CN104878019A - 漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用 - Google Patents
漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用 Download PDFInfo
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Abstract
本发明公开了漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用,JsGLP1基因的核苷酸序列如SEQ ID NO:1所示,编码类萌发素蛋白,本发明通过功能基因组学相关技术研究证实JsGLP1基因具有提高植物抗真菌侵染的功能,将本发明抗真菌JsGLP1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体外抗真菌活性;JsGLP1超表达的转基因烟草对核盘菌、串珠状赤霉菌、胶孢炭疽菌以及尖孢镰刀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相关技术研究领域,特别是具有抗真菌活性的漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用。
背景技术
植物在整个生长发育过程中会时刻受到外界环境信号的调控,遭遇各种逆境胁迫,从而产生各种病害症状,影响植物的生长发育,在众多的植物病害中,真菌病害是最重要的一类,其发病率极高,且大规模发生时导致农作物减产甚至死亡。传统控制真菌病害的方法主要是选育抗性新品种、采用合理的耕作制度和使用化学农药,这些方法虽然取得了一定成效,但均不能从根本上解决真菌病害问题。近年来,随着分子生物学和生物技术的快速发展,利用基因工程方法培育抗真菌病害的新品种取得了一定成效,有望从根本上解决真菌病害问题。
类萌发素蛋白(Germin-like proteins,GLPs)是植物中普遍存在的一类可溶性糖蛋白,与小麦(Triticum aestivum)萌发素(germin)序列高度相似,通过离子键结合以糖蛋白的形式存在于细胞外基质中,大多数GLPs为非常稳定的低聚物(Bernier F, Berna A. Germins and germin-like proteins: Plant do-all proteins. But what do they do exactly Plant Physiology and Biochemistry, 2001, 39: 545-554; Lane BG. Oxalate, germins, and higher-plant pathogens. IUBMB life, 2002, 539: 67-75.)。GLPs最初在小麦中发现(Dunwell JM, Gibbings JG, Mahmood T, et al. Germin and germin-like proteins: evolution, structure, and function. Critical Reviews in Plant Sciences, 2008, 27: 342-375.)。在结构上,GLPs和germin均含有一个“cupin”蛋白结构域,三维结构具有β-折叠桶状结构,共同属于一个功能多样化的超家族,即cupin超家族。目前根据功能的差异将植物的GLPs分为3个亚类:第1亚类为真萌发素(true germin),主要包括小麦、大麦的萌发素及一些其他谷类植物的GLPs,它们具有SOD和OXO的双重活性;第2亚类的GLPs主要是来自于除小麦和大麦之外的其他禾谷类、裸子植物和茄科植物等,这一亚类主要与植物耐氧化胁迫有直接关系,并与锰超氧化物歧化酶(MnSOD)极其相似;第3亚类的GLPs主要包括与生长素代谢相关的一些调节蛋白,它们与植物的生理节律和花期诱导功能有关 (Khuri S, Bakker FT, Dunwell JM. Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins. Molecular Biology and Evolution, 2001, 318: 593-605.)。
GLPs是一种多功能蛋白,主要以酶、受体和结构蛋白的形式参与多种生理生化过程。其中,酶主要包括超氧化物歧化酶(superoxide dismutase, SOD)、草酸盐氧化酶(oxalate oxidase, OXO)、ADP葡萄糖焦磷酸酶/磷酸二酯酶(ADP glucose pyrophosphatase/phosphodiesterase, AGPPase),受体如雄激素结合蛋白(androgen binding protein, ABP19/20)激素受体、Rhicadhesins受体等。OXO和SOD能催化产生H2O2,而H2O2迅速积累导致植物细胞壁结构的增强,触发脂质过氧化反应,乙烯的合成,并激活程序性细胞死亡(programmed cell death,PCD),同时H2O2能够诱导防御基因表达,从而提高植物的抗病性(Dunwell JM, Gibbings JG, Mahmood T, et al. Germin and germin-like proteins: evolution, structure, and function. Critical Reviews in Plant Sciences, 2008, 27: 342-375.)。另外,GLPs还可作为一种信号分子,直接或间接诱导防御反应。从烟草(Nicotiana tabacum)中分离纯化得到的水杨酸结合蛋白,经鉴定确认该蛋白与H2O2酶十分相似,并具有H2O2酶活性,水杨酸在植物体内通过与H2O2酶结合来抑制H2O2酶的活性,使植物内GLPs催化草酸氧化产生的H2O2迅速增加,由于H2O2能够诱导与抗性有关的基因表达,从而使植物产生抗性(Chen Z, Silva H, Klessiq DF. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science, 1993, 262: 1883-1886.)。因此GLPs在植物的防御反应中具有重要作用。
GLPs的表达在不同的组织和不同发育阶段差异较大。Godfrey等人在葡萄 (Vitis vinifera)中发现7个GLPs基因,这些GLPs的ORF所编码蛋白长度从207-205个氨基酸残基不等,并且包含不同的GLP特征结构域[Godfrey D, Able AJ, Dry IB. Induction of a grapevine germin-like protein (VvGLP3) gene is closely linked to the site of Erysiphe necator infection: a possible role in defense. Molecular Plant-Microbe Interactions, 2007, 20: 1112-1125.]。VvGLP2和VvGLP6在葡萄叶和浆果(包括果皮和果肉)中有表达;VvGLP1和VvGLP5分别只在根和成熟之前的浆果中表达;VvGLP3、VvGLP4、VvGLP7则在所有的组织中都表达。白粉病菌(Erysiphenecator)侵染迅速诱导浆果和叶片中VvGLP3和VvGLP4的表达,相对地,白粉病菌侵染的浆果中VvGLP5和发病叶中VvGLP6的表达量都显著减少,VvGLP2的表达却基本不受白粉病菌侵染的影响。VvGLP3的表达受E. necator侵染的诱导,并具有SOD活性。岷江百合(Lilium regale Wilson) LrGLP2基因在正常生长发育的根中有一定量的表达,而在茎和叶中几乎检测不到表达量,水杨酸、茉莉酸以及H2O2处理均不同程度抑制LrGLP2的转录水平,但乙烯处理能明显上调LrGLP2的表达 (刘亚龙, 李红丽,刘迪秋等. 岷江百合类萌发素蛋白基因LrGLP2的克隆及表达谱分析。植物生理学报, 2013, 10: 1063-1070.)。
GLPs是病程相关蛋白(pathogenesis-related protein, PR)家族的一员,病原体或其他外界因子的刺激能够诱导其表达,使其在植物防卫反应中发挥重要作用。BvGLP1是从抗根结线虫甜菜中分离的1个GLP基因,BvGLP1的异源超量表达显著提高了转BvGLP1基因拟南芥对轮枝孢菌(Fusarium verticillioide)和立枯丝核菌(Rhizoctonia solani)的抗性(Knecht K, Seyffarth M, Desel C, et al. Expression of BvGLP-1 encoding a germin-like protein from sugar beet in Arabidopsis thaliana leads to resistance against phyto-pathogenic fungi. Molecular Plant-Microbe Interactions, 2010, 23: 446–457.)。拟南芥中GLP13的敲减突变体 (SAIL-433-H06)对甲基紫精(methyl viologen, MV)胁迫敏感,而超表达GLP13的拟南芥对MV胁迫表现较强的抗性,与野生型相比,转基因拟南芥子叶变绿比率提高,且主根生长受抑制程度减轻,表明GLP13基因参与调控拟南芥应对氧化胁迫,是MV氧化胁迫响应途径的正调控因子(唐源江, 闵伶俐, 高桂兰等. 拟南芥GLP13基因在植物抗氧化胁迫响应中的作用. 植物学报, 2011, 46: 147-154.)。油菜(Brassica napus)类萌发素基因BnGLP3同时具有OXO和SOD活性,还可以在氧爆发时水解活性氧生成H2O2,降低植株对病原菌的敏感性,且在受核盘菌(Sclerotinia sclerotiorum)侵染6 h后表达量迅速增加(Rietz S, Bernsdorff FE M, Cai D. Members of the germin-like protein family in Brassica napus are candidates for the initiation of an oxidative burst that impedes pathogenesis of Sclerotinia sclerotiorum. Journal of experimental botany, 2012, 63: 5507-5519.)。
本发明中类萌发素蛋白基因JsGLP1来自漾濞大泡核桃(Juglans sigillata Dode)。漾濞大泡核桃是目前云南主要的核桃栽培品种,具有果大、壳薄、仁白、味香、出油率高、营养丰富等优点,并且对病原真菌胶孢炭疽菌具有较强的抗性。
发明内容
本发明的目的是提供一种从漾濞大泡核桃中克隆获得具有抗真菌活性的类萌发素蛋白的全长基因JsGLP1,JsGLP1的核苷酸序列如SEQ ID NO:1所示,该基因全长为976 bp,包含一个654 bp的开放阅读框、114 bp的5′非翻译区(untranslated regions,UTR)及208 bp的3′UTR,编码如SEQ ID NO:2所示氨基酸序列的蛋白质。
本发明所述类萌发素蛋白基因JsGLP1的编码区是序列表SEQ ID NO:1中第 115-769 位所示的核苷酸序列。
本发明分离克隆漾濞大泡核桃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JsGLP1。
GLPs是PRs家族中的一类胞外糖蛋白,在植物的生长发育以及对生物和非生物胁迫的防卫反应中起重要作用。GLPs具有OXO、SOD、AGPPase中的至少一种活性。SOD和OXO是植物体内重要的清除ROS的酶类,在植物抵御抗氧化胁迫的过程中,SOD将氧爆发时形成的活性氧转化为过氧化氢和氧分子,降低过多活性氧对植株形成的伤害;OXO可以催化草酸产生二氧化碳和过氧化氢;两种反应所产生的过氧化氢可以通过纤维素交联作用增强细胞壁的结构,并催化细胞壁的氧化交联形成乳突,延缓和阻止病原菌的侵入和扩散,以保护细胞免受再次感染。此外,在激活防卫基因表达过程中,过氧化氢作为一种第二信使引发超敏反应,进而使受感染部位的细胞发生程序性死亡,从而提高植物的抗病性。
本发明涉及分离包含JsGLP1的DNA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入侵的表型。其中所述DNA片段如序列表所示,对该基因进行分析,表明JsGLP1全长cDNA为976bp,包含一个654 bp的开放阅读框、114 bp的5′UTR及208 bp的3′UTR,其中ORF编码一个具有217个氨基酸的蛋白质。JsGLP1编码蛋白具有类萌发素蛋白的保守结构域,与来自胡杨(Populus euphratica) 、野生大豆(Glycine soja)、葡萄(Vitis vinifera)等物种的类萌发素蛋白具有较高的相似性,表明其属于漾濞大泡核桃的类萌发素蛋白。超表达序列表SEQ ID NO:1所示序列可以增强烟草对核盘菌(Sclerotinia sclerotiorum)、串珠状赤霉菌(Gibberella moniliformis)、胶孢炭疽菌(Colletotrichum gloeosporioides)、尖孢镰刀菌(Fusarium oxysporum)的抗性。
上述JsGLP1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JsGLP1的特异引物,从接种胶孢炭疽菌后的漾濞大泡核桃叶中提取总RNA,通过逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)扩增出JsGLP1的全长编码区,然后将其连接到pMD-18T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Pst和EcoR酶切pMD18-T-JsGLP1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JsGLP1基因片段与pCAMBIA2300S载体片段连接,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。本发明中来自漾濞大泡核桃的JsGLP1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JsGLP1转基因烟草基因组DNA的PCR检测结果,其中Marker: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;正对照:质粒pMD18-T-JsGLP1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JsGLP1转基因烟草中JsGLP1转录水平的表达分析结果图,其中Marker:DL2000 DNA Marker(大连宝生物);WT:非转基因烟草总RNA逆转录cDNA为模板的PCR产物;正对照:质粒pMD18-T-JsGLP1为模板的PCR产物;
图3是本发明中JsGLP1转基因烟草体外抗真菌活性的抑菌效果图;其中a、b、c、d图中的真菌分别是核盘菌、串珠状赤霉菌、胶孢炭疽菌以及尖孢镰刀菌;WT为野生型烟草的总蛋白;CK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:JsGLP1全长cDNA克隆以及序列分析
用胶孢炭疽菌接种漾濞大泡核桃,用接种后4 h的叶提取总RNA,用液氮将处理过的漾濞大泡核桃的叶研磨成粉末,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5 μg 总RNA,依次加入50 ng oligo (dT),2 μL dNTP Mix (2.5 mM each),用DEPC水将反应体积补齐至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 (200U)、1 μL M-MLV (200U),混匀并简短离心,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JsGLP1,所用上下游引物序列分别为及。采用AdvantageTM 2 PCR Enzyme (Clontech)扩增出目的基因;PCR反应条件:95℃ 1 min;94℃ 30 s,60℃ 30 s,72℃ 50 s,32个循环;72℃ 5 min。反应体系(20 μL)为1 μL cDNA、2 μL 10×Advantage 2 PCR Buffer、1.8 μL dNTP Mix (10mM 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、14.6 μL PCR-Grade water。PCR结束后,取8 μL进行琼脂糖凝胶电泳,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,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 pMD18-T vector (50 ng/μL)和2.5 μL 2×Ligation solution I,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,摇菌后用扩增JsGLP1的特异引物检测多克隆位点插入JsGLP1的克隆。将得到的阳性克隆进行测序,最终获得的JsGLP1全长cDNA为 976 bp,通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个 654 bp的开放读码框(见序列表)。JsGLP1编码一个含217个氨基酸的蛋白质,分子量约为 22.77 KDa,等电点为7.83。借助生物信息学软件SignalP 4.1分析JsGLP1编码的蛋白序列,检测其是否具有N端信号肽;结果显示在JsGLP1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JsGLP1的大肠杆菌质粒pMD18-T-JsGLP1以及植物表达载体pCAMBIA2300S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EcoRI (TaKaRa)和Pst(TaKaRa)分别对质粒pMD18-T-JsGLP1和pCAMBIA2300S进行双酶切(100 μL体系),反应体系和操作过程为:分别取20 μL pMD18-T-JsGLP1和pCAMBIA2300S质粒、依次加入10 μL 10×K buffer、5 μL EcoRI、5 μL Pst、60 μL ddH2O,混匀后短时离心,置于37℃过夜反应。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JsGLP1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JsGLP1DNA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 JsGLP1DNA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,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JsGLP1的特异引物进行PCR,挑选出JsGLP1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后置于-80℃保存备用。
采用SanPrep柱式质粒抽提试剂盒(上海生工)提取并纯化上述大肠杆菌DH5α中的pCAMBIA2300S-JsGLP1质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-JsGLP1转入所制备的根癌农杆菌LBA4404感受态细胞中。操作步骤为:取2 μg pCAMBIA2300S-JsGLP1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,再冰浴2 min,之后加入500 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℃倒置培养。挑选单菌落摇菌,再用扩增JsGLP1的特异性引物进行PCR反应,检测pCAMBIA2300S-JsGLP1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 L.)。将烟草种子用75%的酒精浸泡30 s,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5-8 d,发芽后转至光照培养箱(25℃,16 h/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-JsGLP1质粒的农杆菌LBA4404菌种,取20 μL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5-8 h以活化农杆菌。
取烟草无菌烟草幼嫩叶片切成约1 cm2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15 min。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℃无光条件下共培养2天。烟草转化的共培养基为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。烟草筛选培养基为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)。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。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,取1 μL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JsGLP1的特异引物进行PCR反应,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,JsGLP1转基因烟草共筛选到35株阳性转基因植株。
实施例4:转基因烟草中JsGLP1的表达分析以及转基因植株抗真菌活性分析
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JsGLP1的特异引物进行PCR,根据PCR结果分析各转基因植株中JsGLP1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8 μL用于琼脂糖凝胶电泳,部分单株的检测结果如图2所示。
将实验室保存的几种真菌接种于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℃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、6、7)及野生型叶片放入研钵中,加入1 mL蛋白提取液(1 M NaCl,0.1 M 乙酸钠,1% PVP,pH6.0),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),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0.2 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(蛋白提取液);28℃培养几天后观察各处理真菌生长的情况,并据此来评价JsGLP1转基因烟草的体外抗真菌活性。结果如图3所示,JsGLP1转基因烟草蛋白对核盘菌、串珠状赤霉菌、胶孢炭疽菌以及尖孢镰刀菌的生长具有明显的抑制作用。
序列表
<110> 昆明理工大学
<120> 漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 949
<212> DNA
<213> JuglanssigillataDode
<220>
<221> mRNA
<222> (1)..(949)
<220>
<221> 5'UTR
<222> (1)..(87)
<220>
<221> CDS
<222> (88)..(741)
<220>
<221> 3'UTR
<222> (742)..(949)
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cttctcgctt tggttttcac tgctgcattg gcatctgata gtagtcctct ccaagatttc 180
tgcgtcgcag atgcaagcag tcaagtggtc gtcaatggtt tagcatgtaa ggatcctaag 240
acggttcaag ccaatgactt ttccgccagt ggacttcata tggctggcaa tacatcaaat 300
ccagtcggtt cgaaggttac cccattgaca gctgctcaaa taccaggact caacactctt 360
ggcatttcac ttgctcgtat tgactatgca ccatggggca tcaaccctcc tcacacccat 420
cctcgtgcct ctgagatctt gttggtctta gaaggtagtc tggaagttgg gttcgtcacc 480
tctaatccag aaaaccgaca gatcacaaaa gtgctacaaa agggtgatgt gtttgtattc 540
ccagttggcc tcatccatta ccaaagaaat gttgtcaatg gaaatgccat cgccattgcc 600
gctctcagca gtcaaaaccc tggtgttatc actattgcta atgctgtgtt tgggtcgaaa 660
ccggatattg caagcgacat tttagtgaag gctttccagg tgcataagaa cgttatcgcc 720
aatgtccagt ccaagttcta gacaagccgt gctcgattat tgctgcttct gctgattgta 780
ttagctagtg catgtgtgca tatattttgt gtgtttcatg tcgtcatacg cagtttgtgt 840
gaaatgttat tttgctattc aattaattgt acgtttctcg ttaaatcaac aataattcaa 900
attccctcac cacatgcata aacaacgtaa aaaaaaaaaa aaaaaaaaa 949
<210> 2
<211> 217
<212> PRT
<213> JuglanssigillataDode
<400> 2
Met Ala Phe Arg Phe Ile Leu Leu Ser Ala Gly Leu Leu Ala Leu Val
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Phe Thr Ala Ala Leu Ala Ser Asp Ser Ser Pro Leu Gln Asp Phe Cys
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Val Ala Asp Ala Ser Ser Gln Val Val Val Asn Gly Leu Ala Cys Lys
35 40 45
Asp Pro Lys Thr Val Gln Ala Asn Asp Phe Ser Ala Ser Gly Leu His
50 55 60
Met Ala Gly Asn Thr Ser Asn Pro Val Gly Ser Lys Val Thr Pro Leu
65 70 75 80
Thr Ala Ala Gln Ile Pro Gly Leu Asn Thr Leu Gly Ile Ser Leu Ala
85 90 95
Arg Ile Asp Tyr Ala Pro Trp Gly Ile Asn Pro Pro His Thr His Pro
100 105 110
Arg Ala Ser Glu Ile Leu Leu Val Leu Glu Gly Ser Leu Glu Val Gly
115 120 125
Phe Val Thr Ser Asn Pro Glu Asn Arg Gln Ile Thr Lys Val Leu Gln
130 135 140
Lys Gly Asp Val Phe Val Phe Pro Val Gly Leu Ile His Tyr Gln Arg
145 150 155 160
Asn Val Val Asn Gly Asn Ala Ile Ala Ile Ala Ala Leu Ser Ser Gln
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Asn Pro Gly Val Ile Thr Ile Ala Asn Ala Val Phe Gly Ser Lys Pro
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Asp Ile Ala Ser Asp Ile Leu Val Lys Ala Phe Gln Val His Lys Asn
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Val Ile Ala Asn Val Gln Ser Lys Phe
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<210> 3
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<212> DNA
<213> 人工序列
<400> 3
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<210> 4
<211> 21
<212> DNA
<213> 人工序列
<400> 4
aagcagcaat aatcgagcac g 21
Claims (3)
1.一种漾濞大泡核桃类萌发素蛋白基因JsGLP1,其特征在于:其核苷酸序列如SEQ ID NO:1 所示。
2.权利要求1 所述的漾濞大泡核桃类萌发素蛋白基因JsGLP1在提高烟草对核盘菌、串珠状赤霉菌、胶孢炭疽菌、尖孢镰刀菌抗性中的应用。
3.根据权利要求2所述的漾濞大泡核桃类萌发素蛋白基因JsGLP1的应用,其特征在于提高烟草的真菌抗性的具体操作如下:
(1)将漾濞大泡核桃类萌发素蛋白基因JsGLP1与植物超表达载体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烟草中;
(3)以重组载体T-DNA上具有的卡那霉素抗性基因来筛选转化子,并通过聚合酶链式反应筛选获得阳性转基因植株,接种特定病原真菌,分析转基因烟草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。
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CN108707610A (zh) * | 2018-05-04 | 2018-10-26 | 昆明理工大学 | 三七defensin抗菌肽基因PnDEFL1及应用 |
CN114835789A (zh) * | 2022-05-25 | 2022-08-02 | 河南科技学院 | 小麦抗白粉病相关蛋白TaGLP-7A及其编码基因与应用 |
CN115521935A (zh) * | 2022-01-28 | 2022-12-27 | 福建农林大学 | 一种花生果皮丰富表达启动子pAhGLP17及其应用 |
CN116496372A (zh) * | 2023-04-17 | 2023-07-28 | 西南大学 | 抑制核盘菌的水稻OsGLP8-11及其应用 |
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CN105505955A (zh) * | 2015-11-25 | 2016-04-20 | 广东省农业科学院水稻研究所 | 一种提高水稻稻瘟病抗性的基因Os02g0532500、蛋白及其应用 |
CN105505955B (zh) * | 2015-11-25 | 2019-04-23 | 广东省农业科学院水稻研究所 | 一种提高水稻稻瘟病抗性的基因Os02g0532500、蛋白及其应用 |
CN108707610A (zh) * | 2018-05-04 | 2018-10-26 | 昆明理工大学 | 三七defensin抗菌肽基因PnDEFL1及应用 |
CN108707610B (zh) * | 2018-05-04 | 2021-01-05 | 昆明理工大学 | 三七defensin抗菌肽基因PnDEFL1及应用 |
CN115521935A (zh) * | 2022-01-28 | 2022-12-27 | 福建农林大学 | 一种花生果皮丰富表达启动子pAhGLP17及其应用 |
CN114835789A (zh) * | 2022-05-25 | 2022-08-02 | 河南科技学院 | 小麦抗白粉病相关蛋白TaGLP-7A及其编码基因与应用 |
CN114835789B (zh) * | 2022-05-25 | 2023-12-22 | 河南科技学院 | 小麦抗白粉病相关蛋白TaGLP-7A及其编码基因与应用 |
CN116496372A (zh) * | 2023-04-17 | 2023-07-28 | 西南大学 | 抑制核盘菌的水稻OsGLP8-11及其应用 |
CN116496372B (zh) * | 2023-04-17 | 2024-03-01 | 西南大学 | 抑制核盘菌的水稻OsGLP8-11及其应用 |
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