CN103194466B - 一种岷江百合谷胱甘肽S-转移酶基因LrGSTU5及其应用 - Google Patents
一种岷江百合谷胱甘肽S-转移酶基因LrGSTU5及其应用 Download PDFInfo
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- CN103194466B CN103194466B CN201310144011.2A CN201310144011A CN103194466B CN 103194466 B CN103194466 B CN 103194466B CN 201310144011 A CN201310144011 A CN 201310144011A CN 103194466 B CN103194466 B CN 103194466B
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Images
Abstract
本发明公开了一种谷胱甘肽S-转移酶基因LrGSTU5,该基因核苷酸序列如SEQ ID NO:1所示,编码谷胱甘肽S-转移酶,本发明采用功能基因组学相关技术证明岷江百合LrGSTU5基因具有提高植物抗真菌的功能,将本发明所述的岷江百合谷胱甘肽S-转移酶LrGSTU5构建到植物表达载体上并转入烟草中使其过量表达,转基因烟草具有很强的体外抗真菌活性,表达LrGSTU5的转基因烟草蛋白对葡萄座腔菌、尖孢镰刀菌、交链孢等多种病原真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领域,尤其是一种岷江百合的具有抗真菌活性的谷胱甘肽S-转移酶基因LrGSTU5及应用。
技术背景
随着人口的逐渐增长,粮食短缺问题日益加剧,因此增加粮食产量是一个迫切需要解决的问题。农作物病害是我国的主要农业灾害之一,也是影响粮食产量的重要因素之一。在农业生产中,植物病害越来越严重,尤其是真菌病害,其引起的病害占植物总病害的80%以上。真菌性病害的种类繁多,引起的病害症状也千变万化,可以出现在植物的任何部位。传统的病害防治主要是依靠化学农药和培育抗性品种,虽然也获得了一定成效,但是农药大量和长期使用已经给人畜健康及生态平衡造成不利影响,同时常规育种因周期长、费时费力,有利变异少等缺点,使得它们不能从根本上解决植物病害难题。随着分子生物学和生物技术的快速发展,可以通过转基因技术在短期内培育出具有优良性状的植物新品种和新材料。获得具有抗真菌活性的基因,并通过转基因技术培育具有抗病性的作物,是提高植物抗病性的新方法。
病原体入侵植物后,激活植物产生多种防御反应。这些防御反应包括细胞新陈代谢的改变、细胞壁功能增强、氧爆发、诱导病程相关蛋白以及产生抗菌化合物,从而抵御病原菌的入侵。氧爆发是植物病原体感染的最早的反应之一,随后产生超敏反应(hypersensitivity response,HR)。氧爆发后,植物体内的活性氧(reactive oxygen species,ROS)类物质的生成量明显增加,活性氧可以损伤蛋白质、膜脂以及其它细胞组分,从而对植株造成氧化损伤。为了防止活性氧的损伤,植物通过超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽S-转移酶(glutathione S-transferase,GSTs)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)和谷胱甘肽还原酶(glutathione reduetase,GR)等解毒酶类降低机体内过量产生的ROS和氧自由基的水平,维持氧化还原状态的平衡,保护机体免受外界刺激的损伤,从而缓解胁迫效应(Dixon DP, Lapthorn A, Edwards R. Plant glutathione transferases. Genome Biol. 2002, 3004: 1-10.)。
GSTs是广泛存在植物体内的一类由多基因家族编码的多功能蛋白,由2个分子量为22-27 kDa的亚基组成的可溶性蛋白,主要以同源二聚体的形式存在。GSTs能催化还原性谷胱甘肽(glutathione,GSH)上的硫原子与亲电化合物结合,生成的谷胱甘肽共轭反应产物水溶性增加,易于被机体清除。从而降低ROS等细胞内有毒物质的水平,维持了胞内氧化还原状态的平衡。另外,GSTs还具有谷胱甘肽过氧化物酶活性,利用GSH降低脂肪酸和核酸中的过氧化氢,形成一元醇,从而提高植物的抗病性(Dixon DP, Cole DJ, Edwards R. Characterisation of a zeta class glutathione transferase from Arabidopsis thaliana with a putative role in tyrosine catabolism. Archives of Biochemistry and Biophysics. 2000, 384: 407-412.)。
GSTs在植物抵抗真菌胁迫中起重要作用。将油菜菌核病菌(Sclerotinia sclerotiorum),烟草野火病菌(Pseudomonas syringae pv. Tabaci),水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae)接种NbGST2基因沉默后的烟草叶片,分析NbGST2基因在抗病调控中的作用(黎飞. 植物GST、DCL基因和泛素-蛋白酶体途径对植物抗性的调控功能. 浙江大学硕士学位论文. 2010.)。结果表明NbGST2被沉默后的叶片更容易被S. sclerotiorum侵染,发病更早更重,可见NbGST2在植物抗真菌侵染中有正调控的作用。从感染白粉病菌(Erysiphe graminis)的小麦中克隆得到GstA1,并转入小麦中高效表达,GstA1转基因小麦抗E. graminis能力有所提高(Mauch F, Dudler R. Differential induction of distinct glutathione S-transferases of wheat by xenobiotics and by pathogen attack. Plant Physiology. 1993, 102: 1193-1201.)。将荧光素酶基因置于GSTF8的启动子之后并转入拟南芥(Arabidopsis thaliana)中表达,用不同的纹枯病菌(Rhizoctonia solani) 菌株ZG1-1、ZG3和ZG5的孢子悬液浸染上述转基因拟南芥,ZG1-1诱导的GSTF8启动子活性在大多数幼苗中明显增加,同时受ZG1-1、ZG3和ZG5浸染的转基因拟南芥幼苗中水杨酸含量明显降低,这表明GSTF8启动子可能会参与水杨酸介导的信号传导,降低水杨酸反应,有助于拟南芥抗R. solani的反应(Rafael PT, Rhonda CF, Chen W, Karam BS. Early induction of the ArabidopsisGSTF8 promoter by specific strains of the fungal pathogen Rhizoctonia solani. Molecular Plant-Microbe Interactions. 2004, 17: 70-80.)。
从感染炭疽病菌(Colletotrichum destructivum)的烟草cDNA文库中获得一个GST基因NbGSTU1,将该基因克隆到诱导基因沉默的PVP病毒载体上,通过根癌农杆菌(Agrobacterium tumefaciens)介导将基因沉默载体转入烟草中。发现NbGSTU1沉默的烟草植株对C. destructivum的敏感性显著增加,叶片中C. destructivum的生物量明显上升,同时RT-PCR(reverse transcription-polymerase chain reaction)结果表明NbGSTU1基因的表达明显降低,NbGSTU1沉默的烟草植株发病率在67%以上。这说明GST基因在烟草抗C. destructivum感染中具有重要的作用(Dean JD, Goodwin PH, Hsiang T. Induction of glutathione S-transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C. orbiculare and involvement of one in resistance. Journal of Experimental Botany. 2005, 56: 1525-1533.)。烟草的GST基因被沉默后能促进SOD、HSR203、MAPK、β-1,3-glucanase等病程相关蛋白基因的表达,反而抑制了烟草黑胫病菌(Phytophthora parasitica)的浸染,说明GST在抗病反应中也具有负调控作用(Hernández I, Chacón O, Rodriguez R, Portieles R, LopeznY, Pujol M, Hiadlgo OB. Black shank resistant tobacco by silencing of glutathione S-transferase. Biochemical and Biophysical Research Communications. 2009, 387: 300-304.)。
发明内容
本发明的目的是提供一种岷江百合谷胱甘肽S-转移酶基因LrGSTU5,该基因是从岷江百合(Lilium regale Wilson)中克隆获得有抗真菌活性的谷胱甘肽硫转移酶基因。
本发明的LrGSTU5基因来自百合野生种岷江百合,岷江百合又名王百合,是我国特有种,仅分布于川西岷江流域海拔800~2700m的河谷到山腰的岩石缝中,对真菌、病毒等具有极强的抗性,是珍稀的百合抗病育种遗传资源。
本发明中所述岷江百合谷胱甘肽S-转移酶基因LrGSTU5核苷酸序列如SEQ ID NO:1所示,LrGSTU5基因全长cDNA为924bp,具有669bp的开放阅读框(ORF)、37bp的5’非翻译区和218bp的3’非翻译区,编码如SEQ ID NO:2所示氨基酸序列的含有222个氨基酸的蛋白质。
本发明中所述谷胱甘肽S-转移酶基因LrGSTU5编码区是序列表SEQ ID NO:1中第38-706位所示的核苷酸序列或是编码SEQ ID NO:2所示氨基酸序列蛋白质的其他DNA序列。
本发明分离克隆岷江百合中携带的一个抗真菌相关基因的完整cDNA片段,利用农杆菌介导使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以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LrGSTU5。
植物受病原真菌胁迫时,植物体内的活性氧大量增加,GSTs催化还原性GSH与亲电化合物结合,生成的谷胱甘肽共轭反应产物水溶性增加,易于被机体清除,从而降低ROS等细胞内有毒物质的水平,维持了胞内氧化还原状态的平衡。此外GSTs还具有谷胱甘肽过氧化物酶活性,因而在植物抗病反应中起重要的作用。
本发明涉及分离LrGSTU5的全长cDNA片段并鉴定其功能,具有该基因片段的植物在一定程度上具有抵抗特定真菌浸染的表型。其中所述DNA片段如SEQ ID NO:1所示。对该基因进行序列分析,表明LrGSTU5全长cDNA为924bp,具有669bp的开放阅读框(Open reading frame,ORF)、37bp的5’非翻译区(untranslated region,UTR)和218bp的3’UTR,编码含有222个氨基酸的蛋白质。LrGSTU5编码蛋白具有GSTs家族的保守结构域,与来自洋葱(Allium cepa)、油棕(Elaeis guineensis)和毛果杨(Populus trichocarpa)以及其他植物的GSTs蛋白高度相似,同时聚类分析将LrGSTU5归于植物tau类GSTs (GSTU),上述分析结果表明LrGSTU5属于岷江百合中的GST基因。超量表达如SEQ ID NO:2所示氨基酸序列蛋白质可以增强烟草和其他植物对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌等多种真菌的抗性。
本发明另一目的是将岷江百合谷胱甘肽S-转移酶基因LrGSTU5应用在提高烟草对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌抗性中,具体操作如下:
(1)基因的获得:采用扩增LrGSTU5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RT-PCR扩增出LrGSTU5的全长编码区,然后将其连接到pMD-18T载体上,经测序验证获得具有目的基因的克隆。
(2)植物表达载体构建与遗传转化:用限制性内切酶BamHⅠ和PstⅠ酶切pMD-18T-LrGSTU5质粒,通过胶回收获得目的基因片段,用同样的内切酶消化植物表达载体pCAMBIA2300s,胶回收获得载体大片段;将目的基因片段与pCAMBIA2300s载体片段连接,构建植物超表达载体pCAMBIA2300s-LrGSTU5;通过液氮冻融法将pCAMBIA2300s-LrGSTU5质粒导入农杆菌菌株LBA4404中;利用农杆菌介导的遗传转化法,将LrGSTU5导入烟草等植物中表达,通过抗生素筛选、基因组DNA PCR和RT-PCR筛选阳性转基因植株。
(3)转基因植株抗真菌活性分析:提取生长健壮的转基因植株和野生型植株(非转基因对照)叶片的蛋白,分析转基因植物蛋白抑制真菌生长的活性,最后筛选出对真菌抗性明显增强的转基因植株。
本发明为增强植物对真菌病害的抗性提供了一种新方法,通过基因工程手段培育抗病植物能克服传统育种的不足,不仅育种周期短、操作简单,而且能产生高抗性植物。将来自岷江百合的LrGSTU5基因导入植物中表达,能产生具有抗真菌特性的新材料和新品种,将该基因导入烟草中,转基因烟草具有很强的体外抗真菌活性;表达LrGSTU5的转基因烟草对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌的生长具有明显的抑制作用。利用基因工程技术防治病害具有显著的优势和不可代替的重要性;它可以为作物、花卉等大规模生产提供方便,大量减少农药的使用,为农业生产节约成本且提高管理水平,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LrGSTU5转基因烟草基因组DNA的PCR检测结果,其中:Marker是DL2000 DNA Marker (大连宝生物),由2,000bp、1,000bp、750bp、500bp、250bp以及100bp六条DNA片段组成;正对照是以质粒pMD-18T-LrGSU5U为模板的PCR产物;WT是以非转基因烟草(即野生型烟草)总DNA为模板的PCR产物;其余泳道为转基因烟草单株;
图2是本发明中部分阳性转基因烟草中LrGSTU5转录水平的表达分析结果,其中: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;正对照是质粒pMD-18T-LrGSTU5为模板的PCR产物;其余泳道为不同的阳性LrGSTU5转基因烟草单株;
图3是本发明中LrGSTU5转基因烟草体外抗真菌活性分析结果,其中:图a、b、c、d中的真菌分别是:葡萄座腔菌、尖孢镰刀菌、拟茎点霉属真菌、交链孢菌;WT为野生型烟草的总蛋白,CK为空白对照,即蛋白提取缓冲液。
具体实施方式
下面通过实施例对本发明作进一步详细说明,但本发明的内容并不局限于此,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:LrGSTU5全长基因的克隆以及序列分析
用尖孢镰刀菌接种岷江百合,用接种24 h后的根提取总RNA,用液氮将处理过的岷江百合的根研磨成粉末以后,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5 μg Total RNA,依次加入50 ng oligo(dT) 15、DEPC水至反应体积为12.5 μL;混匀后,70℃加热变性5min后迅速在冰上冷却5min,然后依次加入4 μL 5×First-stand buffer、0.5 μL RNasin (200U)、2 μL dNTP (2.5mM each)、1 μL 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LrGSTU5,所用引物序列分别为5’- GCTTCTCTCAATAATGGCAGAGGAG- 3’以及5’- GGTTCTTTTCTCCGTGGCTCACT- 3’,采用AdvantageTM 2 PCR Enzyme (Clontech)扩增出目的基因。PCR反应条件:94℃ 2min;94℃ 30s,61℃ 30s,72℃ 1min,30cycles;72℃ 5 min。反应体系(20 μL)为2 μL 第一链cDNA、2 μL 10×Buffer、0.5 μL dNTP (10mM each)、0.3 μL 正向引物(10μM)、0.3 μL 反向引物(10μM)、0.25 μL AdvantageTM 2 PCR Enzyme、14.65 μL PCR-Grade Water,PCR结束后,取5 μL用于琼脂糖凝胶电泳,检测扩增产物的特异性以及大小。
由于PCR产物只有一条DNA带,故直接对PCR产物进行TA克隆,使用的试剂盒为pMD18-T vector kit (大连宝生物),反应体系和操作过程为:取1.5 μL PCR产物,依次加入1 μL pMD-18T vector (50ng/μL) 和2.5 μL 2×Ligation solution I,混匀置于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。用含有X-Gal、IPTG、氨苄青霉素(ampicillin,Amp)的LB固体培养基筛选阳性克隆。挑选若干个白色菌落,摇菌后用扩增LrGSU5T的特异引物鉴定出多克隆位点插入LrGSTU5的克隆。将鉴定的克隆进行测序,最终获得的LrGSTU5全长cDNA为924bp,通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html)分析发现其包含一个669bp的ORF(见序列表)。LrGSTU5编码一个含222个氨基酸的蛋白质,LrGSTU5的分子量约为25.5KDa,等电点为5.77。SignalP 3.0分析结果显示LrGSTU5没有信号肽的存在,而且未发现线粒体、过氧化酶体、溶酶体和细胞核等亚细胞定位序列,可见LrGSTU5属于非分泌蛋白。
实施例2:植物表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取插入LrGSTU5的大肠杆菌质粒pMD-18T-LrGSTU5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和浓度高低。用BamHI (TaKaRa)和PstI (TaKaRa)分别对质粒pMD-18T-LrGSTU5和pCAMBIA2300S进行双酶切(100 μL体系),反应体系和操作过程为:取20 μL pMD-18T-LrGSTU5或pCAMBIA2300S质粒、依次加入10 μL 10×H buffer、5 μLBamHI、5 μL PstI、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LrGSTU5片段和pCAMBIA2300S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LrGSTU5DNA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 LrGSTU5DNA片段依次加入2 μL pCAMBIA2300S载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μL ddH2O,混匀后短时离心,置于16℃金属浴过夜反应。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LrGSTU5的特异引物进行PCR,挑选出LrGSTU5与pCAMBIA2300S成功连接的克隆,所检测的菌株若为阳性,加入20%甘油混匀后置于-80℃保存备用。
用试剂盒提取并纯化上述大肠杆菌中的pCAMBIA2300S-LrGSTU5质粒。制备农杆菌LBA4404菌株的感受态细胞并分装于1.5 mL离心管中,每管200 μL,液氮速冻后置于-80℃保存备用。采用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-LrGSTU5转入所制备的农杆菌LBA4404感受态细胞中。操作步骤为:取200 ng pCAMBIA2300S-LrGSTU5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,接着转入液氮中冷冻1 min,然后迅速置于37℃水浴5min,之后立即冰浴2 min,加入800 μL LB液体培养基,28℃振荡培养4h。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,用扩增LrGSTU5的特异引物进行PCR,检测pCAMBIA2300S-LrGSTU5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表面消毒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-LrGSTU5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48h,将LB固体培养基上的农杆菌刮下适量接种于MGL液体培养基中,附加20 mg/L的乙酰丁香酮,28℃振荡培养2-3h以活化农杆菌。
取烟草无菌苗嫩叶切成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置于室温培养2d,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,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+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℃,16 h/d光照,8 h/d黑暗),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50mg/L Km的MS培养基上使其生根,最后选择生根较好的再生苗进行分子水平的检测。
采用CTAB法提取转基因烟草植株叶片的基因组DNA,取1 μL基因组DNA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LrGSTU5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,部分转基因烟草植株的扩增结果如图1所示,LrGSTU5转化烟草共筛选到62株阳性转基因植株。
实施例4:LrGSTU5表达分析以及转基因植株抗真菌活性分析
随机挑选30株阳性转基因单株和一株非转基因烟草(野生型),分别取嫩叶并提取总RNA,逆转录生成第一链cDNA,并以此为模板扩增LrGSTU5的特异引物,根据RT-PCR结果分析各转基因单株中LrGSTU5转录水平的表达,总RNA提取及RT-PCR的方法和步骤与实施例1中相同,PCR结束后,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22株转基因单株中LrGSTU5在转录水平有表达,这些植株分别编号为1~22。
将几种病原真菌接种于PDA固体培养基(200 g/L 马铃薯,15 g/L 琼脂,20 g/L 葡萄糖)上,28℃倒置培养,待菌落生长至直径约为2cm时添加野生型烟草及转基因烟草叶片总蛋白,分析转基因植株体外抗真菌活性。供试真菌有5种:拟茎点霉属(Phomopsis sp.)真菌、交链孢菌(Alternaria sp.)、灰葡萄孢(Botrytis cinerea)、葡萄座腔菌(Botrosphaeria dothidea)和尖孢镰刀菌(Fusarium oxysporum)。
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在无菌条件下操作,首先取1g转基因单株(1、3、4、8、18)或野生型叶片放入研钵中,加入500 μL蛋白提取液(1 M NaCl,0.1 M乙酸钠,1% PVP,pH5),充分研磨。转入1.5 mL离心管中,充分混匀后4℃静止过夜。4℃离心30min (12,000 g/min),将上清转入新的1.5 mL离心管中,并取适量用紫外分光光度仪测定蛋白浓度,将转基因和野生型植株的总蛋白浓度调整至0.2 μg/μL,然后取20 μl滴于各真菌培养基的滤纸上,在每个平板的滤纸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℃培养几天后观察各处理抑菌真菌生长的情况,并据此评价LrGSTU5转基因烟草的体外抗真菌活性,结果如图3所示,LrGSTU5转基因烟草蛋白对葡萄座腔菌、尖孢镰刀菌、拟茎点霉属真菌和交链孢有明显的抑制作用。
序列表(SEQ ID)
<110> 昆明理工大学
<120> 一种岷江百合谷胱甘肽S-转移酶基因LrGSTU5及其应用
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Claims (4)
1.一种岷江百合谷胱甘肽S-转移酶基因LrGSTU5,其特征在于:其核苷酸序列如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.根据权利要求1所述岷江百合谷胱甘肽S-转移酶基因LrGSTU5,其特征在于:谷胱甘肽S-转移酶基因LrGSTU5编码区是序列表SEQ ID NO:1中第38-706位所示的核苷酸序列。
3.根据权利要求1或2所述的岷江百合谷胱甘肽S-转移酶基因LrGSTU5在提高烟草对葡萄座腔菌、拟茎点霉属真菌、尖孢镰刀菌、交链孢菌抗性中的应用。
4.根据权利要求3所述的岷江百合谷胱甘肽S-转移酶基因LrGSTU5的应用,其特征在于提高烟草的抗真菌能力的具体操作如下:
(1) 将上述谷胱甘肽S-转移酶基因与植物表达载体pCAMBIA2300s连接,构建植物超表达载体;
(2) 将上述构建的重组载体通过根癌农杆菌介导转入目标植物中;
(3) 以重组载体T-DNA上具有的抗生素标记筛选转化子,并通过聚合酶链式反应获得真正的转基因植株,接种特定病原真菌并分析转基因植物蛋白对真菌生长的抑制作用,最后筛选出对真菌抗性明显增强的转基因植株。
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CN1169160A (zh) * | 1995-01-23 | 1997-12-31 | 拜尔公司 | 编码谷胱甘肽s-转移酶的脱氧核糖核酸及其应用 |
CN1687422A (zh) * | 2005-04-27 | 2005-10-26 | 东北林业大学 | 长春花谷胱甘肽转移酶的基因序列 |
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CN1169160A (zh) * | 1995-01-23 | 1997-12-31 | 拜尔公司 | 编码谷胱甘肽s-转移酶的脱氧核糖核酸及其应用 |
CN1687422A (zh) * | 2005-04-27 | 2005-10-26 | 东北林业大学 | 长春花谷胱甘肽转移酶的基因序列 |
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