CN101736024B - 一种具有抗真菌活性的火把梨类甜蛋白基因PpTLP及应用 - Google Patents
一种具有抗真菌活性的火把梨类甜蛋白基因PpTLP及应用 Download PDFInfo
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- CN101736024B CN101736024B CN2010101003475A CN201010100347A CN101736024B CN 101736024 B CN101736024 B CN 101736024B CN 2010101003475 A CN2010101003475 A CN 2010101003475A CN 201010100347 A CN201010100347 A CN 201010100347A CN 101736024 B CN101736024 B CN 101736024B
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Images
Abstract
本发明涉及具有抗真菌活性的火把梨类甜蛋白基因PpTLP及应用。PpTLP基因具有SEQID所述的碱基序列,编码类甜蛋白。本发明通过功能基因组学相关技术证实PpTLP基因具有提高植物抗真菌的功能。将本发明抗真菌PpTLP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PpTLP的转基因烟草蛋白对核盘菌、烟草疫霉、拟茎点霉属真菌等多种真菌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,特别是一种具有抗真菌活性的火把梨类甜蛋白基因PpTLP及应用。
背景技术
在农业生产中,植物病害是一个非常棘手的问题,尤其是真菌病害,由其引起的病害占植物总病害的80%以上。各类病害中以真菌病害的症状类型最多,可以出现在植物的各个部位。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,但是存在严重的弊端。常规育种方法周期长、费时费力,化学农药的高残留、易对环境造成污染、危害人畜健康等缺点限制了传统病害防治方法的大规模应用。
随着重组DNA技术的创立和发展,通过转基因培育抗真菌植物新品种可望从根本上解决真菌病害问题。首先,抗真菌病害基因的获得及其抗性机理的研究非常重要。近年来,对植物-病原菌相互识别机制、信号传导途径,防卫反应的调控以及抗病和致病基因本质的研究不断深入,一系列专化抗性和广谱抗性的抗真菌基因陆续被克隆,通过转基因改良植物对真菌病害抗性已取得初步成效。
病程相关蛋白(pathogenesis-related protein,PR)是普遍存在于高等植物中的一类防御蛋白,病原体或其他外界因子的刺激能诱导其表达,在植物抵御病原体、响应外界压力以适应不良环境过程中发挥重要作用。根据序列相似性、血清或免疫特性以及酶功能将PRs分为17个家族,其中第5家族PR-5是一组很独特的蛋白,具有β-1,3葡聚糖酶活性、蛋白酶活性、抗真菌、抑制α淀粉酶以及抑制反转录酶等多种生物学活性(Van Loon LC,VanStrien EA.The families of pathogenesis-related proteins,their activities,andcomparative analysis of PR-1 type proteins.Physiological and Molecular PlantPayhology,1999,22:5585-5597.)。
类甜蛋白(thaumatin-like proteins,TLPs)属于PR-5家族,其氨基酸序列与非洲植物西非竹竽(Thaumatococcus danielli)的甜蛋白(thaumatin)高度同源,故称为类甜蛋白。两种蛋白的同源性很高,但其功能完全不一样,甜蛋白具有甜味无抗真菌活性,而类甜蛋白无甜味具有抗真菌活性。目前发现双子叶植物葡萄、番茄等,单子叶植物大麦、小麦、燕麦等以及部分真菌都能产生TLPs。TLPs的分子量范围在21-26kDa之间,并且不同植物的TLPs在功能上具有一定的差异性。
从芭蕉中提取的分子量为20kDa的TLP,在琼脂糖扩散实验中能抑制尖孢镰刀菌和褐斑病菌菌丝体的生长。该TLP对小鼠肿瘤细胞增殖无抑制作用,而对HIV-1的反转录酶有轻微的抑制作用(Ho VS,Wong JH,Ng TB.A thaumatin-like antifungal protein fromthe emperor banana.Peptides,2007,28:760-766.)。胶孢炭疽菌能引起胡椒的炭疽病,该真菌附着孢的生长范围以及菌丝的侵染程度在成熟胡椒果实中明显低于未成熟果实,实验表明PepTLP在成熟胡椒的防御中起着重要的作用(Kim YS,Park JY,Kim KS,etal.A thaumatin-like gene in nonclimacteric pepper fruits used as molecular markerin probing disease resistance,ripening,and sugar accumulation.Plant MolecularBiology,2002,49:125-135.)。长穗决明的类甜蛋白CdTLP,分子量为23kDa,不具有类β-1,3葡聚糖酶活性,但对假丝酵母属微生物具有抑制作用(Vitali A,Pacini L,BordiE,et al.Purification and characterization of an antifungal thaumatin-likeprotein from Cassia didymobotrya cell culture.Plant Physiology and Biochemistry,2006,44:604-610.)。竹笋TLP与已知TLPs在序列上的同源性较低,分子量也较低,但该蛋白能抑制尖孢镰刀菌、褐斑病菌以及灰霉菌的菌丝体生长(Wang HX,Ng TB.Dendrocin,a distinctive antifungal protein from bamboo shoots.Biochemical and BiophysicalResearch Communications,2003,307:750-755.)。
从感染镰刀菌的小麦中克隆得到tlp,并通过基因枪法获得高效表达tlp的小麦,tlp转基因小麦表现出延缓病原体扩散(Anand A,Zhou T,Trick HN,et al.Greenhouse andfield testing of transgenic wheat plants stably expressing genes forthaumatin-like protein,chitinase and glucanase against Fusarium graminearum.Journal of Experimental Botany.2003,54:1101-1111.)。将水稻tlp转入大麦中表达,转基因植株的抗真菌能力有所提高[Tobias DJ,Manoharan M,Pritsch C,et al.Co-bombardment,integration and expression of rice chitinase and thaumatin-likeprotein genes in barley(Hordeum vulgare cv.Conlon).Plant Cell Reports,2007,26:631-639.]。通过转基因手段获得转入大麦tlp-1的小麦植株,温室和大田的实验研究结果表明转基因小麦对于赤霉病的抗性有所增加(Mackintosh CA,Lewis J,Radmer LE,et al.Overexpression of defense response genes in transgenic wheat enhancesresistance to Fusarium head blight.Plant Cell Reports,2007,26:479-488.)。
本发明的TLP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。
发明内容
本发明的目的是从云南火把梨(Pyrus pyrifolia Nakai)中克隆获得有抗真菌活性的类甜蛋白的全长基因PpTLP,以及它的应用。
本发明分离克隆云南火把梨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PpTLP。
TLPs通过改变真菌细胞膜的渗透压来破坏其细胞膜的完整性,具有抑制真菌生长、抑制孢子裂解以及减少孢子数目或降低孢子发育能力的作用。TLPs同时也具有葡聚糖酶活性,即结合并切断多聚葡聚糖(polymeric glucans)的能力。葡聚糖酶活性使TLPs能结合并降解真菌细胞壁的主要组分-β-1,3葡聚糖,为TLPs进一步破坏真菌细胞膜奠定了基础。
本发明涉及分离包含PpTLP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浸染的表型。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PpTLP全长cDNA为973bp,具有744bp的开放阅读框(ORF)、44bp的5’非翻译区和185bp的3’非翻译区,编码含有248个氨基酸的蛋白质。PpTLP编码蛋白具有甜蛋白超家族的保守结构域,与来自苹果(Malusx domestica)以及其他物种的TLPs和PR5蛋白高度相似,表明其属于火把梨中的TLP。超量表达序列表SEQ ID所示序列可以增强烟草和其他植物对特定真菌的抗性。
上述基因可以应用于提高植物的真菌抗性,具体操作如下:
(1)将上述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)将上述构建的重组载体通过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,接种特定真菌或是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。
本发明为增强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PpTLP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,大量减少抗病剂的使用,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。
本发明通过功能基因组学相关技术证实PpTLP基因具有提高植物抗真菌的功能。将本发明抗真菌PpTLP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PpTLP的转基因烟草蛋白对核盘菌、烟草疫霉、拟茎点霉属真菌等多种真菌具有明显的抑制作用。
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-pPTLP为模板的PCR反应;WT:非转基因烟草总DNA为模板进行的PCR;空白对照:PCR体系中不包含DNA的反应。
图2是部分阳性转基因烟草中PpTLP转录水平的表达分析结果。Marker:DL2000 DNAMarker(大连宝生物);6、7、8、9、10、11、12、13分别为不同的阳性转基因烟草单株;WT:非转基因烟草总RNA逆转录的cDNA为模板进行的PCR;空白对照:PCR体系中不包含DNA的反应。正对照:质粒pMD-18T-pPTLP为模板的PCR反应;
图3是PpTLP转基因烟草体外抗真菌活性分析结果。A、B、C、D、E图示中的真菌分别是烟草疫霉、核盘菌、交链孢菌、派伦霉属真菌、拟茎点霉属真菌,WT为野生型烟草的总蛋白,空白对照为无蛋白对照(用于提取蛋白的缓冲液)。
具体实施方式
实施例
1)PpTLP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5μg Total RNA,依次加入50ng oligo(dT)15、2μL dNTP(2.5mM each)、ddH2O(RNase-free)至反应体积为13.5μL;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-stand buffer、0.5μL RNasin(200U)、1μLM-MLV(200U)、1μL 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PpTLP,所用引物序列分别为5’-GGATCCGGCAATTAAGACATATTCAATGTCG-3’和5’-GAATTCCTGCATATATAATCCCATTTCGTGC-3’。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,60℃ 30s,72℃ 1min,32个循环;72℃ 10min。反应体系(20μL)为1μL cDNA、2μL 10×Long taq Buffer、0.4μL dNTP(10mM each)0.1μL正向(20μM)、0.1μL反向引物(20μM)、0.2μL ex Taq DNA polymerase(2U/μL)16.2μL ddH2O。PCR结束后,取5μL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1.5μL PCR产物,依次加入1μL pMD18-T vector(50ng/μL)和2.5μL 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PpTLP的特异引物鉴定出多克隆位点插入PpTLP的克隆。将鉴定的克隆进行测序,最终获得的PpTLP全长cDNA为791bp,通过NCBI ORF finder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个744bp的开放读码框(见序列表)。PpTLP编码一个含248个氨基酸的蛋白质,PpTLP的分子量为26KDa,等电点4.45。就整个蛋白质的氨基酸组成而言,疏水氨基酸丙氨酸(A)的含量最高,约为11%。所有中性氨基酸含量为81%,酸性氨基酸含量为13%,碱性氨基酸含量为6%。半胱氨酸(C)的含量较高,有16个,占6%。借助生物信息学软件SignalP 3.0分析PpTLP编码的蛋白序列,检测它们是否具有N端信号肽。结果表明该蛋白的氨基酸残基1-27是一段信号肽,说明PpTLP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PpTLP的大肠杆菌质粒pMD-18T-PpTLP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和浓度高低。用EcoRI(Fermentas)和BamHI(Fermentas)分别对质粒pMD-18T-PpTLP和pCAMBIA2300S进行双酶切(100μL体系),反应体系和操作过程为:取10μLpMD-18T-PpTLP或pCAMBIA2300S质粒、依次加入10μL 10×Tango buffer、2μL EcoRI、2μL BamHI、76μL 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PpTLP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1.5mL离心管中,按400μL/100mg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2mL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500μL WashSolution,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1.5mL离心管中,在膜中央加入预热的20μL 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(大连宝生物),将回收的PpTLP 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 PpTLPDNA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,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PpTLP的特异引物进行PCR,挑选出PpTLP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-PpTLP质粒。并制备农杆菌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2mL LB液体培养基,28℃振荡培养至混浊;取5mL菌液转接于含20mg/L利福平的100mL 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10mL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4mL预冷的含15%甘油的0.1M CaCl2溶液,充分悬浮后分装于1.5mL离心管中,每管200μL,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-PpTLP转入所制备的农杆菌LBA4404感受态细胞中。操作步骤为:取2μg pCAMBIA2300S-PpTLP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,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800μL 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PpTLP的特异引物进行PCR,检测pCAMBIA2300S-PpTLP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1/2MS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-PpTLP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。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+2mg/L NAA+30g/L sucrose,培养时间为2d。
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。烟草筛选培养节为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分子水平检测。
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PpTLP的特异引物进行PCR。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。PpTLP转基因烟草共筛选到20株阳性转基因植株,分别编号为1~20。
4)PpTLP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)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PpTLP的特异引物进行PCR,根据PCR结果分析各转基因单株中PpTLP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5μL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PpTLP在转录水平有表达,这些单株的编号为5、8、9、10、12、14、16、18、19、21。
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5种:烟草疫霉(Phytophthora parasitica var.nicotianae)、核盘菌(Sclerotinia sclerotiorum)、拟茎点霉属(Phomopsis sp.)真菌、派伦霉属(Peyroneuaea)真菌和交链孢菌(Alternaria sp.)。
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1g转基因烟草单株(5、8、9、10、12、14、16、18、19、21)或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,pH5),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6mg/mL,然后分别取20μL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PpTLP转基因烟草的体外抗真菌活性。结果如图3所示,PpTLP转基因烟草蛋白对烟草疫霉、核盘菌、拟茎点霉属真菌、派伦霉属真菌和交链孢菌均有明显的抑制作用。
序列表(SEQ ID)
<110>昆明理工大学
<120>一种具有抗真菌活性的火把梨类甜蛋白基因PpTLP及应用
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gcaggtagtt aattagcagc aaacaggcaa ttaagacata ttca atg tcg atg atg 56
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tac act gtc tgg cca gga acc tta acc ggt gac caa aaa cct cag tta 200
Tyr Thr Val Trp Pro Gly Thr Leu Thr Gly Asp Gln Lys Pro Gln Leu
40 45 50
tca ctc act ggc ttc gaa cta gca tcc aaa gct agc caa tca gtg gac 248
Ser Leu Thr Gly Phe Glu Leu Ala Ser Lys Ala Ser Gln Ser Val Asp
55 60 65
gct cca ttt cca tgg tct ggt cgc ttc tgg ggt cga acc aga tgc tcc 296
Ala Pro Phe Pro Trp Ser Gly Arg Phe Trp Gly Arg Thr Arg Cys Ser
70 75 80
acg aac gcc gct gga aaa ttc act tgt gaa act gca gac tgt ggc tct 344
Thr Asn Ala Ala Gly Lys Phe Thr Cys Glu Thr Ala Asp Cys Gly Ser
85 90 95 100
ggc cag gta gca tgc aac ggg gca ggt gca att cca cca gca act tta 392
Gly Gln Val Ala Cys Asn Gly Ala Gly Ala Ile Pro Pro Ala Thr Leu
105 110 115
gtt gaa atc aca att gca gca aac ggg ggt caa gat ttt tac gat gtt 440
Val Glu Ile Thr Ile Ala Ala Asn Gly Gly Gln Asp Phe Tyr Asp Val
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agc ctt gtt gac ggc ttc aac ttg cct atg tct gtt gcc cca caa ggt 488
Ser Leu Val Asp Gly Phe Asn Leu Pro Met Ser Val Ala Pro Gln Gly
135 140 145
ggc acg ggc gac tgc aag ccc tca tct tgc cct gcc aat gtt aat gcg 536
Gly Thr Gly Asp Cys Lys Pro Ser Ser Cys Pro Ala Asn Val Asn Ala
150 155 160
gcg tgc cca gct caa ctt caa gta aaa gcg gct gat ggg act gtc atc 584
Ala Cys Pro Ala Gln Leu Gln Val Lys Ala Ala Asp Gly Thr Val Ile
165 170 175 180
gct tgc aaa agc gct tgc ctt gcg ttt ggt gat tcg aag tac tgc tgc 632
Ala Cys Lys Ser Ala Cys Leu Ala Phe Gly Asp Ser Lys Tyr Cys Cys
185 190 195
act ccg ccg aat aat acg ccg gag aca tgt cct ccc aca gag tac tct 680
Thr Pro Pro Asn Asn Thr Pro Glu Thr Cys Pro Pro Thr Glu Tyr Ser
200 205 210
cag ttc ttt gag cag cag tgc cct caa gct tat agc tac gct tat gat 728
Gln Phe Phe Glu Gln Gln Cys Pro Gln Ala Tyr Ser Tyr Ala Tyr Asp
215 220 225
gat aaa aac agc aca ttt acc tgc agt ggt gga cct gac tac gtc att 776
Asp Lys Asn Ser Thr Phe Thr Cys Ser Gly Gly Pro Asp Tyr Val Ile
230 235 240
act ttc tgc cca taagcacgaa atgggattat atatgcagat gatcatatct 828
Thr Phe Cys Pro
245
gtttctttat gtaacaataa tggagaagaa taaattcctc gaagcttgac acagtgctgt 888
tgtcaagaat ttgtaatact aattacatga tcaaataaag gaacaaatat tatttttaga 948
aaaaaaaaaa aaaaaaaaaa aaaaa 973
<210>2
<211>248
<212>PRT
<213>Pyrus pyrifolia Nakai
<400>2
Met Ser Met Met Lys Asn Gln Val Ala Ser Leu Leu Gly Leu Thr Leu
1 5 10 15
Ala Ile Leu Phe Phe Ser Gly Ala His Ala Ala Lys Ile Thr Phe Thr
20 25 30
Asn Asn Cys Pro Tyr Thr Val Trp Pro Gly Thr Leu Thr Gly Asp Gln
35 40 45
Lys Pro Gln Leu Ser Leu Thr Gly Phe Glu Leu Ala Ser Lys Ala Ser
50 55 60
Gln Ser Val Asp Ala Pro Phe Pro Trp Ser Gly Arg Phe Trp Gly Arg
65 70 75 80
Thr Arg Cys Ser Thr Asn Ala Ala Gly Lys Phe Thr Cys Glu Thr Ala
85 90 95
Asp Cys Gly Ser Gly Gln Val Ala Cys Asn Gly Ala Gly Ala Ile Pro
100 105 110
Pro Ala Thr Leu Val Glu Ile Thr Ile Ala Ala Asn Gly Gly Gln Asp
115 120 125
Phe Tyr Asp Val Ser Leu Val Asp Gly Phe Asn Leu Pro Met Ser Val
130 135 140
Ala Pro Gln Gly Gly Thr Gly Asp Cys Lys Pro Ser Ser Cys Pro Ala
145 150 155 160
Asn Val Asn Ala Ala Cys Pro Ala Gln Leu Gln Val Lys Ala Ala Asp
165 170 175
Gly Thr Val Ile Ala Cys Lys Ser Ala Cys Leu Ala Phe Gly Asp Ser
180 185 190
Lys Tyr Cys Cys Thr Pro Pro Asn Asn Thr Pro Glu Thr Cys Pro Pro
195 200 205
Thr Glu Tyr Ser Gln Phe Phe Glu Gln Gln Cys Pro Gln Ala Tyr Ser
210 215 220
Tyr Ala Tyr Asp Asp Lys Asn Ser Thr Phe Thr Cys Ser Gly Gly Pro
225 230 235 240
Asp Tyr Val Ile Thr Phe Cys Pro
245
Claims (2)
1.一种具有抗真菌活性的火把梨类甜蛋白基因PpTLP在提高烟草对烟草疫霉抗性中的应用,其特征在于所述的火把梨类甜蛋白基因PpTLP的序列如序列表SEQ ID NO:1所示。
2.根据权利要求1所述的具有抗真菌活性的火把梨类甜蛋白基因PpTLP在提高烟草对烟草疫霉抗性中的应用,其特征在于提高植物的真菌抗性的具体操作如下:
(1)将上述基因与适宜的植物超表达载体pCAMBIA1301、pK2GW7、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PCR获得真正的转基因植株,接种特定真菌烟草疫霉或是分析转基因植物蛋白对烟草疫霉生长的抑制活性,最后筛选出对烟草疫霉抗性明显增强的转基因植株。
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CN102344952B (zh) * | 2010-07-30 | 2013-06-19 | 中国检验检疫科学研究院 | 用于样品中苹果源性成分检测的引物及方法和试剂盒 |
CN102344953B (zh) * | 2010-07-30 | 2013-07-24 | 中国检验检疫科学研究院 | 用于样品中桃源性成分检测的引物及方法和试剂盒 |
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CN102174547B (zh) * | 2011-01-14 | 2013-04-03 | 昆明理工大学 | 一种火把梨β-1,3-葡聚糖酶基因PpGlu及应用 |
CN107488669B (zh) * | 2017-09-25 | 2021-06-25 | 南开大学 | 花椰菜BoTLP1基因的编码序列及其在培育耐盐抗旱转基因植物中的应用 |
CN109112144B (zh) * | 2018-08-28 | 2021-11-23 | 信阳师范学院 | 茶树类甜蛋白基因CsTHA1在增强作物抗逆性中的应用 |
CN109234284B (zh) * | 2018-09-14 | 2021-04-09 | 昆明理工大学 | 一种三七类甜蛋白基因PnTLP5及应用 |
CN109295068B (zh) * | 2018-09-14 | 2021-04-09 | 昆明理工大学 | 一种三七类甜蛋白基因PnTLP2及应用 |
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