRPL1编码的蛋白序列，检测其是否具有N端信号肽。结果显示在PnPRPL1的N端存在信号肽，因此推测该蛋白是分泌蛋白。
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PnPRPL1的大肠杆菌质粒pGEM-T-PnPRPL1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BamHI(TaKaRa)和EcoRI(TaKaRa)分别对质粒pGEM-T-PnPRPL1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-PnPRPL1和pCAMBIA2300S质粒、依次加入7.5μL 10×K buffer、2.5μL BamHI、2.5μL EcoRI、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PnPRPL1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T4 DNA Ligase(TaKaRa)，将回收的PnPRPL1 DNA片段和pCAMBIA2300S载体片段连接起来，反应体系(20μL)和操作过程为：取10μL PnPRPL1 DNA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，Kana)的固体培养基筛选阳性克隆。挑选单菌落含有50mg/L Kana的LB液体培养基震荡培养，以菌液为模板用扩增PnPRPL1的特异引物进行PCR，挑选出PnPRPL1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-PnPRPL1质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PnPRPL1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-PnPRPL1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PnPRPL1的特异性引物进行PCR反应，检测pCAMBIA2300S-PnPRPL1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)。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-PnPRPL1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℃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PnPRPL1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，PnPRPL1转基因烟草共筛选到42株阳性转基因植株。
实施例4：转基因烟草中PnPRPL1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PnPRP1的特异引物进行PCR，根据PCR结果分析各转基因植株中PnPRPL1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。
将实验室保存的弯孢霉接种于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7天。取温室中生长良好、大小均一且完全伸展的WT烟草和PnPRPL1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约相同位置形成大小一致的伤口，分别接种大小相等的弯孢霉菌丝块。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，接种弯孢霉后，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，PnPRPL1转基因烟草对弯孢霉具有很明显的抗性。
Claims (2)
1.一种三七富含脯氨酸蛋白基因PnPRPL1，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的三七富含脯氨酸蛋白基因PnPRPL1在提高烟草对弯孢霉(Curvularia clavata)抗性中的应用。
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