CN106589086A - 三七抗病相关蛋白PnPR10‑2及其编码基因与应用 - Google Patents
三七抗病相关蛋白PnPR10‑2及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了一种三七抗病相关蛋白PnPR10‑2及其编码基因与应用,其氨基酸序列如SEQ ID NO:2所示,编码该蛋白的基因的核苷酸序列如SEQ ID NO:1所示;本发明的三七抗病蛋白PnPR10‑2在植物抗根腐病领域具有广阔的应用前景,可用于培育抗根腐病园艺植物,有一定的经济效益潜力。
Description
技术领域
本发明涉及一种三七抗病相关蛋白PnPR10-2及其编码基因与应用。
背景技术
三七受到致病菌的侵染后,植物中的一系列基因响应而上调表达,其中的一些基因与防御反应相关,包括病程相关蛋白(PR)和抑菌基因,其通过增强其蛋白活性来提高抗病能力。PR蛋白被认为可被病原菌和非生物胁迫诱导。根据PR蛋白的结构和功能,目前PR蛋白在单子叶植物和双子叶植物中被发现并已确认了17个家族。PR-10是种子植物中广泛分布的一种PR蛋白,它与树木花粉过敏原和食物过敏源十分相似,属于Bet v 1-like超家族,这类基因编码的蛋白分子量在15~19kD,等电点偏酸性,无信号肽、属胞内蛋白(intracellular pathogenesis-related proteins,IPR)。PR10除了与植物的生物及非生物胁迫密切相关,还在植物的生长发育,次级代谢等过程中起着重要作用。证据表明,PR10直接参与植物的防卫反应,具有体外抗菌活性或核酶活性。
三七(Panaxnotoginseng(Burk.)F.H.Chen)为我国传统的名贵中药材,多年来,其规模化种植受到以根腐病、白粉病等病害的严重限制,其中,根腐病常年发病率为5%~20%,严重的达70%以上,甚至绝收,且生长年限越长病害越严重,目前根腐病的控制手段主要以抗菌性农药的大量施用为主,根腐病的无害化防治已经成为了三七栽培技术研究的热点和难点问题。三七根腐病病原菌比较复杂,致病菌包括细菌中的假单胞杆菌(pseudomonas sp.)、真菌中的坏损柱孢菌(Cylindrocarpondestructans,C.didynum)、镰刀菌(Fusariumsolani,Fusariumsolani f.sp.Radicicola,F.oxysporumSchlecht.,F.scirpi lamb.)等,通常以坏损柱孢菌、镰刀菌或假单胞菌中某一类为主,数种病原菌复合侵染,加速根系腐烂。
目前根腐病的防治主要通过化学农药大量施用的方法,对药材品质和安全性有一定程度的影响,而目前尚未发现对根腐病有较好抗病潜力的三七品种,因此,通过克隆三七中的抗根腐病病PR基因,研究其抗病能力,对于培育和筛选抗病品种,利用分子育种方法培育抗根腐病三七等园艺作物新品种具有重要意义。
发明内容
本发明的目的是提供三七抗病相关蛋白PnPR10-2及其编码基因。
本发明所提供的三七病程相关蛋白,名称为PnPR10-2,来源于三七,是具有下述氨基酸序列之一的蛋白质:
(1)序列表中的SEQ ID NO:2的氨基酸残基序列;由154个氨基酸残基组成;
(2)将序列表中SEQ ID NO:2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物抗病相关的蛋白质。
所述一个或几个氨基酸残基的取代和/或缺失和/或添加是指不多于十个氨基酸残基的取代和/或缺失和/或添加。
上述三七病程相关蛋白的编码基因,具有下述核苷酸序列之一:
(1)序列表中SEQ ID NO:1的DNA序列;
(2)编码SEQ ID NO:2蛋白质序列的多核苷酸;
含有本发明基因的表达载体、细胞系及宿主菌均属于本发明的保护范围。
本发明中的PnPR10-2基因可用现有的方法构建到现有的原核表达载体或植物表达载体中,可在其转录起始核苷酸前加上包括组成型启动子、增强型启动子、诱导型启动子、组织特异性启动子、发育阶段特异型启动子在内的任何一种启动子,为了便于对转PnPR10-2基因细胞或植物进行鉴定及筛选,可对所使用的载体进行加工,如加入具有抗性的抗生素标记物(氨苄青霉素、庆大霉素、卡那霉素等)或加入植物可选择性标记(GUS基因、荧光素酶基因等)。被转化的植物宿主既可以是单子叶植物,也可以是双子叶植物,如水稻、小麦、玉米、黄瓜、烟草等。携带有本发明的PnPR10-2基因的表达载体可通过施用植物病毒载体、直接DNA转化、农杆菌介导等常规生物学方法转化植物细胞或组织,并将转化的植物经组织培养成植株,获得抗病性提高的植株。
实验表明,PnPR10-2的重组纯化蛋白,对典型根腐病病菌腐皮镰刀菌(Fusariumsolani)和人参锈腐病菌(Cylindrocarpon destructans)有抑制作用;本发明的基因可以转入植物中提高植物的抗病性,同时还为植物抗病育种打下良好的工作基础。
附图说明
图1为从根腐病菌诱导的三七主根中扩增PnPR10-2基因片段的电泳图;
图2为PnPR10-2蛋白保守结构域预测;
图3为PnPR10-2与其它相似物种的PR蛋白氨基酸序列比较,其中三七(P.notoginseng)PnPR10-2(KY129859);人参(P.ginseng)PR10(ACY36943.1);胡萝卜(Daucuscarota subsp.sativus)PR蛋白(XP_017234488.1)、香芹菜(P.crispum)PR1(CAA67246.1)、山芹菜(S.brachycarpa)PR蛋白(AAC31957.1)、马铃薯(S.tuberosum)PR蛋白(XP_006349827.1)、丹参(S.miltiorrhiza)PR10(AFJ05109.1)、猕猴桃(A.chinensis)PR蛋白(CAM31908.1);
图4为PnPR10-2基因进化树分析图;
图5为PnPR10-2蛋白的表达及纯化,图中:1为pET-32a-PnPR10-2蛋白原液(上清);2为洗脱条件(80%结合液+20%洗脱液);3为洗脱条件(60%结合液+40%洗脱液);4为洗脱条件(40%结合液+60%洗脱液);5为洗脱条件(20%结合液+80%洗脱液);
图6为PnPR10-2纯化蛋白的抑制根腐病菌试验结果,其中A图为对腐皮镰刀菌处理0h和24h后的抑菌效果图;B图为对人参锈腐病菌处理4h和24h后的抑菌效果图。
具体实施方式
下面通过实施例和附图对本发明作进一步详细说明,但本发明保护范围不局限于所述内容。实施例中方法如无特殊说明,按常规操作进行,如无特殊说明使用试剂均为常规购试剂或按常规方法配制的试剂。
实施例1、PnPR10-2保守片段及其cDNA全长的获得
三七根部总RNA提取采用上海生工柱式植物总RNA抽提纯化试剂盒(SK8662),提取后进行RNA电泳验证。RNA提取成功后,采用TaKaRa PrimeScript RT reagent Kit进行逆转录反应,用于后续实验。
根据转录组cDNA数据库中预测的三七PR10-2基因序列,设计含酶切位点的全长特异性引物PR10-2-F(5’-GGATCCatgggtgtccaaaagaccgaaac-3’,GGATCC为BamHI酶切位点)和PR10-2-R(5’-GAATTCctaatttgctaggaggtaagcttcaacagc-3’,GAATTC为EcoRI酶切位点)。
使用二年生三七主根的cDNA进行扩增,PCR反应条件为:94℃3min,94℃30s,54℃30s,72℃90s,35cycles,72℃l0min。PCR产物使用1%琼脂糖凝胶电泳检测(图1)。然后对PnPR10-2基因进行T/A克隆,转化Trans1-T1感受态细胞,涂Amp平板,12h后挑取三个单菌落,37℃Amp-LB培养基摇菌4h后,送上海生工生物技术公司进行测序。将测序结果取出载体序列后进行序列比对,最终确定PnPR10-2基因的开放阅读框并获得含酶切位点的cDNA片段(见SEQ ID NO:1)。
将PnPR10序列输入NCBI中的ORF(Open Reading Frame Finder)(www.ncbi.nlm.nih.gov/gorf/gorf.html)和DNAMAN软件分析序列,结果显示PnPR10-2全长465bp,编码154个氨基酸(见SEQ ID NO:2)。同时,用ExPASY(ProtParam)软件分析PnPR10-2编码的蛋白质分子量为16582.8道尔顿,理论pI值为4.41,分子式为C743H1180N182O239S3。PnPR10的154个氨基酸(SEQ ID NO:2)中碱性氨基酸(R,H,K)13个,酸性氨基酸(D,E)22个,非极性氨基酸(A,V,L,I F,W,M,P)有66个,非电离的极性氨基酸(G,S,T,C,Y,N,Q)有53个。其中,带负电荷的氨基酸包括D和E,共计22个,带正电荷的氨基酸包括R和K,共11个。
利用在线工具InterPro(http://www.ebi.ac.uk/interpro/search/sequence-search)对PnPR10-2蛋白预测保守结构域(采用默认参数),详见图2,在PnPR10-2蛋白中发现其含有1个保守结构域家族,Bet v I型过敏源(Bet v I type allergen,IPR024949),该家族中含有病程相关蛋白Bet v I结构PATHOGENESIS_BETVI,PS00451),位于89~120位,以及7个Major pollen allergen Bet V I乳胶蛋白过敏原结构(Major pollen allergenBet V I,PR00634),分别位于3~23位,26~36位,5-~59位,66~85位,85~98位,109~125位,143~153位;此外,还在PnPR10-2蛋白中发现START类结构域(START-like domain,IPR023393),Bet v I乳胶蛋白结构域(Bet v I/Major protein,IPR000916);两个Bet_v 1类似结构域(SSF55961和cd07816);两个未命名的结构域(PTHR31213)和PTHR31213:SF14)。Bet v I型过敏原(Bet v I type allergen)的基因本体(GO)预测表明,该蛋白参与防御反应(GO:0006952)和生物应激反应(GO:0009607)的生物学过程。
三七PnPR10-2序列经Blast比对后,检测与之覆盖率相对较高的几种植物,并获得与三七PnPR10-2蛋白相似其它植物PR蛋白质序列。选择与三七PnPR10-2蛋白亲缘关系相对较近的人参、胡萝卜、香芹菜、山芹菜、马铃薯、丹参、猕猴桃等植物的PR蛋白进行氨基酸序列比对(详见图3),同源性分别为97%,62%,61%,57%,52%,51%,50%。以上基因编码的氨基酸序列长度相似,在N端和C端有较高的保守性。
利用DNAMAN软件进行同源性分析,将三七PnPR10-2蛋白与人参、胡萝卜、香芹菜、山芹菜、马铃薯、丹参、猕猴桃、芝麻、葡萄的蛋白构建了系统发育树,见图4。三七PnPR10-2蛋白与人参PR10蛋白(登录号:ACY36943.1)聚为一类,表现较近的亲缘关系。
实施例2、PnPR10-2原核表达载体的构建、重组蛋白的表达及纯化
1、pET32a-PnPR10-2原核表达载体的构建
(1)质粒提取:挑取含pET-32a空载体的的单克隆菌落及测序正确的pMD-18T-PnPR10-2阳性克隆菌落摇菌后提取质粒;
(2)酶切与胶回收:将测序检测正确的pMD-18T-PnPR10-2载体质粒用限制性内切酶BamHI和EcoRI双酶切,琼脂糖凝胶电泳检测并用DNA回收试剂盒回收;
(3)连接:将回收的目的片段与酶切后的pET32a空载体用DNA ligation kit进行连接,转化Trans1T1感受态细胞,涂Amp平板,12h后挑取三个单菌落,进行PCR检测并送上海生工测序验证;
(4)将测序正确的菌,抽取5μL,37℃下10mL Amp-LB培养基摇菌12h,提取pET-32a-PnPR10-2质粒,转化大肠杆菌BL21感受态细胞,涂Amp平板,12h后挑取三个单菌落,Amp-LB培养基摇菌12h,PCR检测,加入30%甘油,将菌种保存于-80℃备用。
2、pET32a-PR10-2重组蛋白的表达和纯化
将pET32a-PnPR10-2重组工程菌在含Amp100mg/L的LB培养基中培养至OD值达到0.5~0.7之间,加入终浓度为1mmol/L的IPTG(Isopropyl-beta-D-thiogalactoside)分别在20℃、28℃和37℃诱导8h,并对诱导蛋白进行超声破碎,将获得的蛋白上清和沉淀进行SDS-PAGE电泳鉴定表达情况。
A、SDS-PAGE步骤
(1)用酒精彻底清洗玻璃板,自然晾干,组装电泳槽;
(2)根据表6.1配制分离胶,将分离胶倒入玻璃板之间,然后用异丁醇覆盖;
(3)待分离胶凝固后,倒去上层的异丁醇,按配方配制浓缩胶,插上梳子;
(4)待浓缩胶凝固后,拔去梳子,向电泳槽中加入电泳缓冲液;
(5)蛋白样品处理:蛋白样品100μL,蛋白上样缓冲液20μL(Tris-HCl(pH6.8)250mM,SDS 10%,BPB 0.5%,甘油50%,β-巯基乙醇:5%),沸水浴5min后置于冰上;
(6)电泳条件:开始时的电压为60V、100mA,蛋白样品再进入分离胶后,电压改成120V、150mA。然后继续电泳一直到染料从下部流出,断开电源;
(7)考马斯亮蓝染色,用刀片分离玻璃板,切除浓缩胶,将分离胶置于染色液中,50rpm,30min,染色后回收染色液,将分离胶置于清水中,脱色2h,将脱色后的凝胶拍照。
B、pET32a-PR重组蛋白的纯化步骤
(1)大量培养含重组质粒pET32a-PR的表达菌株,按照优化条件大量诱导表达PR蛋白,经10000rpm/min离心15min,收集上清液,加入20mL PBS(pH8.0)洗脱缓冲液重悬浮,超声破碎5min,4℃,12000rpm,离心15min;
(2)上清利用0.22μm的滤器进行过滤除菌;
(3)His-Trap HP柱预处理:先使用10倍柱体积纯水洗柱;再用5倍柱体积的结合缓冲液(磷酸钠缓冲液20mM,NaCl 0.5M,咪唑30mM,pH7.4).平衡柱子,流速为1mL/min;
(4)将破碎过滤后的蛋白的上清液过柱,流速控制在1mL/min;
(5)洗脱,使用1倍柱体积的洗脱缓冲液(磷酸钠缓冲液20mM,NaCl 0.5M,咪唑200mM,pH7.4)进行洗脱,收集洗脱液;
(6)His-Trap HP柱后处理:用5倍体积的洗脱缓冲液(磷酸钠缓冲液20mM,NaCl0.5M,咪唑200mM,pH7.4)进行洗脱,再用5倍体积的纯水洗脱柱子,最后再用5倍体积的20%的乙醇洗脱,保存于4℃、20%的乙醇中;
(7)将纯化后的蛋白进行SDS-PAGE分析。
结果表明,将纯化后的蛋白进行SDS-PAGE,结果如图5所示,当洗脱液含80%结合液及20%洗脱液时,纯化蛋白浓度最高,但纯度较低;而当洗脱液含40%结合液及60%洗脱液时,蛋白纯化纯度较高,但浓度不高。蛋白纯化实验效果较好,测定了纯化后蛋白液的浓度后,-80℃保存。
实验例3:纯化PnPR10-2重组蛋白抑菌试验
1、带菌平板制备
筛选适宜菌株腐皮镰刀菌(Fusarium solani)和人参锈腐病菌(Cylindrocarpondestructans)的改良PDA培养基,筛选得到最佳培养条件。制备改良PDA固体平板(直径90mm),用接种针挑取菌丝,接种于PDA培养基上,于28℃培养箱中培养72h,即制备好带菌平板。
2、重组PnPR10-2蛋白对病菌的抑制作用实验
采用纸片法进行抑菌试验,取10μL纯化重组PnPR10-2蛋白滴到直径6mm的灭菌滤纸片上,放到已制备好的带菌斑的PDA固体培养基平板上进行共培养,磷酸盐缓冲液作阴性对照,观察和测定滤纸片抑菌环的宽度,并进行拍照,确定纯化重组PnPR10-2蛋白是否有抑菌活性。由于两种菌株的生长速率不同,采用不同处理。对腐皮镰刀菌(Fusarium solani)采用直径为15mm纸片,加入纯化PnPR10-2重组蛋白溶液量为50μL,浓度分别为467μg/mL和93.4μg/mL两个处理,处理24h。对人参锈腐病菌(Cylindrocarpon destructans)采用直径为4mm纸片,加入纯化PnPR10-2重组蛋白溶液量为10μL,浓度为467μg/mL,处理24h。
结果表明,可见清晰的抑菌环,如图6,表明PnPR10-2重组纯化蛋白对典型根腐病病菌腐皮镰刀菌(Fusarium solani)和人参锈腐病菌(Cylindrocarpon destructans)有抑制作用。
实验例4:PnPR10-2植物表达载体的构建和转基因株系抗根腐病能力分析
1、PnPR10-2植物表达载体的构建
(1)入门载体的构建
用BamHI和EcoRI酶切空pENTR2B载体和pMD18-T-PnPR10-2载体,并用胶回收试剂盒回收PnPR10-2目的基因片段和pENTR2B,配制连接体系(目的片段2μl,pENTR2B 1μl,ligation solution 3μl),于16℃连接8-12h。热激转化DH5α感受态,并将其涂在Kan抗性的LB平板上,挑取单菌落,提取质粒,进行PCR、酶切检测,并测序。
(2)Gateway LR反应
测序正确的质粒pENTR2B-PnPR10-2通过Gateway技术与植物表达载体pK2GW7进行连接,进行热击转化,将其转入DH5α感受态细胞中,并将其涂在Spe抗性的LB平板上,挑单菌落,进行菌液PCR检测。
2、烟草的转化
根据已知的方法把植物表达载体pK2-35S-PnPR10-2转入农杆菌LBA4404中。通过农杆菌介导,用叶盘法将该植物表达载体转化烟草(Nicotiana tabacum cv.Xanthi)。转化后的叶盘置于25℃恒定光照(100μmol·m-2·s-1)下培养,在含有卡那霉素(Km,50μg/ml)的培养基上筛选转基因小苗。
3、转基因烟草的培养和鉴定
将筛选的具有Km抗性的烟草小苗转移到含有3%蔗糖的MS培养基上继续培养,期间通过剪切叶片,提取基因组DNA进行PCR检测。同时,提取RNA,逆转录cDNA进行realtimePCR检测,鉴定得到转基因烟草幼苗并获得其PnPR10-2表达水平信息。将检测后的转基因烟草小苗转移到含有1/2珍珠岩和1/2有机土的花盆中培养,获得土壤栽培的转基因烟草植株。
4、转基因烟草对根腐病病菌抗性能力分析
由于根腐病主要为真菌病害,因此采用针刺法开展抗病性研究。首先将灭菌的牙签与腐皮镰刀菌(Fusarium solani)在PDA培养基上进行共培养10d,使牙签布满根腐病病菌菌丝体。
选取经分子鉴定的转基因株系,以野生型烟草为对照,将植株移入植物培养箱。植物适应3d后,用针刺法在植株根基部接种腐皮镰刀菌(Fusarium solani),每个转基因株系接种3株苗,在接种10d和20d后进行病情调查。病情指数=(发病级数×叶片数/最高发病级数×总叶片数)×100%。
病情指数分级标准:叶片没有明显变化为0级,小于1/4叶片出现轻微变黄为1级,小于1/4叶片叶片出现大面积变黄为3级,约1/2叶片变黄或萎蔫定为5级,叶片全部变黄或萎蔫定为7级。
在接种根腐病病菌腐皮镰刀菌(Fusarium solani)10d,与野生型烟草相比,发现2个转基因株系病情指数差异达到显著水平。接种根腐病病菌腐皮镰刀菌(Fusariumsolani)20d,与野生型烟草相比,发现2个转基因株系病情指数达到显著水平,2个转基因株系达到极显著水平。
结果表明,PnPR10-2基因在植物中的过量表达能够赋予烟草对典型根腐病菌腐皮镰刀菌(Fusarium solani)有一定程度的抗性。
序列表
<110> 昆明理工大学
<120> 三七抗病相关蛋白PnPR10-2及其编码基因与应用
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 464
<212> DNA
<213> 三七
<400> 1
atgggtgtcc aaaagaccga aaccacggcc ttttccccag tcccggcaga aaagctgttc 60
aagggttctt tccttgacat ggacaccgtc gtccccaagg cttttccaga aggtattaag 120
agtgtccaag ttcttgaggg cgacggcgga gttggaacca tcaaaaacgt cactctaggg 180
gatgccaccc cattcaacac catgaatacc cggatagatg caattgacga gcatgcattg 240
acttacactt acaccattat aggaggtgat atccttttgg acattattga atccatagag 300
aatcatttca agattgtgcc tactgatgga gggagcacca tcacacagac taccatatat 360
aacaccatag gtgatgctgt aattccagaa gagaatatca aggatgccac cgataagtca 420
atccaactat tcaaggctgt tgaagcttac ctcctagcaa attag 465
<210> 2
<211> 154
<212> PRT
<213> 三七
<400> 2
Met Gly Val Gln Lys Thr Glu Thr Thr Ala Phe Ser Pro Val Pro Ala Glu Lys Leu Phe
1 10 20
Lys Gly Ser Phe Leu Asp Met Asp Thr Val Val Pro Lys Ala Phe Pro Glu Gly Ile Lys
30 40
Ser Val Gln Val Leu Glu Gly Asp Gly Gly Val Gly Thr Ile Lys Asn Val Thr Leu Gly
50 60
Asp Ala Thr Pro Phe Asn Thr Met Asn Thr Arg Ile Asp Ala Ile Asp Glu His Ala Leu
70 80
Thr Tyr Thr Tyr Thr Ile Ile Gly Gly Asp Ile Leu Leu Asp Ile Ile Glu Ser Ile Glu
90 100
Asn His Phe Lys Ile Val Pro Thr Asp Gly Gly Ser Thr Ile Thr Gln Thr Thr Ile Tyr
110 120
Asn Thr Ile Gly Asp Ala Val Ile Pro Glu Glu Asn Ile Lys Asp Ala Thr Asp Lys Ser
130 140
Ile Gln Leu Phe Lys Ala Val Glu Ala Tyr Leu Leu Ala Asn *
150
<210> 3
<211> 29
<212> DNA
<213> 人工序列
<400> 3
ggatccatgg gtgtccaaaa gaccgaaac 29
<210> 4
<211> 36
<212> DNA
<213> 人工序列
<400> 4
gaattcctaa tttgctagga ggtaagcttc aacagc 36
Claims (4)
1.一种三七抗病相关蛋白PnPR10-2,其氨基酸序列如SEQ ID NO:2所示。
2.权利要求1所述的三七抗病相关蛋白PnPR10-2的编码基因,其核苷酸序列如SEQ IDNO:1所示。
3.权利要求1所述的三七抗病相关蛋白PnPR10-2在提高植物抗病性中的应用。
4.权利要求2所述的三七抗病相关蛋白PnPR10-2基因在提高植物抗病性中的应用。
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