CN113603757A - 一种岷江百合Dirigent类似蛋白基因LrDIR1及应用 - Google Patents
一种岷江百合Dirigent类似蛋白基因LrDIR1及应用 Download PDFInfo
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- CN113603757A CN113603757A CN202110957491.9A CN202110957491A CN113603757A CN 113603757 A CN113603757 A CN 113603757A CN 202110957491 A CN202110957491 A CN 202110957491A CN 113603757 A CN113603757 A CN 113603757A
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Abstract
本发明公开了一种岷江百合Dirigent类似蛋白基因LrDIR1，其核苷酸序列如SEQ ID NO:1所述，编码如SEQ ID NO:2所示氨基酸序列的蛋白质，本发明通过功能基因组学相关技术研究证实LrDIR1基因具有提高植物抗真菌的功能，将本发明抗真菌的LrDIR1基因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抗真菌侵染能力，实验结果显示过表达LrDIR1的转基因烟草对尖孢镰刀菌、草茎点霉的侵染具有抗性。
Description
技术领域
本发明涉及分子生物学以及基因工程相关技术领域，特别是一种具有抗真菌侵染能力的岷江百合Dirigent类似蛋白基因LrDIR1及应用。
背景技术
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，不同程度地限制其生长发育，从而影响最终植物经济性状和产量性状的形成。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***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。例如，植物生成木质素加固细胞壁，合成植物抗毒素(如有毒的酚类化合物)，并积累各种病程相关蛋白（pathogenesisrelated protein）。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。
Dirigent蛋白首先是由Davin等在连翘（Forsythia intermedia）中发现，它可以捕获木质素单体的自由基，并指导其形成多聚的木质素和寡聚的木酚素，木酚素进一步参与植物的防御反应，而木质素则可以强化植物细胞壁的木质化程度，增加细胞壁的机械强度，进而抑制植物病原菌的侵染（Davin L B, Wang H B, Crowell A L, et al.Stereoselective bimolecular phenoxy radical coupling by an auxiliary(dirigent) protein without an active center. Science, 1997, 275: 362-366）。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，存在木质部中，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。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，因此除了DIR-a之外的亚家族也被称为DIR-like（DIR类似）基因（Ralph S G , Jancsik S , Bohlmann J. Dirigent proteins inconifer defense II: Extended gene discovery, phylogeny, and constitutive andstress-induced gene expression in spruce (Picea spp.). Phytochemistry, 2007,68(14): 1975-1991）。
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（Weidenbach D , Esch L , Mller C , et al. Polarized defenseagainst fungal pathogens is mediated by the jacalin-related lectin domain ofmodular Poaceae - specific proteins. Molecular Plant, 2016, 9(4): 514-527）。小麦TaDIR13在烟草中的过表达上调了对丁香假单胞菌（Pseudomonas syringae）和烟草疫霉菌（Phytophthora nicotianae）的抗性（Qinghu Ma, Yunchao Liu. TaDIR13, a dirigentprotein from wheat, promotes lignan biosynthesis and enhances pathogenresistance. Plant Molecular Biology Reporter, 2015, 33(1): 143-152）。当芜菁（Brassica rapa）接种尖孢镰刀菌（Fusarium oxysporum）后，BrDIR-like-2的表达量显著上调，为未接种的5倍（Arasan S T , Park J I , Ahmed N U , et al.Characterization and expression analysis of dirigent family genes related tostresses in Brassica. Plant Physiol Biochem, 2013, 67: 144-153）。在抗病橄榄（Olea europaea）品种中，DIR基因的转录水平在黄萎病菌（Verticillium dahliae）侵染的早期阶段大幅增加（Leyva-Pérez María de la O, Jiménez-Ruiz Jaime, Gómez-LamaCabanás Carmen, et al. Tolerance of olive (Olea europaea) cv Frantoio toVerticilliumdahliae relies on both basal and pathogen-induced differentialtranscriptomic responses. The New phytologist, 2018, 217217: 671-686）。
百合是百合科（Liliaceae）百合属（Lilium）植物的总称，属多年生球根类花卉。在种球繁殖及鲜切花生产过程中，百合易受到真菌、病毒、细菌等多种病原菌的危害。目前发现的百合病害达几十种之多，其中由镰刀属（Fusarium spp.）真菌引起的枯萎病（又称为基腐病、茎腐病）是百合生产中危害最严重的病害。镰刀菌侵染百合种球后引起基盘坏死、鳞片腐烂脱落，造成种球质量下降；植株感染镰刀菌后叶片变黄、萎蔫下垂，植株提早枯萎死亡，严重影响百合切花的产量和品质。其中尖孢镰刀菌（F. oxysporum）致病性最强、分离频率最高，是百合枯萎病的主要致病菌。岷江百合（L. regale Wilson）为我国特有种，仅分布于岷江流域海拔800-2700m的河谷到山腰的岩石缝中，具有强的抗枯萎病性，是现代百合育种的重要种质资源。Dirigent参与应答多种生物胁迫，是植物防御***的重要组成部分，因此对岷江百合中Dirigent基因的发掘以及功能分析具有重要的研究及其应用价值。
发明内容
本发明目的是提供一种岷江百合Dirigent类似蛋白基因LrDIR1及其在提高烟草对尖孢镰刀菌（F. oxysporum）、草茎点霉（Phoma herbarum）抗性中的应用。
本发明从岷江百合中克隆获得的具有抗真菌活性的dirigent类似蛋白基因LrDIR1的全长基因，LrDIR1核苷酸序列如SEQ ID NO:1所示，该基因cDNA全长序列为867bp，包含一个504bp的开放阅读框、146 bp的5’非翻译区、217 bp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LrDIR1基因的编码区是序列表SEQ ID NO:1中第147-650位所示的核苷酸序列。
本发明分离克隆岷江百合的一个抗真菌相关基因的完整cDNA片段，利用根癌农杆菌(Agrobacterium tumefaciens)介导将目的基因转入受体植物中并过量表达，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LrDIR1。
上述LrDIR1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LrDIR1的特异引物，从接种尖孢镰刀菌后的岷江百合根中提取总RNA，通过逆转录-聚合酶链式反应（reverse transcription-polymerase chainreaction，RT-PCR）扩增出LrDIR1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-LrDIR1载体，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LrDIR1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，分析转基因植物叶片总蛋白体外抑制病原真菌生长的能力，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。本发明来自岷江百合的LrDIR1基因能增强植物对真菌的抗性，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农作物、药材、园艺植物等提供方便，大量减少化学农药的使用，还可以为农业生产节约成本、减小环境污染，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LrDIR1转基因烟草基因组DNA的PCR检测结果图，图中：Marker为DL2000 DNA Marker （大连宝生物）；阳性对照为质粒pGEM-T-LrDIR1为模板的PCR结产物；WT为非转基因烟草（野生型）总DNA为模板PCR的产物；
图2是本发明阳性LrDIR1转基因烟草中LrDIR1转录水平的表达分析结果图；图中：Marker是DL2000 DNA Marker（大连宝生物）；WT是非转基因烟草总RNA逆转录cDNA为模板的PCR产物；阳性对照是质粒pGEM-T-LrDIR1为模板的PCR产物；
图3是本发明LrDIR1转基因烟草体外抗真菌活性的分析结果图；图a、b中所示真菌分别是尖孢镰刀菌、草茎点霉；WT为野生型烟草总蛋白，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作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1：LrDIR1基因克隆以及序列分析
用尖孢镰刀菌接种岷江百合，用接种24 h后的根提取总RNA，用液氮将接种后的岷江百合根研磨成粉末，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5μg TotalRNA，依次加入50ng oligo（dT）、2μL dNTP（2.5mM each）、DEPC水加至反应体积为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、0.5μL RNasin（200U）、1μL M-MLV（200U），混匀并短时离心，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LrDIR1，所用上下游引物序列分别为5’ CGCTAGCTGCAAAAAAGTTGA 3’及5’ GAAACGATGAATAACCCATGATTTT 3’。采用AdvantageTM 2PCR Enzyme（Clontech）扩增出目的基因；PCR反应条件：94℃ 5min；94℃ 30s，60℃ 30s，72℃ 1min，32个循环；72℃ 10min；反应体系（20μL）为0.5μL cDNA、2μL 10×Advantage 2PCR Buffer、0.4μL 50×dNTP Mix （10mM each）、0.4μL 正向引物（10μM）、0.4μL 反向引物（10μM）、0.4μL Advantage 2 PCR Polymerase Mix、15.9μL PCR-Grade water；PCR结束后，取5μL用于琼脂糖凝胶电泳，以检测扩增产物的特异性以及大小。
对PCR产物进行TA克隆，使用的试剂盒为pGEM-T Vector SystemⅠ（Promega,USA），反应体系和操作过程为：取1.5μL PCR产物，依次加入1μL pGEM-T Vector（50ng/μL）和2.5μL 2×Ligation solution I，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LrDIR1的特异引物鉴定出多克隆位点***LrDIR1的克隆，将所鉴定的克隆进行测序，最终获得的LrDIR1全长cDNA为867bp，通过NCBI ORFfinder （http://www.ncbi.nlm.nih.gov/gorf/gorf.html）分析发现其包含一个504bp的开放读码框（见序列表），LrDIR1编码一个含167个氨基酸的蛋白质，其分子量约为17.69KDa，等电点约为9.25。借助生物信息学软件SignalP5.0分析LrDIR1编码的蛋白序列，检测其是否具有N端信号肽；结果显示在LrDIR1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***LrDIR1的大肠杆菌质粒pGEM-T-LrDIR1以及植物表达载体pCAMBIA2300s的质粒，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XbaⅠ（TaKaRa）和EcoRⅠ（TaKaRa）分别对质粒pGEM-T-LrDIR1和pCAMBIA2300s进行双酶切（100μL体系），反应体系和操作过程为：分别取20μL pGEM-T-LrDIR1和pCAMBIA2300s质粒、依次加入10μL 10×K buffer、4μL XbaI、6μL EcoRI、60μL 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LrDIR1片段和pCAMBIA2300s载体大片段分别进行胶回收，整个过程使用SanPrep柱式DNA胶回收试剂盒（上海生工）；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LrDIR1 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 LrDIR1 DNA片段依次加入2μLpCAMBIA2300s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，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LrDIR1的特异引物进行PCR，挑选出LrDIR1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-LrDIR1质粒，随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300s-LrDIR1转入根癌农杆菌LBA4404感受态细胞中。操作步骤为：取2μg pCAMBIA2300s-LrDIR1质粒加入含有200μL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1min，然后迅速置于37℃水浴5min，之后立即冰浴2min，加入800μL 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 Km的LB固体培养基上，28℃下静止培养。挑选单菌落摇菌，再用扩增LrDIR1的特异性引物进行PCR，检测pCAMBIA2300s-LrDIR1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8min，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℃，16 h/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-LrDIR1质粒的农杆菌LBA4404菌种，接种于5mL含有50mg/L Km和20mg/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。
取烟草无菌苗叶片切成1cm2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15min，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+0.02mg/L 6-BA+2.1mg/L NAA+30g/L sucrose+6g/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+0.5mg/L 6-BA+0.1mg/L NAA+30g/L sucrose+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℃，16 h/d光照，8 h/d黑暗），待烟草长出芽后用含有50mg/L Km和200mg/L Cef的MS培养基继代培养，因烟草愈伤分化率较高，故需要对再生植株进行进一步筛选，将烟草再生苗移至含有50mg/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LrDIR1的特异引物进行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，LrDIR1转基因烟草共筛选到40株阳性转基因植株。
实施例4：转基因烟草中LrDIR1的表达分析以及转基因植株抗真菌侵染的功能分析
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LrDIR1的特异引物进行PCR，根据PCR结果分析各转基因单株中LrDIR1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8μL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35个转基因单株中LrDIR1在转录水平大量表达，这些单株的编号为1～35。
将实验室保存的几种病原真菌接种于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时添加植物蛋白，分析转基因植株体外抗真菌活性。为了防止其他杂菌污染提取的蛋白，整个植物蛋白提取的过程均是无菌操作。首先取1 g转基因烟草单株（编号分别为L-4、L-7、L-8) 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，pH6.0)，充分研磨。转入1.5mL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的生长情况，并据此来评价LrDIR1转基因烟草的体外抗真菌活性，结果如图3所示，LrDIR1转基因烟草蛋白对尖孢镰刀菌和草茎点霉的生长具有明显的抑制作用。
序列表
<110> 昆明理工大学
<120> 一种岷江百合Dirigent类似蛋白基因LrDIR1及应用
<160> 4
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<212> DNA
<213> 岷江百合(Lilium regale Wilson)
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cgctagctgc aaaaaagttg accaagacag cttcatccta cctgccggtt catacaagag 60
aataatcgtt tgtatcctct atatatatct cacaacataa cgaacctctc tcatctcatc 120
tctcaagctt cccactactt gttccaatgg ccttcaattt ccccagcctc ctcctactct 180
tattagccgc caccacctcc tttgcagtac tagctacagc caagaagacc cagttgcagt 240
tctacgtaca cgtcataaac agcggcccca atgccaccac cgccgtagta gcaggcctca 300
acaagacatc ttccgcattc ggaaacatcg acgtgtacga caacatactg cgagtaggga 360
cagatccaag ctcagcaatc atcggaagga tccagggaat cgatgcccag gcttcactgg 420
gttcgccagc agtgacagcg gtgtacaatt ttgtgttcac aggtggtgcg tacaacggga 480
gcactcttgg catgatgggc taccatatca tgagccagcc gacatgggag cagagcgtga 540
ttgcagggac agggcagttc cgtctcgctc gaggatatgc aatagcgcag ctcgtcagct 600
acacccccgc ctacatagtc atcaacttca acgcttatgt tcttcattag atcttcatta 660
cagtataatg tttctgtttc tgttatgtat gttaaaacat caataaatct gtgtgccctt 720
tcatcaataa aactgttaca gaaaattcac tttgccgggc caaaactatg tacttaacac 780
caactttgat gacattactc gataaactca tttgagagaa tgtaaacaca gattgacaat 840
ataaaatcat gggttattca tcgtttc 867
<210> 2
<211> 167
<212> PRT
<213> 岷江百合(Lilium regale Wilson)
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Met Ala Phe Asn Phe Pro Ser Leu Leu Leu Leu Leu Leu Ala Ala Thr
1 5 10 15
Thr Ser Phe Ala Val Leu Ala Thr Ala Lys Lys Thr Gln Leu Gln Phe
20 25 30
Tyr Val His Val Ile Asn Ser Gly Pro Asn Ala Thr Thr Ala Val Val
35 40 45
Ala Gly Leu Asn Lys Thr Ser Ser Ala Phe Gly Asn Ile Asp Val Tyr
50 55 60
Asp Asn Ile Leu Arg Val Gly Thr Asp Pro Ser Ser Ala Ile Ile Gly
65 70 75 80
Arg Ile Gln Gly Ile Asp Ala Gln Ala Ser Leu Gly Ser Pro Ala Val
85 90 95
Thr Ala Val Tyr Asn Phe Val Phe Thr Gly Gly Ala Tyr Asn Gly Ser
100 105 110
Thr Leu Gly Met Met Gly Tyr His Ile Met Ser Gln Pro Thr Trp Glu
115 120 125
Gln Ser Val Ile Ala Gly Thr Gly Gln Phe Arg Leu Ala Arg Gly Tyr
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Ala Ile Ala Gln Leu Val Ser Tyr Thr Pro Ala Tyr Ile Val Ile Asn
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Phe Asn Ala Tyr Val Leu His
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gaaacgatga ataacccatg atttt 25
Claims (2)
1.一种岷江百合Dirigent类似蛋白基因LrDIR1，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的岷江百合Dirigent类似蛋白基因LrDIR1在提高烟草对尖孢镰刀菌（Fusarium oxysporum）、草茎点霉（Phoma herbarum）抗性中的应用。
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