CN102174547B - 一种火把梨β-1,3-葡聚糖酶基因PpGlu及应用 - Google Patents
一种火把梨β-1,3-葡聚糖酶基因PpGlu及应用 Download PDFInfo
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Abstract
本发明涉及具有抗真菌活性的火把梨β-1,3-葡聚糖酶基因PpGlu及应用。PpGlu基因具有SEQID所述的碱基序列,编码β-1,3-葡聚糖酶。本发明通过功能基因组学相关技术证实PpGlu基因具有提高植物抗真菌的功能。将本发明抗真菌PpGlu基因构建到植物表达载体上并转入烟草中过量表达,转基因烟草具有很强的体外抗真菌活性。表达PpGlu的转基因烟草的蛋白提取液对拟茎点霉属真菌、交链孢、镰刀菌有不同程度的抑制作用。
Description
技术领域
本发明涉及分子生物学以及基因工程技术领域,特别是一种火把梨的具有抗真菌活性的β-1,3-葡聚糖酶(β-1,3-glucanase)基因PpGlu及应用。
背景技术
真菌病害是造成农作物减产的主要因素之一,由植物病原真菌引起的病害约占植物病害的70~80%,一种作物上可发现几种甚至几十种真菌病害。目前,控制真菌病害主要有三种方法:一是选育并采用抗性品种,二是使用化学杀菌剂,三是采取合理的耕作制度,如轮作、避免已感染土壤和带病原植物材料的传播等。随着重组DNA技术的创立和发展,能将动物、植物、微生物的基因相互转移,突破了物种之间难以杂交的天然屏障,开辟了植物育种的新途径。近年来,一些科学家致力于利用基因工程方法,采用基因转移技术培育抗真菌病害的作物品种。
当植物受到病原菌攻击或处于其他逆境胁迫时,会在体内产生大量的病程相关蛋白(pathogenesis-related protein,PR),它们与植物的防卫反应关系密切。植物的β-1,3葡聚糖酶属于PR-2家族,是植物防卫反应中合成的一种重要水解酶类,能降解β-1,3葡聚糖,而葡聚糖正是真菌细胞壁的主要成分之一,因此β-1,3葡聚糖酶具有一定的抗菌作用。β-1,3-葡聚糖酶参与植物对病原真菌的防卫反应,并调节植物的生长发育,它们被认为是一类重要的抗真菌蛋白(Mauch F, Hadwiger LA, Boller T. Ethylene: symptom, not signal for the induction of chitinase and beta-1,3-glucanase in pea pods by pathogens and elicitors. Plant Physiol. 1984, 76:607-611)。已知的β-1,3-葡聚糖酶均属于糖基水解酶第十七家族,分为外切酶和内切酶,目前主要研究的是内切酶。它的分子量为32-37kD,等电点从酸性到碱性。它的作用底物为以β-1,3-苷键连接起来的多聚糖,以随机作用方式将多聚糖分解成为糊精或寡聚糖。β-1,3-葡聚糖酶主要通过2个途径增强植物对真菌的抗性:1)β-1,3-葡聚糖是大多数植物病原真菌细胞壁的主要成分之一,β-1,3-葡聚糖酶以随机作用方式将以β-1,3-糖苷键连接起来的多聚糖分解成为糊精或寡聚糖,因此可以破坏很多植物病原真菌的细胞壁,使真菌细胞失去支撑,破裂死亡;2)水解真菌细胞壁所产生的多糖或寡聚糖能作为激发子激活或者上调植物防御系统,如促使植物分泌植物抗毒素,从而间接提高植物的抗病能力(Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J. 1995, 8:97-109)。
平皿抑菌实验表明,β-1,3-葡聚糖酶对大豆疫霉(Phytophthora sojae)、炭疽菌(Colletotrichum lagenarium)的菌丝生长以及孢子的萌发有明显的抑制作用(Ji C, Kúc J. Purification and characterization of an acidic β-1,3-glucanase from cucumber and its relationship to systemic disease resistance induced by Colletotrichum lagenarium and tobacco necrosis virus. Mol Plant Microbe In. 1995, 8: 899-905; 娄树宝, 彭东君, 王辉. 大豆β-1,3-葡聚糖酶的抑菌活性. 黑龙江八一龙垦大学学报. 2008, 20:27-29),对小麦纹枯病菌(Rhizoctonia longipes)和烟草赤星病菌(Alternaria cerealis)的菌丝生长也有抑制作用,同时对大丽轮枝菌(Verticillium dahliae)、黄瓜枯萎病病原菌(Fusarium oxysporum f. sp. cucumerinum)的孢子萌发或芽管伸长也有一定程度的抑制作用(孙斌, 李多川, 慈晓燕, 郭润芳, 王颖. 小麦叶片β-1,3-葡聚糖酶的诱导、纯化与抗菌活性. 植物生理与分子生物学学报. 2004, 30:399-404)。大豆在接种大豆疫霉后,β-1,3-葡聚糖酶和几丁质的活性增强,β-l,3-葡聚糖酶和几丁质酶混合液对大豆疫霉菌的菌丝生长、孢子囊形成和孢子萌发的抑制作用明显,表明β-1,3-葡聚糖酶和几丁质酶可受大豆疫霉的诱导,且大豆对大豆疫霉根腐病的抗性与β-1,3-葡聚糖酶和几丁质酶的活性呈正相关(左豫虎, 康振生, 杨传平, 芮海英, 娄树宝, 刘惕若. β-1,3-葡聚糖酶和几丁质酶活性与大豆对疫霉根腐病抗性的关系. 植物病理学报. 2009, 39:600-607)。葡萄霜霉病菌能诱导葡萄叶片几丁质酶和β-1,3-葡聚糖酶活性增高,而且两种酶活性增高的速度和幅度与品种的抗病性呈正相关(史娟, 胡景江, 王红玲, 满丽梅,张永丽. 霜霉病菌对葡萄细胞壁水解酶的诱导作用与寄主抗病性的关系. 西北林学院学报. 2002, 17:42-44)。
通过基因工程的方法,转入β-1,3-葡聚糖酶基因可提高植物对真菌病害的抵抗力。转入马铃薯β-1,3-葡聚糖酶基因的亚麻(Linum usitatissimum L.),对两种镰刀菌F. oxysporum、F. culmorum的抗性相对于野生型增强了3倍(Wróbel-Kwiatkowska M, Lorenc-Kukula K, Starzycki M, Oszmianski J, Kepczynska E, Szopa J. Expression of β-1,3-glucanase in flax causes increased resistance to fungi. Physiol Mol Plant Pathol. 2004, 65:245-256)。含有大豆β-1,3-葡聚糖酶基因的转基因烟草能有效地抵御赤星病菌和黑胫病菌(Phytophthora parasitica var. nicotianae)的侵染(Yoshikawa M, Tsuda M, Takeuchi Y. Resistance to fungal diseases in transgenic tobacco plants expressin the phytodexin elicitor-releasing factor, β-1,3-endogulcanase, from soybean. Naturwiss. 1993,
80:417-420)。
发明内容
本发明的目的是从云南火把梨(Pyrus pyrifolia Nakai)中克隆获得编码β-1,3-葡聚糖酶的全长基因PpGlu以及它的应用。
本发明的β-1,3-葡聚糖酶基因来自云南火把梨。火把梨是云南本地的沙梨品种,具有稳定遗传的红色果皮表型。火把梨对土壤适应性强,抗黑星病、腐烂病,对梨木虱有较强的抗性,并且抗晚霜,耐低温能力也很强。
本发明分离克隆云南火把梨的一个抗真菌相关基因的完整cDNA片段,利用根癌农杆菌介导法将目的基因转入受体植物中并过量表达,通过进一步试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PpGlu。
β-1,3-葡聚糖酶通过水解真菌细胞壁重要成分β-1,3-葡聚糖,使细胞失去支撑,破裂死亡,从而使植物具有抵抗真菌侵染的能力。β-1,3-葡聚糖酶作为一种病程相关蛋白还在小孢子形成、养料供给、开花、花粉形成、种子萌发、果实成熟、芽休眠和伤口愈合等方面起作用。
本发明涉及分离包含PpGlu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侵染的表型。其中所述DNA片段如序列表SEQ ID所示,或者基本相当于SEQ ID所示的DNA序列,或者其功能相当于SEQ ID所示序列的部分片段。对该基因进行序列分析,表明PpGlu全长cDNA为1217bp,具有1047bp的开放阅读框(Open reading frame,ORF)、29bp的5’非翻译区(untranslated region,UTR)和141bp的3’UTR,编码含有348个氨基酸的蛋白质。PpGlu编码蛋白具有β-1,3-葡聚糖酶的保守结构域,与来自草莓(Fragaria x ananassa)、长喙田菁(Sesbania rostrata)、苹果(Malus x domestica)以及其它物种的β-1,3-葡聚糖酶蛋白高度相似,表明其属于火把梨中的β-1,3-葡聚糖酶。超量表达序列表SEQ ID所示序列可以增强烟草对茎点霉属真菌、交链孢、镰刀菌的抗性。
上述PpGlu基因可以应用于提高植物的真菌抗性,具体操作如下:
(1) 将基因与适宜的植物超表达载体如pCAMBIA1301、pK2GW7、pCAMBIA2300s等连接,构建植物超表达载体。
(2) 将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上具有的抗性标记筛选转化子,并通过聚合酶链式反应(Polymerase Chain Reaction,PCR)获得真正的转基因植株,接种特定真菌或分析转基因植物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。
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能克服传统育种的不足,不仅育种周期短,而且操作简单,容易获得高抗材料。本发明来自火把梨的PpGlu基因能增强植物对真菌的抗性,将该基因导入烟草、康乃馨、洋桔梗等植物中,可以产生具有真菌抗性的种质和品种。利用基因工程技术防治真菌病害具有明显的优势和不可取代的重要性。它可以为作物、花卉等大规模生产提供方便,大量减少化学农药的使用,为农业生产节约成本、减小环境污染且提高管理水平,因此本发明具有广阔的市场应用前景。
附图说明
图1是部分转基因烟草基因组DNA的PCR检测结果。Marker: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:质粒pMD-18T-PpGlu为模板的PCR反应;WT:非转基因烟草总DNA为模板进行的PCR;负对照:PCR体系中不包含DNA的反应。
图2是部分阳性转基因烟草中PpGlu转录水平表达分析的凝胶电泳图谱。
Marker:DL2000 DNA Marker (大连宝生物);1-18分别为不同的阳性转基因烟草单株;WT:非转基因烟草总RNA逆转cDNA为模板的PCR产物;负对照:PCR体系中不包含模板DNA的反应产物;正对照:质粒pMD-18T-PpGlu为模板的PCR产物。
图3是PpGlu转基因烟草体外抗真菌活性分析抑菌效果图。
A、B、C图示中的真菌分别是拟茎点霉属真菌、交链孢和镰刀菌;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
实施例
1)PpGlu全长基因克隆以及序列分析
用液氮将火把梨的红色果皮研磨成粉末以后,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 (天根)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、2 μL dNTP (2.5mM each)、ddH2O (RNase-free)至反应体积为13.5 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)、1 μL M-MLV (200U)、1 μL DTT (0.1M),混匀并短时离心,42℃温浴1.5h,取出后95℃加热5min,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PpGlu,所用上下游引物序列分别为5’GGATCCTTCTCCAATAGCTTCATGCATTT3’和5’GAATTCTGATTCTGTGACTCC TTCTATCCA3’。采用大连宝生物的高保真DNA聚合酶Ex Taq扩增出目的基因。PCR反应条件:94℃ 3min;94℃ 30s,58℃ 30s,72℃ 1.5min,32个循环;72℃ 10min。反应体系(20 μL)为1 μL cDNA、2 μL 10×Ex Taq Buffer、0.4 μL dNTP (10mM each)、0.1 μL上游引物(20μM)、0.1 μL下游引物(20μM)、0.2 μL Ex Taq DNA polymerase (5U/μL)、16.2μL ddH2O。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 pMD18-T vector (50ng/μL) 和2.5 μL 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PpGlu的特异引物鉴定出多克隆位点插入PpGlu的克隆。将鉴定的克隆进行测序,最终获得的PpGlu全长cDNA为1217bp,通过NCBI ORF finder (http://www.ncbi. nlm.nih.gov/gorf/gorf.html)分析发现其包含一个1047bp的开放读码框(见序列表)。PpGlu编码一个含348个氨基酸的蛋白质PpGlu,其分子量约为37.5 KDa,等电点5.07。就整个蛋白质的氨基酸组成而言,三种疏水氨基酸丙氨酸(A)、亮氨酸(L)、缬氨酸(V)的含量最高,分别为11.5%、9.2%、8%。所有中性氨基酸含量为83.4%,酸性氨基酸含量为8.9%,碱性氨基酸含量为7.7%。还含有1个半胱氨酸残基(C),位于第39位。借助生物信息学软件SignalP 3.0分析PpGlu编码的蛋白序列,检测其是否具有N端信号肽。结果表明该蛋白的氨基酸残基1-33为信号肽,可能的剪切位点为33-34,说明PpGlu是一种分泌蛋白。
2)植物表达载体构建
采用碱裂解法提取插入PpGlu的大肠杆菌质粒pMD-18T-PpGlu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EcoRI (Fermentas)和BamHI (Fermentas)分别对质粒pMD-18T-PpGlu和pCAMBIA2300S进行双酶切(100 μL体系),反应体系和操作过程为:取10 μL pMD-18T-PpGlu或pCAMBIA2300S质粒、依次加入10 μL 10×Tango buffer、2 μL EcoRI、2 μL BamHI、76 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PpGlu片段和pCAMBIA2300S大片段分别进行胶回收,整个过程使用UNIQ-10柱式DNA胶回收试剂盒(上海生工)。具体操作流程为:切下含有目的DNA条带的琼脂糖凝胶块置于1.5 mL离心管中,按400 μL/100 mg琼脂糖凝胶的比例加入Binding Buffer Ⅱ,置于60℃水浴10min,使胶彻底融化;将融化的凝胶溶液转移至2 mL收集管内的UNIQ-10柱中,室温放置2min后离心1min (6,000g/min);取下UNIQ-10柱,倒掉收集管中的废液,再将UNIQ-10柱放入收集管中,加入500 μL Wash Solution,室温离心1min (8,000g/min),再重复此步骤一次;取下UNIQ-10柱,倒掉收集管中的废液,再将UNIQ-10柱放入收集管中,室温离心2min (12,000g/min);取下UNIQ-10柱放入新的1.5 mL离心管中,在膜中央加入预热的20 μL Elution Buffer,室温放置2min,室温离心1min (12,000g/min),离心管中收集液体即为回收的DNA片段。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 (大连宝生物),将回收的PpGlu DNA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 PpGlu DNA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PpGlu的特异引物进行PCR,挑选出PpGlu 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。
用碱裂解法提取并纯化上述大肠杆菌中的pCAMBIA2300S-PpGlu质粒。并制备根癌农杆菌LBA4404菌株的感受态细胞,操作过程为:将实验室保存的LBA4404菌株在LB固体培养基(含利福平20mg/L)上划线,28℃培养至长出单菌落后,挑取一个单克隆于含20mg/L利福平的2 mL LB液体培养基,28℃振荡培养至混浊;取5 mL菌液转接于含20mg/L利福平的100 mL LB液体培养基中,28℃振荡培养至OD600为0.5;4℃离心5min (5,000g/min)收集菌体,弃上清,加入10 mL预冷的0.1M CaCl2,轻轻充分悬浮菌体,冰浴20min;接着4℃离心5min (5,000g/min)收集菌体,弃上清,加入4 mL预冷的含15%甘油的0.1M CaCl2溶液,充分悬浮后分装于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-PpGlu转入所制备的农杆菌LBA4404感受态细胞中。操作步骤为:取2 μg pCAMBIA2300S-PpGlu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5min,接着转入液氮中冷冻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50mg/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PpGlu的特异引物进行PCR,检测pCAMBIA2300S-PpGlu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。
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-PpGlu质粒的农杆菌LBA4404菌种,接种于5 mL含有50mg/L Km和20mg/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50mg/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,附加一定量的乙酰丁香酮,28℃振荡培养2-3h以活化农杆菌。
取烟草无菌苗叶子切成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 mg/L NAA+30g/L sucrose,无光条件下22℃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+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℃,16h/d光照,8h/d黑暗)。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和200mg/L Cef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1 μL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PpGlu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。部分烟草转基因植株的扩增结果如图1所示。PpGlu转基因烟草共筛选到18株阳性转基因植株,分别编号为1~18。
4)PpGlu表达分析以及转基因植株抗真菌活性分析
取阳性转基因单株以及非转基因烟草(野生型,WT)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PpGlu的特异引物进行PCR,根据PCR结果分析各转基因单株中PpGlu转录水平的表达。总RNA提取以及RT-PCR的方法与步骤与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10株转基因单株中PpGlu在转录水平有表达,这些单株的编号为1、3、4、8、11、13、15、16、17、18。
将实验室保存的几种真菌接种于PDA固体培养基(200g/L 马铃薯,15g/L 琼脂,20g/L葡萄糖)上,28℃倒置培养,待菌落生长至直径约为3cm时添加蛋白,分析转基因植株体外抗真菌活性。供试真菌共有6种:拟茎点霉属真菌、交链孢、镰刀菌、黑曲霉、青霉、烟草疫霉。
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、3、4、8、11)或野生型叶片放入研钵中,加入1 mL蛋白提取液(1M NaCl,0.1M 乙酸钠,1% PVP,pH5),充分研磨。转入1.5 mL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1.5 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6mg/mL,然后分别取20 μL滴于各真菌培养基的滤纸上。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(提取蛋白所用的溶液)。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PpGlu转基因烟草的体外抗真菌活性。结果如图3所示,PpGlu转基因烟草蛋白对拟茎点霉属真菌、交链孢、镰刀菌有不同程度的抑制作用,而对实验所用其它真菌无抑制作用。
序列表(SEQ ID)
<110> 昆明理工大学
<120> 一种火把梨β-1,3-葡聚糖酶基因PpGlu及应用
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1217
<212> DNA
<213> Pyrus pyrifolia
<220>
<221> mRNA
<222> (1)..(1217)
<220>
<221> 5'UTR
<222> (1)..(29)
<220>
<221> CDS
<222> (30)..(1076)
<220>
<221> 3'UTR
<222> (1077)..(1217)
<400> 1
acgcggggat atatttctcc aatagcttc atg cat ttt tcc aac ata tat aaa 53
Met His Phe Ser Asn Ile Tyr Lys
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Thr Gly Lys Ala Leu Met Ala Ser Ile Leu Leu Leu Leu Val Val Leu
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Met Pro Ala Leu Gln Ile Thr Gly Ala Gln Ser Val Gly Val Cys Tyr
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gga cga aac ggc aac aat tta cca gct gaa gga gaa gtg gtc gac ttg 197
Gly Arg Asn Gly Asn Asn Leu Pro Ala Glu Gly Glu Val Val Asp Leu
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tac aaa agc aat ggc atc ggg cgg atg agg atc tat gaa cca aat gaa 245
Tyr Lys Ser Asn Gly Ile Gly Arg Met Arg Ile Tyr Glu Pro Asn Glu
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Ala Thr Leu Gln Ala Leu Arg Gly Ser Asn Ile Glu Leu Thr Val Thr
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Ile Leu Asn Asn Glu Leu Gln Ala Leu Asn Asp Ala Ala Ala Ala Thr
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gcc tgg gtc cag aag aac gta caa ccc tat tca gct gat gtt aaa ttc 389
Ala Trp Val Gln Lys Asn Val Gln Pro Tyr Ser Ala Asp Val Lys Phe
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aaa tac atc gct gtt gga aac gaa gtg cgc ccc ggt gct gca gag gtc 437
Lys Tyr Ile Ala Val Gly Asn Glu Val Arg Pro Gly Ala Ala Glu Val
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ggg ttt ctc ctg cct gcc atc caa aac atc cac agt gca att gta gca 485
Gly Phe Leu Leu Pro Ala Ile Gln Asn Ile His Ser Ala Ile Val Ala
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gcc aat ctg caa ggc caa atc aag gtc tct aca gca att gac aca act 533
Ala Asn Leu Gln Gly Gln Ile Lys Val Ser Thr Ala Ile Asp Thr Thr
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Leu Val Thr Asn Ala Tyr Pro Pro Ser Asp Gly Val Tyr Thr Asp Pro
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Ala Asn Gln Phe Ile Lys Pro Val Ile Asp Phe Leu Val Ser Asn Gly
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Ala Pro Leu Leu Val Asn Val Tyr Pro Tyr Phe Ser Tyr Asn Asp Asn
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Pro Gly Ser Ile Asp Leu Ala Tyr Ala Leu Phe Thr Ser Gln Gly Val
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Val Val Pro Asp Gly Thr Arg Tyr Pro Ser Leu Phe Asp Ala Leu Leu
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Asp Ala Gln Tyr Ala Ala Leu Glu Lys Ala Gly Ala Pro Asn Val Glu
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Ile Val Val Ser Glu Ser Gly Trp Pro Phe Glu Gly Gly Asn Gln Ala
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acc cct gag aac gca gcc aca ttt tac cag aat ttg atc aag cat gtg 917
Thr Pro Glu Asn Ala Ala Thr Phe Tyr Gln Asn Leu Ile Lys His Val
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acg agt act act ggg act cca aag agg cct ggt aaa gct ata gag act 965
Thr Ser Thr Thr Gly Thr Pro Lys Arg Pro Gly Lys Ala Ile Glu Thr
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Tyr Leu Phe Ala Met Phe Asp Glu Asn Leu Lys Ala Gly Asn Ala Asp
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gag aaa cac ttt gga att ttt acc cct gac aaa caa ccc aag tac caa 1061
Glu Lys His Phe Gly Ile Phe Thr Pro Asp Lys Gln Pro Lys Tyr Gln
330 335 340
ctc aca ttt gga tag aaggagtcac agaatcatgt ttgatgtaag ttttagttga 1116
Leu Thr Phe Gly
345
ataaggatgc cttgtgccca gttctctcac cccatatata tgagatacaa taaaaagaat 1176
acacatggtt atcacgaaaa aaaaaaaaaa aaaaaaaaaa a 1217
<210> 2
<211> 348
<212> PRT
<213> Pyrus pyrifolia
<400> 2
Met His Phe Ser Asn Ile Tyr Lys Thr Gly Lys Ala Leu Met Ala Ser
1 5 10 15
Ile Leu Leu Leu Leu Val Val Leu Met Pro Ala Leu Gln Ile Thr Gly
20 25 30
Ala Gln Ser Val Gly Val Cys Tyr Gly Arg Asn Gly Asn Asn Leu Pro
35 40 45
Ala Glu Gly Glu Val Val Asp Leu Tyr Lys Ser Asn Gly Ile Gly Arg
50 55 60
Met Arg Ile Tyr Glu Pro Asn Glu Ala Thr Leu Gln Ala Leu Arg Gly
65 70 75 80
Ser Asn Ile Glu Leu Thr Val Thr Ile Leu Asn Asn Glu Leu Gln Ala
85 90 95
Leu Asn Asp Ala Ala Ala Ala Thr Ala Trp Val Gln Lys Asn Val Gln
100 105 110
Pro Tyr Ser Ala Asp Val Lys Phe Lys Tyr Ile Ala Val Gly Asn Glu
115 120 125
Val Arg Pro Gly Ala Ala Glu Val Gly Phe Leu Leu Pro Ala Ile Gln
130 135 140
Asn Ile His Ser Ala Ile Val Ala Ala Asn Leu Gln Gly Gln Ile Lys
145 150 155 160
Val Ser Thr Ala Ile Asp Thr Thr Leu Val Thr Asn Ala Tyr Pro Pro
165 170 175
Ser Asp Gly Val Tyr Thr Asp Pro Ala Asn Gln Phe Ile Lys Pro Val
180 185 190
Ile Asp Phe Leu Val Ser Asn Gly Ala Pro Leu Leu Val Asn Val Tyr
195 200 205
Pro Tyr Phe Ser Tyr Asn Asp Asn Pro Gly Ser Ile Asp Leu Ala Tyr
210 215 220
Ala Leu Phe Thr Ser Gln Gly Val Val Val Pro Asp Gly Thr Arg Tyr
225 230 235 240
Pro Ser Leu Phe Asp Ala Leu Leu Asp Ala Gln Tyr Ala Ala Leu Glu
245 250 255
Lys Ala Gly Ala Pro Asn Val Glu Ile Val Val Ser Glu Ser Gly Trp
260 265 270
Pro Phe Glu Gly Gly Asn Gln Ala Thr Pro Glu Asn Ala Ala Thr Phe
275 280 285
Tyr Gln Asn Leu Ile Lys His Val Thr Ser Thr Thr Gly Thr Pro Lys
290 295 300
Arg Pro Gly Lys Ala Ile Glu Thr Tyr Leu Phe Ala Met Phe Asp Glu
305 310 315 320
Asn Leu Lys Ala Gly Asn Ala Asp Glu Lys His Phe Gly Ile Phe Thr
325 330 335
Pro Asp Lys Gln Pro Lys Tyr Gln Leu Thr Phe Gly
340 345
Claims (2)
1.一种火把梨β-1,3-葡聚糖酶基因PpGlu,其特征在于其序列是序列表中SEQ ID No.1所述的碱基序列,PpGlu的编码区是序列表SEQ ID No.1中第30-1076位所示的核苷酸序列,PpGlu全长1217bp,具有1047bp的开放阅读框、29bp的5’非翻译区和141bp的3’非翻译区,编码含有348个氨基酸的蛋白质。
2.权利要求1所述的火把梨β-1,3-葡聚糖酶基因PpGlu在提高烟草对镰刀菌抗性和交链孢菌抗性中的应用。
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CN109295031B (zh) * | 2018-10-23 | 2020-06-19 | 南京工业大学 | 一种抗真菌蛋白β-1,3-葡聚糖酶和含有该基因的工程菌及其应用 |
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Yanlin Shi,et al.Cloning and expression analysis of two beta-1,3-glucanase genes from strawberry..《Journal of Plant Physiology》.2006, * |
李文娴 等.类甜蛋白的结构特征以及功能研究进展.《中国生物工程杂志》.2010, * |
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