CN116676330A - StATP6V1C1在提高马铃薯对晚疫病抗性中的应用 - Google Patents
StATP6V1C1在提高马铃薯对晚疫病抗性中的应用 Download PDFInfo
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Abstract
本发明涉及植物生物技术领域,具体涉及一种马铃薯抗晚疫病基因StATP6V1C1的功能验证和应用。分别利用强启动子驱动和VIGS技术在马铃薯Désirée中超量及抑制StATP6V1C1基因的表达。StATP6V1C1瞬时表达和稳定转基因株系对马铃薯晚疫病的抗性显著增强,StATP6V1C1沉默株系更加感病,且抗病蛋白R2、Rpi‑mcq1对致病疫霉效应蛋白Avr2(PITG_22870)、AL3(PITG_23008)的识别依赖StATP6V1C1的表达,证明StATP6V1C1基因在马铃薯抗晚疫病中发挥重要作用。
Description
技术领域
本发明涉及植物基因工程技术领域,提供了一个马铃薯抗病相关基因StATP6V1C1的功能验证,StATP6V1C1正向调控马铃薯对晚疫病的抗性,并且两个抗病蛋白R2、Rpi-mcq1对致病疫霉效应蛋白Avr2、AL3的识别依赖StATP6V1C1的表达,证明StATP6V1C1基因在马铃薯抗晚疫病过程中发挥重要作用。
背景技术
植物固生性生长,即使遇到逆境环境也不能从栖息地自由移动趋避,必须直接承受非生物和生物胁迫,因此,植物发展出更为复杂的抵抗逆境胁迫的***,包括抵抗非生物胁迫和生物胁迫等。植物在与病原微生物长期协同进化的过程中形成了双层免疫防御***(Dangl and Jones,2006)。第一层,植物通过细胞膜定位的模式识别受体(patternrecognition receptor,PRR)直接识别病原菌的病原相关分子模式(pathogen associatedmolecular patterns,PAMPs),触发植物的第一层免疫***(Pattern-triggeredimmunity,PTI)来抵抗病原菌的侵染。为了对抗植物的第一层防御***,病原菌会通过特定的转运***向植物细胞内分泌效应因子(Effector),直接靶向抑制PRR或PTI免疫信号途径,使植物对病原体变得易感,我们称之为效应子触发的敏感性(Effector-triggeredsusceptibility,ETS)(Sáket al.,2021)。第二层,植物胞内抗病蛋白(R)或细胞膜上某些类受体蛋白(receptor-like proteins,RLPs)识别病原体效应蛋白的存在时,触发更为强烈的免疫反应,如超敏反应(Hypersensitive response,HR)和***性获得抗性(Systemicacquired resistance,SAR),一般通过局部细胞快速死亡限制病原的扩展,通常被称为效应器触发的免疫反应(effector-triggered immunity,ETI)。ETS也成为病原微生物抑制ETI,逃逸抗病反应的重要途径(Ngou et al.,2022)。植物和病原微生物,分别围绕PTI/ETI和效应分子不断进化各自武器库,形成一轮又一轮的军备竞赛(Arm race)。当前农业生产上,抗病品种培育主要针对主效抗病基因R介导的ETI反应进行作物改良,因此,R基因以及R蛋白识别病原菌效应分子的机制对于抗病品种改良具有重要的价值。
植物R蛋白与病原菌无毒基因(Avr)蛋白的识别是多数ETI产生的机制,一直也是植物免疫领域研究热点。已有报导R蛋白可以直接识别效应蛋白,例如水稻CC-NBS-LRR类R蛋白Pi-ta与稻瘟病原菌无毒蛋白AVR-Pita直接互作(Jia et al.,2000);亚麻TIR-NBS-LRR类R蛋白M与亚麻锈病病原效应因子AvrM直接识别(Catanzariti et al.,2010)。同时R蛋白也可以通过中间蛋白间接识别效应因子,早期深入研究的中间蛋白RIN4是一种重要的保卫蛋白,它可以被效应蛋白AvrPto和AvrRpt2诱导发生降解(Mackey et al.,2003;Luoet al.,2009),也能被AvrRpm1诱导进行磷酸化修饰(Mackey et al.,2002)。R蛋白RPS2可以监控AvrRpt2诱导的RIN4降解(Mackey et al.,2003);另外一个R蛋白RPM1也可以监控效应蛋白AvrB和AvrRpm1诱导的RIN4磷酸化(Mackey et al.,2002)。中间蛋白的存在增加了R-Avr识别的复杂度,使相对较小的植物R基因库能够有效抵御各种病原体的侵袭。目前,在马铃薯与致病疫霉的互作***中已知的最复杂抗性识别模型为效应蛋白Avr2可以通过依赖中间蛋白BSLs的方式被两个独立进化的抗病蛋白R2、Rpi-mcq1识别(Aguileragalvez etal.,2018),尚未有报导是否存在新的效应蛋白以及中间蛋白参与它们之间复杂的抗性识别过程。
马铃薯(Solanum tuberosum L.)是世界第三大主粮作物,迄今,晚疫病仍然是马铃薯生产中最具毁灭性的经济病害,具有发生范围广、流行速度快、为害损失重和窖藏损失大等特点。据不完全统计,全球平均每年晚疫病导致的损失和化学防治相关的成本总计超过90亿欧元(Haverkort et al.,2016)。
与抗病基因的应用相比,中间蛋白的应用能提供植物更为广谱及长效的抗性,因此如何筛选和利用抗病中间蛋白来防治马铃薯晚疫病是亟待解决的问题。
发明内容
本申请针对上述现有技术的情况,鉴定到一个马铃薯晚疫病正向调控因子StATP6V1C1,并且证明StATP6V1C1作为中间蛋白介导两个马铃薯R蛋白R2(ACU65456)、Rpi-mcq1(GN043560)交叉识别两个致病疫霉效应蛋白Avr2(XM_002902940)、AL3(XM_002899560),从双层面共同提高马铃薯对晚疫病的抗性。
为解决上述技术问题,本申请采用如下技术方案:
在本发明的第一方面,提供了StATP6V1C1在提高马铃薯对晚疫病抗性中的应用。
首先,本申请发明人克隆了StATP6V1C1的完整编码区序列,连入pCXSN-Myc载体,在马铃薯Désirée中瞬时表达,发现StATP6V1C1瞬时表达显著提高马铃薯对晚疫病的抗性(图1)。为了进一步确定StATP6V1C1的抗性功能,发明人创制了StATP6V1C1的稳定转基因株系,挑选三个阳性株系进行抗病性检测,发现以野生型为对照,三个StATP6V1C1-OE转基因株系水渍状病斑面积均小于对照组,菌丝生长量则分别下调26.39%、68.32%和52.84%;相比于野生型病情指数68.33%,三个超量株系病情指数分别为40%、50%和41.07%,显著低于对照组(图2)。
然后,本申请发明人克隆StATP6V1C1部分编码区序列(781-943bp),使用病毒介导的基因沉默(VIGS)技术在马铃薯中沉默该基因,相较于对照TRV::00株系,病毒介导的沉默株系TRV::StATP6V1C1(抑制效率77.32%)在接种晚疫病3天后病斑面积显著增大(图3)。
在本申请的第二方面,提供了StATP6V1C1与抗病蛋白R2、Rpi-mcq1以及致病疫霉效应蛋白Avr2、AL3形成的复合体在提高马铃薯、烟草对晚疫病抗性中的应用,所述StATP6V1C1作为桥梁蛋白介导两个抗病蛋白R2、Rpi-mcq1对两个致病疫霉效应蛋白Avr2、AL3的识别,是相关抗病基因产生抗病性的必需因子。
本申请发明人发现了ATP6V1C1影响R2、Rpi-mcq1介导的抗性识别,以R3a识别Avr3a产生的HR反应为对照,分别在TRV::00株系和TRV::NbATP6V1C1株系中交叉共表达两个抗病蛋白R2、Rpi-mcq1和两个效应蛋白Avr2、AL3,发现这四组交叉抗性识别激发的HR在TRV::StATP6V1C1株系中全部消失,说明ATP6V1C1是抗病蛋白R2、Rpi-mcq1发挥抗性功能的必需中间蛋白。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本申请发明人通过瞬时表达、稳定转基因以及VIGS技术鉴定该基因在抗病反应过程中所起作用,在国际上首次发现StATP6V1C1正向调控马铃薯对晚疫病的抗性。同时发明人证明StATP6V1C1作为中间蛋白介导两个抗病蛋白R2、Rpi-mcq1识别两个Avr2家族效应蛋白的抗性功能,从两个层面揭示了StATP6V1C1对马铃薯抗晚疫病的必需性。
附图说明
图1为瞬时表达StATP6V1C1提高马铃薯对晚疫病的抗性示意图;(A)使用携带pCX-StATP6V1C1-Myc的农杆菌GV3101侵染马铃薯,侵染24h后接种马铃薯晚疫病菌EC1孢子悬浮液,接种4天后观察发病情况,n=30。(B)病情指数统计数据。
图2为StATP6V1C1稳定转基因株系对马铃薯晚疫病抗性增强示意图;(A)StATP6V1C1-OE转基因株系增强马铃薯对晚疫病的抗性。以Actin为内参,使用anti-Myc标签抗体检测转基因株系阳性,接种晚疫病菌EC1孢子悬浮液3天后观察发病情况,n=30。(B)致病疫霉菌丝生长量(5dpi),t-test(P<0.05)。
(C)病情指数统计数据。
图3为抑制StATP6V1C1基因表达的VIGS株系接种致病疫霉EC1孢子悬浮液3天后发病结果示意图。(A)TRV::StATP6V1C1株系更感晚疫病。(B)病情指数统计,n=30。
图4为抑制StATP6V1C1基因表达对R2、Rpi-mcq1介导抗性识别的影响示意图;(A)TRV::00对照组中,R3a对Avr3a的识别以及R2、Rpi-mcq1与Avr2、AL3的四组交叉识别均能触发HR反应。(B)抑制StATP6V1C1基因表达的TRV::StATP6V1C1株系中,R3a对Avr3a的识别不受影响,R2、Rpi-mcq1与Avr2、AL3的四组交叉识别均消失。(C)HR坏死出现的概率。
具体实施方式
以下实施例中进一步定义本发明,根据以上的描述和这些实施例,本领域技术人员可以确定本发明的基本特征,并且在不偏离本发明精神和范围的情况下,可以对本发明作出各种改变和修改,以使其适用各种用途和条件。除特殊注明外,本发明所采用的均为本领域现有技术;
实施例1:StATP6V1C1基因的瞬时表达显著提高马铃薯对晚疫病的抗性
1、克隆StATP6V1C1基因
根据NCBI数据库中的基因序列(登录号LOC102595740)设计引物扩增StATP6V1C1基因(见表1中的引物1和引物2)。使用Plant RNA kit(OMEGA Bio-tek,Norcross,USA),取新鲜马铃薯叶片材料50-100mg提取RNA,采用HiFiScript gDNA Removal cDNA SynthesisKit(CWBIO,北京)试剂盒进行cDNA第一链合成。目的片段扩增使用KOD-Plus-Neo(TOYOBO,Osaka,Japan),使用诺为赞公司的DNA回收试剂盒回收并纯化扩增片段,将纯化DNA片段连接中间载体,连接反应使用T4 DNA ligase,使用热激法转化大肠杆菌DH5ɑ感受态细胞,挑选阳性克隆提质粒,测序由生工生物工程有限公司完成。结果以马铃薯Désirée的cDNA为模板,经过PCR扩增后,获得一个长度为1131bp的DNA片段。
表1
引物名称 | 序列(5’-3’) |
引物1 | ATGGCTTCCAGGTATTGGGTGGTTTCTC(SEQ ID NO.1) |
引物2 | TCAAACAAGATTTATTGTGAAGGAGACGT(SEQ ID NO.2) |
引物3 | GGCTTCCAGGTATTGGGTGGTTTCTC(SEQ ID NO.3) |
引物4 | TCAAACAAGATTTATTGTGAAGGAGACGT(SEQ ID NO.4) |
2、构建并转化StATP6V1C1-Myc载体
为了确定StATP6V1C1对马铃薯抗性的影响,发明人使用TA克隆方法将StATP6V1C1全长CDS序列***pCXSN-Myc载体,使用KOD-Plus-Neo(TOYOBO,Osaka,Japan)进行目的片段扩增(见表1中的引物3和引物4),载体酶切使用NEB公司购买的限制性内切酶Xcm I,连接反应使用T4 DNA ligase,挑取阳性克隆测序成功后转化农杆菌GV3101用于瞬时表达,转化农杆菌AGL1用于创制稳定转基因株系。
3、StATP6V1C1瞬时表达显著提高马铃薯对晚疫病的抗性
致病疫霉于18℃黑暗倒置培养14d,加入无菌水刮洗菌丝,冰浴2-3h使孢子囊充分破裂释放游动孢子。用无菌蒸馏水稀释并用血球计数板于生物显微镜下计数,调节孢子浓度至3.4-6.8×105/mL区间。发明人使用成功转化StATP6V1C1-Myc的GV3101注射侵染马铃薯Désirée叶片,侵染1天后摘取叶片进行离体接种,接种EC1菌株3天后观察发病表型并统计病情指数。病情等级按照发病面积占全株或全叶面积划分5级:0级(0%),1级(0-25%),2级(25-50%),3级(50-75%),4级(75-100%)。经过检测,StATP6V1C1瞬时表达株系对马铃薯晚疫病的抗性显著增强(图1)。
实施例2:StATP6V1C1的稳定转基因株系对晚疫病抗性增强
1、StATP6V1C1-OE稳定转基因株系创制及阳性鉴定
使用茎段法进行马铃薯的遗传转化,将携带StATP6V1C1-Myc表达载体的农杆菌AGL1扩繁培养,切取无生长点的马铃薯组培苗茎段,置于MS培养基于22℃预培养2-3d。用MS液体培养液重悬,调至OD600=0.5,将茎段浸泡于侵染液,于水平摇床40rpm震荡侵染15min,用无菌滤纸吸干水分后将茎段均匀放置在共培养培养基(MS培养基,含添加GA3 0.2mg/L,NAA 0.02mg/L,ZT 2.5mg/L)上,于18℃暗培养2d。使用添加特美汀(50μg/mL)的无菌水清洗完成共培养的茎段5次,每次5min,将茎段用无菌滤纸吸干水分后置于愈伤培养基(MS培养基,添加GA3 0.2mg/L,NAA0.02mg/L,trans-ZT 2.5mg/L,特美汀50μg/mL)培养两周左右直至形成良好愈伤组织,将愈伤组织转入分化培养基(MS培养基,添加GA3 0.02mg/L,NAA0.02mg/L,trans-ZT 2.5mg/L,特美汀50μg/mL)培养至分化出芽,待幼芽长到1cm左右,切取幼芽转移到MS生根培养基中诱导生根和进行抗性筛选,移栽成苗后使用Western Blot对其进行阳性鉴定。本研究中组培室环境为光照16h,黑暗8h,22℃,光照强度2000Lux,本发明挑选三株阳性株系分别命名为Line1、Line2和Line3。
2、StATP6V1C1-OE稳定转基因株系对马铃薯晚疫病的抗性增强
以野生型为对照,检测了三株StATP6V1C1-OE阳性株系的抗病性。结果如图2所示,以野生型为对照,三个StATP6V1C1转基因株系水渍状病斑面积均小于对照组,菌丝生长量则分别下调26.39%、68.32%和52.84%;相比于野生型病情指数68.33%,三个超量株系病情指数分别为40%、50%和41.07%,显著低于对照组。综上,StATP6V1C1稳定转基因株系抗病性增强。
实施例3:StATP6V1C1沉默株系更易感马铃薯晚疫病
为了进一步证实StATP6V1C1基因的抗性功能,发明人使用VIGS技术对马铃薯Désirée的StATP6V1C1进行基因沉默(见表3的引物5和引物6,引物5中带下划线碱基为限制性内切酶BamH I识别位点,引物6中带下划线碱基为限制性内切酶Kpn I识别位点)。
表2
引物名称 | 序列(5’-3’) |
引物5 | CGGGATCCAGTCGGAAACAAGAGCTGGA(SEQ ID NO.5) |
引物6 | GGGGTACCGAGGCAACCCGTATCTCAAA(SEQ ID NO.6) |
引物7 | AGAGTTTCCGGCGTTCTCAG(SEQ ID NO.7) |
引物8 | TTGGACAGATCACGGACAGC(SEQ ID NO.8) |
使用qRT-PCR技术(所用引物见表2的引物7和引物8)检测基因沉默效率为77.32%。离体接种致病疫霉菌株EC1孢子悬浮液3天后,发病结果如图3所示,可见TRV::StATP6V1C1株系病斑面积显著增大,抑制StATP6V1C1基因表达的马铃薯株系更易感晚疫病。
实施例4:本氏烟沉默NbATP6V1C1基因影响两个抗病蛋白R2、Rpi-mcq1对致病疫霉效应蛋白Avr2、AL3的抗性识别
1、在本氏烟中利用VIGS技术敲低NbATP6V1C1的表达,具体操作步骤如下:
提取本氏烟萌发幼苗的RNA,反转录获得本氏烟cDNA;
采用引入BamH I酶切位点和Kpn I酶切位点的特异性引物(见表3的引物5和引物6)进行本氏烟cDNA片段PCR扩增,纯化获得本氏烟NbATP6V1C1基因干扰片段;
利用VIGS病毒载体构建含有本氏烟NbATP6V1C1基因的重组质粒,转化成功后,即构建得到病毒介导的沉默株系TRV::NbATP6V1C1。未沉默的即为TRV::00(对照株系)。
四周后使用qRT-PCR方法(所用引物见表2的引物7和引物8)检测抑制效率为88.25%。
2、以共表达AVR3a和R3a为对照,在TRV::00(对照株系)和病毒介导的沉默株系TRV::NbATP6V1C1交叉组合表达抗病基因R2/Rpi-mcq1和效应因子Avr2/AL3,表达7天后拍照并统计出现HR的概率。具体操作步骤为:分别克隆抗病蛋白R2(ACU65456)、Rpi-mcq1(GN043560)、R3a(JN609622)的全长CDS序列以及效应分子Avr2(XM_002902940)、AL3(XM_002899560)和Avr3a(HQ639930)的羧基端CDS序列;R2/Rpi-mcq1/R3a基因片段分别克隆进载体pCXSN-Myc(Chen et al.,2009),载体pCXSN-Myc采用Xcm1单酶切,后与所述基因片段通过TA克隆连接,获得重组载体;Avr2/AL3/Avr3a基因片段分别克隆进载体CXSN-HA(Chenet al.,2009),载体CXSN-HA采用Xcm1单酶切,后与所述基因片段通过TA克隆连接,获得重组载体;
分别将所述重组载体转化GV3101农杆菌感受态,于28℃摇床培养,使用侵染液(氯化镁10mM,乙酰丁香酮0.1mM)调整菌悬液浓度至OD600=1,分别按照图4中a-e组合1:1混合菌液并注射到烟草叶片中,注射七天后统计HR坏死出现的概率。
结果如图4所示,表明:敲低NbATP6V1C1,R3a对Avr3a的识别不受影响,R2/Rpi-mcq1对Avr2/AL3的4组识别完全消失,证明除了自身的抗性功能,NbATP6V1C1同时介导了R2/Rpi-mcq1与Avr2/AL3间的4种交叉识别,帮助植物更好地抵御致病疫霉的侵染。
Claims (2)
1.StATP6V1C1在提高马铃薯对晚疫病抗性中的应用。
2.StATP6V1C1与抗病蛋白R2、Rpi-mcq1以及致病疫霉效应蛋白Avr2、AL3形成的复合体在提高马铃薯或烟草对晚疫病抗性中的应用,其特征在于,所述StATP6V1C1作为桥梁蛋白介导两个抗病蛋白R2、Rpi-mcq1对两个致病疫霉效应蛋白Avr2、AL3的识别,以提高马铃薯或烟草对晚疫病的抗性。
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