CN107267525A - 三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用 - Google Patents
三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用 Download PDFInfo
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Abstract
本发明公开了三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用,即三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP在提高烟草对葡萄座腔菌、串珠状赤霉菌、人参链格孢抗性中的应用,所述三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的核苷酸序列如SEQ ID NO:1所示;本发明通过分子生物学和功能基因组学相关技术研究证实PnPGIP基因具有抑制真菌侵染的功能,将本发明三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP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,PnPGIP转基因烟草蛋白对葡萄座腔菌、串珠状赤霉菌和人参链格孢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研究领域,特别是一种三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用。
背景技术
植物在生物或非生物因子的影响下,发生一系列形态、生理和生化上的病理变化,阻碍了正常生长、发育的进程,从而影响了人类的经济效益。在多种逆胁迫因子中,细菌、真菌、病毒等是引起植物病害的主要因素,尤其是真菌病害能大规模导致农作物减产甚至死亡。筛选抗病品种、改进栽培技术和利用农药等方法对真菌病害的防治做出了重要贡献。但这些传统的防治措施周期长、效果差、甚至会给环境和人畜的生命健康带来危害,不能彻底解决真菌病害所带来的问题。近年来,分子生物学和基因工程技术的发展迅速,利用DNA重组技术培育出抗真菌的植物新品种,为解决真菌病害问题开辟了新途径。
植物在生长发育过程中常会受到真菌、细菌、病毒等病原物的危害,在与病原物长期的相互作用、相互选择、协同进化的过程中,植物获得了一系列复杂的防御机制来保护自己,例如防御相关蛋白的产生。细胞壁是植物抵抗病原物侵染的重要屏障,病原菌在侵染植物时分泌多聚半乳糖醛酸酶(polygalacturonases, PGs)降解植物细胞壁,达到侵染植物的目的。植物多聚半乳糖醛酸酶抑制蛋白(polygalacturonase inhibiting proteins,PGIPs)能够抑制真菌产生的多聚半乳糖醛酸酶(polygalacturonases, PGs)的活性,激活植物的防御系统,在植物抗病方面发挥着重要的作用。
PGIPs 是一种胞外的细胞壁结合蛋白,并且能够与多聚半乳糖醛酸酶特异性结合,抑制其活性。PGIPs在与PGs相互作用的过程中使植物体内产生并积累长链寡聚半乳糖醛酸(oligogalacturonides,OGs),激活防御反应,抑制真菌的侵染(De Lorenzo G, D’Ovidio R, Cervone F. The role of polygalacturonase-inhibiting proteins(PGIPs) in defense against pathogenic fungi. Annu Rev Phytopathol, 2001, 39:313-335)。PGIP能够专一性地抑制PGs的活性,维护植物细胞壁的完整性,使细胞壁不易被细胞壁降解酶降解,减缓了病原菌的生长速度,进一步增强了植物的抗病性,有效的阻断了病菌的侵染和抑制相应病程的发生与发展(王秀菊. 辣椒多聚半乳糖醛酸酶抑制蛋白基因克隆及功能研究. 硕士研究生毕业论文 2011,6)。PGIP是富含亮氨酸重复(Leucine-richRepeat,LRR)的蛋白质超家族,含有包括20~30个氨基酸的共有重复序列,重复次数一般为10,形成β-折叠/β-转角/β-折叠/α-螺旋结构,即LRR基序,被认为是参与蛋白质与蛋白质相互作用的区域,这也是多数植物PGIP抗病基因表达蛋白特有的保守序列(Jones D A,JonesI D G. The role of leucine-rich repeats in plant defences[J]. Advances inBotanical Research,1997,24:89-167)。PGIPs通过LRR基序中暴露于外表面的氨基酸残基与PG活性位点处的氨基酸残基相互作用,从而抑制PG的活性(Maulik A,Ghosh H,Basu S.Comparative study of protein-protein interaction observed inpolygalacturonase-inhibiting proteins from Phaseolus vulgaris and glycine maxand polygalacturonase from Fusarium moniliforme. BMC Genomics,2009,10(Suppl3):3-19)。
根腐病菌(Bipolaris sorokiniana)侵染小麦(Triticum aestivum)后,小麦中TaPGIP基因的表达水平上调,过表达TaPGIP基因的转基因烟草植株在受根腐病菌侵染12 h时,TaPGIP转基因烟草对小麦根腐病菌的抗性显著增强,表明TaPGIP参与小麦对根腐病菌的抗性(Janni M, Bozzini T, Moscetti I, et al. Functional characterisation ofwheat PGIP genes reveals their involvement in the local response to wounding.Plant Biol, 2013, 15(6): 1019-1024)。油菜(Brassica napus)BnPGIP基因的表达受死体营养型真菌核盘菌(Sclerotinia sclerotiorum)的诱导,过表达BnPGIP1和BnPGIP2的拟南芥(Arabidopsis thaliana)植株延迟了核盘菌侵染引起的病害症状(Bash Z D,RimmerS R, Khachatourians G G, et al. Brassica napus polygalacturonase inhibitorproteins inhibit Sclerotinia sclerotiorum polygalacturonase enzymatic andnecrotizing activities and delay symptoms in transgenic plants. Can JMicrobiol, 2013, 59(2): 79-86),过表达Bnpgip2的转基因油菜显著减轻了核盘菌侵染造成的腐烂症状(Huang F H, Guan C, Jin F, et al. Prokaryotic expression andprotein function of Brassica napus PGIP2 and its genetic transformation.Plant Biotechnol Rep, 2013, 8(2): 171-181)。此外,拟南芥PPGIP2的表达受伤口、茉莉酸的诱导,可见PGIP基因参与拟南芥对生物和非生物胁迫的保护作用。桃(Prunus persica)经外源水杨酸处理后,与对照组相比,其PPGIP1基因的表达水平明显增加,说明PPGIP1与桃抗非生物胁迫能力有关(Liang F S, Zhang K C, Zhou C J, et al.Cloning, characterization and expression of the gene encodingpolygalacturonase-inhibiting proteins (PGIPs) of peach [Prunus persica (L.)Batch] . Plant Sci, 2005, 168(2): 481-486)。
三七(Panax notoginseng)是我国的传统名贵中药材,其种植区域主要分布于我国云南省,由于三七性喜温暖阴湿,对光敏感,生长周期长。三七独特的生长坏境易诱发多种病害,尤其是真菌病害,其中主要由茄腐镰刀菌引起的根腐病是三七的主要病害,严重影响了三七原药材的产量和品质。
发明内容
本发明的目的是提供一种三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用,即在提高烟草对葡萄座腔菌(Botrosphaeria dothidea)、串珠状赤霉菌(Gibberella moniliformis)、人参链格孢(Alternaria panax whetz)抗性中的应用。
本发明从三七中克隆获得的具有抗真菌活性的多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的全长基因,PnPGIP的核苷酸序列如SEQ ID NO:1所示,该基因全长为1171bp,包含一个981bp的开放阅读框、13bp的5′非翻译区(untranslated region, UTR)及177bp的3′UTR,编码如SEQ ID NO:2所示氨基酸序列的蛋白质。
本发明所述三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的编码区是序列表SEQID NO:1中第14-994位所示的核苷酸序列。
本发明分离克隆三七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PnPGIP。
本发明涉及分离包含PnPGIP的DNA片段并鉴定其功能;对该基因的序列进行分析,表明PnPGIP全长cDNA为1171bp,包含一个981bp的开放阅读框、13bp的5′非翻译区(untranslated region, UTR)及177bp的3′UTR,其中ORF编码一个具有326个氨基酸的蛋白质。BLASTn分析表明PnPGIP的部分序列与黄瓜(Cucumis sativus) PGIP (XM_017371813.1)具有75%的相似性,与芝麻(Sesamum indicum)的PGIP基因 (XM_011088934.2)具有74%的相似性。蛋白质同源性分析表明PnPGIP编码的蛋白质序列与黄瓜PGIP蛋白具有70%的相似性,与葡萄(Vitis vinifera)、向日葵(Helianthus annuus)的PGIP蛋白分别具有62%和59%的相似性。PnPGIP编码的蛋白质序列具有PGIP超家族的保守结构域,这表明其属于三七中的PGIP类似蛋白。超表达序列表SEQ ID NO:1所示序列可以增强烟草对葡萄座腔菌(Botryosphaeria dothidea)、人参链格孢(Alternaria panax whetz)和串珠状赤霉菌(Gibberella moniliformis)的抗性。
上述PnPGIP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PnPGIP的特异引物,从接种茄腐镰刀菌后12 h的三七根中提取总RNA,通过逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)扩增出PnPGIP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BamHI和EcoRI酶切pGEM-T-PnPGIP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PnPGIP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)重组载体T-DNA上具有卡那霉素抗性基因,用添加卡那霉素的分化培养基筛选转化子,并通过PCR以及RT-PCR检测得到真正的转基因植株,分析转基因植株对于植物病原真菌的抗性,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;本发明中来自三七的PnPGIP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、药用植物等提供方便,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PnPGIP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;阳性对照:质粒pGEM-T-PnPGIP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PnPGIP转基因烟草中PnPGIP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;阳性对照:质粒pGEM-T-PnPGIP为模板的PCR产物;
图3是本发明中PnPGIP转基因烟草体外抗真菌活性的抑菌效果图;其中a、b、c图示中的真菌分别是串珠状赤霉菌、葡萄座腔菌和人参链格孢;WT为野生型烟草的总蛋白;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:PnPGIP全长cDNA克隆以及序列分析
用茄腐镰刀菌接种三七的根,用接种后12 h的根提取总RNA,用液氮将处理过的三七的根研磨成粉末,然后转入离心管中,采用异硫氰酸胍法提取总RNA;采用M-MLV逆转录酶(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),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 (200 U)、1 μL M-MLV (200 U),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PnPGIP,所用上下游引物序列分别为5’GGAAGACCAATGTTGTTACTCCTCT3’及5’TCCATTGAGTACTAGGGAAACCATG3’。采用AdvantageTM 2PCR Enzyme (Clontech)扩增出目的基因。PCR反应条件:95℃ 1 min;94℃ 30 s,60℃ 30s,72℃ 1 min,32个循环;72℃ 5 min。反应体系(20μL)为1 μL cDNA、2 μL 10×Advantage2 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,直接对PCR产物进行TA克隆,使用的试剂盒为pGEM-T vector kit (Promega),反应体系和操作过程为:取1.5 μL PCR产物,依次加入1 μL pGEM-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PnPGIP的特异引物检测多克隆位点插入PnPGIP的克隆。将得到的阳性克隆进行测序,最终获得的PnPGIP全长cDNA为1171bp,通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个981bp的开放读码框(见序列表)。PnPGIP编码一个含326个氨基酸的蛋白质PnPGIP,其分子量约为36.77KDa,等电点为5.83。借助生物信息学软件SignalP 4.1分析PnPGIP编码的蛋白序列,检测其是否具有N端信号肽。结果显示在PnPGIP中有信号肽,因此推测PnPGIP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PnPGIP的大肠杆菌质粒pGEM-T-PnPGIP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BamHI和EcoRI分别对质粒pGEM-T-PnPGIP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 μLpGEM-T-PnPGIP和pCAMBIA2300S质粒、依次加入10 μL 10×H buffer、5 μL EcoRI、5 μLBamHI、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,然后使用试剂盒对PnPGIP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PnPGIP 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 PnPGIP DNA片段依次加入2 μLpCAMBIA2300S载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μLddH2O,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PnPGIP的特异引物进行PCR,挑选出PnPGIP与pCAMBIA2300S成功连接的克隆,在得到的阳性菌株中加入甘油并置于-80℃保存备用。
采用试剂盒提取并纯化上述大肠杆菌DH5α中的pCAMBIA2300S-PnPGIP质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PnPGIP转入所制备的根癌农杆菌LBA4404感受态细胞中。操作步骤为:取0.2 μg pCAMBIA2300S-PnPGIP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500μL 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PnPGIP的特异性引物进行PCR反应,检测pCAMBIA2300S-PnPGIP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℃,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-PnPGIP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。
取烟草无菌苗幼嫩叶切成约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+50mg/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(MS+30 g/L蔗糖+6 g/L琼脂+)继代培养。
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PnPGIP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,PnPGIP转基因烟草共筛选到56株阳性转基因植株。
实施例4:转基因烟草中PnPGIP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PnPGIP的特异引物进行PCR,根据PCR结果分析各转基因植株中PnPGIP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,共检测到29个转基因单株中PnPGIP在转录水平大量表达,这些单株的编号为1~49。
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,20g/L葡萄糖)上,28℃暗培养,待菌落生长至直径约为2~3 cm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6、11、25、38)及野生型叶片放入研钵中,加入1mL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/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℃培养几天后观察真菌生长的情况,并据此来评价PnPGIP转基因烟草的体外抗真菌活性,结果如图3所示,PnPGIP转基因烟草蛋白对串珠状赤霉菌、葡萄座腔菌和人参链格孢的生长具有明显的抑制作用。
序列表
<110> 昆明理工大学
<120> 三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1171
<212> DNA
<213> Panax notoginseng
<220>
<221> mRNA
<222> (1)..(1171)
<220>
<221> 5'UTR
<222> (1)..(13)
<220>
<221> CDS
<222> (14)..(994)
<220>
<221> 3'UTR
<222> (995)..(1171)
<400> 1
gcggggaaga ccaatgttgt tactcctctt cttgatctac ggcctctctc cggtgaaccc 60
ttgccaccca aatgacttat ccgccatttt acacttcaaa aatagcttct ccaattcgga 120
tctcttgtct gaatggtcat ctgatatgaa ttgttgtcac atatttacct gtgaaaataa 180
tcgtattatt gagtttacat tagatattta tgatctttcc ggttcactta aaccggacgc 240
cctagccggt ttgacatacc tcaagagact tcggcttcac aagatgccat ttcttatcgg 300
tgaaatccct ccggcaatag aaaatttgac tcacttaact taccttgaaa taacttggac 360
caatatctct ggacctatac cccaaaatct cgccaacctc ataaatctca atactctcga 420
tttctcgttt aataaactct atggctcaat tccactctcc cttcttacat taccaaatat 480
tttttctata gaccttagta gaaatcaact aaccggaccg atacctgaat cttatggtca 540
aattccaaat acacacaatt tactcgttct catactatct cataacaagc tttcgggtaa 600
attaccggcg tctttaggca gtatgaattt ctctcgaatt gatctctcga gaaataatct 660
ctccgacgat gcatccatgc tttttggtgc gactaaaggt tgtcaagtgc ttgatatctc 720
acgaaataac tttgagtttg atttctcacg agtggagttc atggacgagt cattgcaaat 780
attggatata tctcataaca agatatatgg gaaaataccg caattgatca cggaagcttc 840
catgttgcaa gggttgaatt ttagttataa tagattgtgc ggcgagatac ccaatggatg 900
gaagttaagg tatcggtcgg agggatttga taactcgtca ttttctcata atcgttgctt 960
gtgtggagct ccacttgatc cttggaaatt ttgatttagt tattaggaca tggtttccct 1020
agtactcaat ggaggatttg gctgcaagcc cgtgtaaaca ttttttttat atttatttat 1080
aagaattata atttcgagat tataatttat ttgattataa tttcaatact actattaatt 1140
ttagaaaaaa aaaaaaaaaa aaaaaaaaaa a 1171
<210> 2
<211> 326
<212> PRT
<213> Panax notoginseng
<400> 2
Met Leu Leu Leu Leu Phe Leu Ile Tyr Gly Leu Ser Pro Val Asn Pro
1 5 10 15
Cys His Pro Asn Asp Leu Ser Ala Ile Leu His Phe Lys Asn Ser Phe
20 25 30
Ser Asn Ser Asp Leu Leu Ser Glu Trp Ser Ser Asp Met Asn Cys Cys
35 40 45
His Ile Phe Thr Cys Glu Asn Asn Arg Ile Ile Glu Phe Thr Leu Asp
50 55 60
Ile Tyr Asp Leu Ser Gly Ser Leu Lys Pro Asp Ala Leu Ala Gly Leu
65 70 75 80
Thr Tyr Leu Lys Arg Leu Arg Leu His Lys Met Pro Phe Leu Ile Gly
85 90 95
Glu Ile Pro Pro Ala Ile Glu Asn Leu Thr His Leu Thr Tyr Leu Glu
100 105 110
Ile Thr Trp Thr Asn Ile Ser Gly Pro Ile Pro Gln Asn Leu Ala Asn
115 120 125
Leu Ile Asn Leu Asn Thr Leu Asp Phe Ser Phe Asn Lys Leu Tyr Gly
130 135 140
Ser Ile Pro Leu Ser Leu Leu Thr Leu Pro Asn Ile Phe Ser Ile Asp
145 150 155 160
Leu Ser Arg Asn Gln Leu Thr Gly Pro Ile Pro Glu Ser Tyr Gly Gln
165 170 175
Ile Pro Asn Thr His Asn Leu Leu Val Leu Ile Leu Ser His Asn Lys
180 185 190
Leu Ser Gly Lys Leu Pro Ala Ser Leu Gly Ser Met Asn Phe Ser Arg
195 200 205
Ile Asp Leu Ser Arg Asn Asn Leu Ser Asp Asp Ala Ser Met Leu Phe
210 215 220
Gly Ala Thr Lys Gly Cys Gln Val Leu Asp Ile Ser Arg Asn Asn Phe
225 230 235 240
Glu Phe Asp Phe Ser Arg Val Glu Phe Met Asp Glu Ser Leu Gln Ile
245 250 255
Leu Asp Ile Ser His Asn Lys Ile Tyr Gly Lys Ile Pro Gln Leu Ile
260 265 270
Thr Glu Ala Ser Met Leu Gln Gly Leu Asn Phe Ser Tyr Asn Arg Leu
275 280 285
Cys Gly Glu Ile Pro Asn Gly Trp Lys Leu Arg Tyr Arg Ser Glu Gly
290 295 300
Phe Asp Asn Ser Ser Phe Ser His Asn Arg Cys Leu Cys Gly Ala Pro
305 310 315 320
Leu Asp Pro Trp Lys Phe
325
<210> 3
<211> 25
<212> DNA
<213> 人工序列
<400> 3
ggaagaccaa tgttgttact cctct 25
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<400> 4
tccattgagt actagggaaa ccatg 25
Claims (1)
1.一种三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP在提高烟草对葡萄座腔菌(Botrosphaeria dothidea)、串珠状赤霉菌(Gibberella moniliformis)、人参链格孢(Alternaria panax whetz)抗性中的应用,所述三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的核苷酸序列如SEQ ID NO:1所示。
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CN109295069A (zh) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | 珠子参转录因子基因PjMYB1的应用 |
CN112662679A (zh) * | 2020-12-14 | 2021-04-16 | 宁波大学 | 一种桃果实多聚半乳糖醛酸酶抑制蛋白PpPGIP1基因及其克隆方法和应用 |
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