CN116479015B - 葡萄白粉菌效应因子、其互作蛋白及其应用 - Google Patents
葡萄白粉菌效应因子、其互作蛋白及其应用 Download PDFInfo
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Abstract
本申请公开了分离的葡萄白粉菌En.NAFU1效应因子CSEP080及其互作蛋白VviB6f和VviPE。本申请还涉及通过在葡萄中沉默互作蛋白VviB6f和VviPE,提高葡萄的抗白粉病能力。
Description
技术领域
本发明属于植物抗逆基因鉴定及基因工程技术领域,尤其是葡萄响应白粉菌侵染的基因的分离和鉴定、其互作蛋白及其抗白粉病的应用。
背景技术
白粉病是严重危害葡萄生产的重要真菌病害,常导致严重减产甚至绝收,造成巨大经济损失。为防控该病,生产上每年需要喷洒农药20-30次,严重影响葡萄产品的食用安全,威胁人体健康,污染生态环境。目前,葡萄育种者越来越强烈地认识到,尽快挖掘白粉菌致病机理,并积极开展葡萄精准抗病分子育种,是从根本上解决这一问题的重要途径。
植物与病原菌互作过程中,病原菌保守的病原体相关分子模式PAMP被植物细胞表面的模式识别受体PRR蛋白识别并激发植物的免疫系统(PTI),为了抑制植物的免疫系统,病原菌会进化出大量编码效应因子的基因。效应因子与传递PTI信号的关键蛋白互作,从而操控植物的PTI。于此同时,植物细胞也会进化出一系列NB-LRR抗病蛋白,识别病原菌分泌的效应因子,并激发植物的免疫系统(ETI)。同时,为了躲避植物的ETI,病原菌再次进化出新的效应因子,有效控制ETI。如此,植物和病原菌会彼此不断进化,从而形成“军备竞赛”。
白粉菌在侵染葡萄在侵染葡萄过程中向其寄主细胞内释放大量的效应因子,但其作用机理尚不明确。其主要原因是缺少葡萄白粉菌的稳定遗传转化体系,而且葡萄的遗传转化又是一份消耗时间较长的工作。基于这样的研究现状,所以研究葡萄与白粉菌互作的研究不够深入。
前期研究表明在病原菌与植物互作过程中,植物叶绿体在其中发挥重要作用。如,在疫霉菌侵染马铃薯过程中,叶绿体会移动至侵染位置,阻止病原菌的侵染。而且植物叶绿体是光合作用和初级代谢产物产生场所,其会产生NO,过氧化氢等物质。但对于在白粉菌侵染葡萄过程中,叶绿体所发挥的作用知之甚少。本申请涉及白粉菌侵染葡萄的一种效应因子及其互作蛋白,这对于葡萄抗白粉病精准分子育种提供基因资源和分子标记,具有重要理论价值和实践意义。
发明内容
本申请的一个方面涉及与白粉菌En.NAFU1侵染葡萄相关的一个效应因子CSEP080,所述效应因子定位于植物叶绿体和细胞膜。CSEP080与寄主叶绿体蛋白VviB6f和水解果胶蛋白VviPE互作,抑制叶绿体产生过氧化氢和促进植物果胶的降解,从而促进白粉菌侵染。
因此,本专利申请的另一个方面涉及效应因子CSEP080在葡萄中的互作蛋白VviB6f和VviPE。
本申请的第三个方面涉及涉及效应因子CSEP080、互作蛋白VviB6f和VviPE用于葡萄抗白粉病的用途。
在一个实施方案中,从葡萄白粉菌En.NAFU1分离的效应因子CSEP080具有如SEQID NO:1所示的核苷酸序列。
在本申请的另一个实施方案中,互作蛋白VviB6f的核苷酸序列如SEQ ID NO:2所示;VviPE的核苷酸序列如SEQ ID NO:3所示。
本申请的效应因子CSEP080的氨基酸序列如SEQ ID NO:4所示;互作蛋白VviB6f的氨基酸序列如SEQ ID NO:5所示;互作蛋白VviPE的氨基酸序列如SEQ ID NO:6所示:
本申请还提供用于检测葡萄白粉菌En.NAFU1的效应因子CSEP080的实时荧光定量PCR检测引物以及内参基因EF1的引物,引物序列如下:
q-CSEP080-F:ATGTGGCTCCAAACACGTGT
q-CSEP080-R:CGTGAACACGCCGGAGCAGC
q-EF1-F:AAAGGATCATTCAAATATGC
q-EF1-R:GCAATGATTAAAATAGCACA
本申请还提供构建CSEP080-GFP及CSEP080-RNAi的引物,引物序列如下:
CSEP080-GFP-F:GGGACGAGCTCGGTACATGTGGCTCCAAACACGTGT
CSEP080-GFP-R:CTAGAGGATCCCCGGGTCCACTCAGTTTTTTCGCATT
CSEP080-RNAi-F:
AGCATGGCCGCGGGATATCACAAGTTTGTACAAAAAAGCCCCTCGCTAAAACAGCTGCT
CSEP080-RNAi-R:
GCGGCCGCACTAGTGATATCACCACTTTGTACAAGAAAGCCCACTCAGTTTTTTCGCATT
本申请提供验证CSEP080是分泌蛋白的引物,引物序列如下:
CSEP080SP-PSUC2-F:ATGAATTATGTGGCTCCAAACACGTGT
CSEP080SP-PSUC2-R:ATCTCGATCCCAAAACCATGGAGATAAAAAA
CSEP080ΔSP-PSUC2-F:ATGAATTGATCCAACCGAACAGACAATCAG
CSEP080ΔSP-PSUC2-R:ATCTCGACCACTCAGTTTTTTCGCATT
本申请提供验证CSEP080与VviB6f和VviPE互作的引物,引物序列如下:
CSEP080-BD-F:
GCCATGGAGGCCGAATTCATGTGGCTCCAAACACGTGT
CSEP080-BD-R:
GCCGCTGCAGGTCGACGGATCCCCCACTCAGTTTTTTCGCATT
VviB6f-AD-F:
GGTGGGCATCGATACGGGATCCATATGGCTGCCTCCACTCTCTC
VviB6f-AD-R:
CTACGATTCATCTGCAGCTCGAGCGGACCACCATGGATCTTCAC
VviPE-AD-F:
GGTGGGCATCGATACGGGATCCATATGTACAACCACAGAGCAAG
VviPE-AD-R:
CTACGATTCATCTGCAGCTCGAGCAACGATGGTCAAATTGGAAT
CSEP080-cYFP-F:
GTACTGTCGACCTCGAGGGTACATGTGGCTCCAAACACGTGT
CSEP080-cYFP-R:
TATGGGTACATCCCGGGAGCGGTCCACTCAGTTTTTTCGCATT
VviB6f-nYFP-F:
ATCCGTCGACCTCGAGGGTACCATGGCTGCCTCCACTCTCTCCT
VviB6f-nYFP-R:
TCGAGCTCCTACCCGGGAGCGGTCGGACCACCATGGATCTTCACCT
VviPE-nYFP-F:
ATCCGTCGACCTCGAGGGTACCATGTACAACCACAGAGCAAGAG
VviPE-nYFP-R:
TCGAGCTCCTACCCGGGAGCGGTCAACGATGGTCAAATTGGAATAA
CSEP080-nLuc-F:
GAACACGGGGGACGAGCTCGGArGTGGCTCCAAACACGTGT
CSEP080-nLuc-R:
GGGACGCGTACGAGATCTGGTCCCACTCAGTTTTTTCGCATT
VviB6f-cLuc-F:
CTCGTACGCGTCCCGGGGCGGTATGGCTGCCTCCACTCTCTCCT
VviB6f-cLuc-R:
GAACGAAAGCTCTGCAGGTCGAGGACCACCATGGATCTTCACCT
VviPE-cLuc-F:
CTCGTACGCGTCCCGGGGCGGTATGTACAACCACAGAGCAAGAG
VviPE-cLuc-R:
GAACGAAAGCTCTGCAGGTCGAAACGATGGTCAAATTGGAATAA
本申请提供VviB6f和VviPE的引物,引物序列如下:
VviB6f-GFP-F:
CACGGGGGACGAGCTCGGTACATGGCTGCCTCCACTCTCTC
VviB6f-GFP-R:TCTAGAGGATCCCCGGGTGGACCACCATGGATCTTCACVviB6f-RNAi-F:
TAGCATGGCCGCGGGATATCACAAGTTTGTACAAAAAAGCATGGCTGCCTCCACTCTCTCCT
VviB6f-RNAi-R:
GCGGCCGCACTAGTGATATCACCACTTTGTACAAGAAAGCTCCTTGGCCACAATACCACC
VviPE-GFP-F:
CACGGGGGACGAGCTCGGTACATGTACAACCACAGAGCAAG
VviPE-GFP-R:TCTAGAGGATCCCCGGGTAACGATGGTCAAATTGGAAT
VviPE-RNAi-F:
TAGCATGGCCGCGGGATATCACAAGTTTGTACAAAAAAGCATGTACAACCA
CAGAGCAAG
VviPE-RNAi-R:
GCGGCCGCACTAGTGATATCACCACTTTGTACAAGAAAGCCTCCGAATGCC
GCTGCAGTT
本申请构建了CSEP080-GFP载体,通过烟草表达系统首次确定葡萄白粉菌效应因子定位于植物的叶绿体和质膜。
本申请阐述了CSEP080在白粉菌和葡萄互作中作用机理,并且通过农杆菌介导的基因表达方法确定CSEP080,VviB6f和VviPE调控葡萄对白粉病的抗性。
本申请的有益效果:
(1)本申请通过生物技术从葡萄白粉菌中克隆到效应因子CSEP080并揭示其在侵袭葡萄中的作用机理。葡萄白粉菌分泌效应因子CSEP080侵袭到植物细胞后被转运至叶绿体和质膜,在叶绿体中,CSEP080与VviB6f互作,抑制植物过氧化物的产生。在质膜上,CSEP080与VviPE互作,促进果胶的降解,从而加速白粉菌的入侵。
(2)本申请通过在葡萄中沉默VviB6f和/或VviPE的表达,降低了葡萄中白粉菌的致病力,提高了葡萄对抗白粉病的抗病性。
附图说明
图1CSEP080转运至植物细胞膜和叶绿体并影响植物光合作用。A,构建CSEP080融合GFP蛋白载体;B,CSEP080具有效应因子分泌信号肽和叶绿体转运肽;C,CSEP080定位于植物细胞膜和叶绿体;D,CSEP080影响植物光合作用。
图2CSEP080是葡萄白粉菌效应因子。A,CSEP080在白粉菌侵染葡萄过程中的表达模式;B,通过酵母分泌系统证实CSEP080具有分泌功能;;C,CSEP080具有抑制INF1诱导的超敏反应;D,CSEP080降低了INF1诱导的电导率。
图3CSEP080是葡萄白粉菌致病的必要效应因子。A,通过农杆菌介导的基因表达沉默和过表达CSEP080后白粉菌生长状态;B,统计沉默和过表达CSEP080后白粉菌菌丝长度;C,统计沉默和过表达CSEP080后白粉菌吸器指数。
图4CSEP080与VviB6f和VviPE互作。A,通过酵母双杂交验证CSEP080与VviB6f互作;B,通过萤火虫素证实CSEP080与VviB6f互作;C,基于双分子荧光互补确定CSEP080与VviB6f互作;D,通过酵母双杂交和短截VviB6f,确定CSEP080与VviB6f含有Rieske结构域的C端互作;E通过酵母双杂交验证CSEP080与VviPE互作;F,通过萤火虫素证实CSEP080与VviPE互作;G,基于双分子荧光互补确定CSEP080与VviPE互作;H,通过酵母双杂交和短截VviPE,确定CSEP080不与VviPE的片端互作。
图5CSEP080促进VviB6f和VviPE的积累。A,构建CSEP080-mCherry,VviB6f-Luc和VviPE-Luc载体;B,通过萤火虫素荧光确定CSEP080对VviB6f的调控;C,统计CSEP080调控VviB6f的萤火虫素荧光强度;D,通过萤火虫素荧光确定CSEP080对VviPE的调控;E,统计CSEP080调控VviPE的萤火虫素荧光强度。
图6VviB6f是叶绿体蛋白且调控植物叶绿体。A,构建VviB6f融合GFP蛋白载体;B,VviB6f具有叶绿体转运肽;C,VviB6f定位于植物细胞膜和叶绿体;D,VviB6f定位于葡萄原生质体叶绿体中;E,VviB6f影响植物光合作用。
图7VviB6f沉默负调控葡萄对白粉病抗性和过氧化氢。A,通过农杆菌介导的基因表达沉默和过表达VviB6f后白粉菌生长状态;B,统计沉默和过表达VviB6f后白粉菌菌丝长度;C,统计沉默和过表达VviB6f后白粉菌吸器指数。D,测定沉默和过表达VviB6f后葡萄中过氧化氢含量、POD、SOD和CAT酶活性。
图8VviPE沉默负调控葡萄对白粉病抗性。A,通过农杆菌介导的基因表达沉默和过表达VviPE后白粉菌生长状态;B,统计沉默和过表达VviPE后白粉菌菌丝长度;C,统计沉默和过表达VviPE后白粉菌吸器指数。D,测定未受白粉菌侵染和受白粉菌侵染的葡萄叶片中果胶含量;E,统计未受白粉菌侵染和受白粉菌侵染的葡萄叶片中果胶含量。
具体实施方式
以下结合实验及附图对本发明做进一步详细描述:
实施例1:葡萄白粉菌En.NAFU1效应因子CSEP080定位分析
通过软件LOCALIZER预测了En.NAFU1编码的CSEPs的亚细胞定位,其中预测到CSEP080定位于宿主的叶绿体中,其中43-83个氨基酸是叶绿体转移肽。为了确定CSEP080在宿主的叶绿体中发挥作用,利用Vector NTI软件设计CSEP080特异引物:CSEP080-F和CSEP080-R。已提取的白粉菌cDNA为模板,加入设计好的引物,用TAKARA公司的高保真酶PrimeSTAR HS DNA Polymerase进行PCR扩增,具体扩增体系为:0.5μL HS Taq,6.0μL 5XPCR buffer,3.0μL dNTP,1.0μL cDNA模板,1.0μL CSEP080-F引物,1.0μL CSEP080-R引物,17.5μL ddH2O。PCR扩增程序为:98℃预变性10s,循环参数为98℃变性10s,57℃退火10s,72℃延伸1min 30s,进行34个循环,72℃充分延伸10min。PCR反应产物在1%的琼脂糖凝胶中电泳检测,在紫外凝胶成像系统下成像并拍照。拍照完后,切取单一的目的条带并用Genstar公司的胶回收试剂盒回收,然后连接至克隆载体pMD19-T,构建为pMD19T-CSEP080质粒,然后转化大肠杆菌感受态细胞。将已转化的感受态细胞挑取白色单克隆,在37℃恒温摇床180rpm/min培养16-18h,经菌液PCR鉴定为阳性的克隆,送北京奥科杨凌测序部测序验证。在测序正确的情况下,再利用Vector NTI软件设计CSEP080特异引物:CSEP080-GFP-F,CSEP080-GFP-R。利用上述方法构建了CSEP080-GFP载体,并将其转化至农杆菌GV3101中。在30℃恒温摇床180rpm/min培养10-12h,利用烟草瞬时表达体系,将农杆菌注射在烟草叶片中并成功表达融合蛋白CSEP080-GFP。注射3天后,在共聚焦显微镜下观察烟草叶片表皮细胞中荧光信号,并确定其具体定位(植物细胞膜和叶绿体)。
由于叶绿体是植物光合作用重要场所,且CSEP080-GFP部分荧光定位于叶绿体,那么,CSEP080是否会影响植物的光合作用。将CSEP080表达于烟草叶片,并测量植物的光合速率。其包括胞间CO2浓度,光合作用效率,气孔导度,蒸腾速率。最后结果显示植物表达CSEP080后胞间C02浓度降低,光合作用速率提高。
实施例2:CSEP080是葡萄白粉菌En.NAFU1分泌的关键效应因子
为了确认CSEP080是否为葡萄白粉菌En.NAFU1分泌效应因子,对CSEP080的表达模式和分泌功能进行分析。
白粉菌处理:在培养室移栽的欧亚葡萄赤霞珠健康叶片上接种葡萄白粉菌En.NAFU1(Erysiphe necator NAFU1)(Gao等人2016),并在处理后0、24、48、72、96和120h采集接菌的叶片,液氮迅速冷冻后提取RNA。
根据CSEP080基因序列设计如下实时荧光定量PCR检测引物:
q-CSEP080-F:ATGTGGCTCCAAACACGTGT
q-CSEP080-R:CGTGAACACGCCGGAGCAGC
q-EF1-F:AAAGGATCATTCAAATATGC
q-EF1-R:GCAATGATTAAAATAGCACA
利用Takara的实时荧光定量PCR试剂盒在Bio-Rad IQ5实时荧光定量PCR仪上进行RT-qPCR试验。反应体系为:SYBR Premix Ex Taq II 10.5μL,cDNA模板1.0μL,Forward-引物0.8μL,Reverse-引物0.8μL,ddH2O 7.4μL。PCR扩增程序:95℃预变性3min,40个循环(95℃30s,58℃30s)。PCR循环后,50℃保持1min,然后以每10秒逐渐增加0.5℃。用IQ5软件标准化表达方法分析基因的相对表达水平。
结果表明,CSEP080在白粉菌侵染过程中诱导上调表达。在白粉菌En.NAFU1接种后0-48h,CSEP080在白粉菌中急剧上调表达,这意味着CSEP080响应白粉菌侵染。
CSEP080是由152个氨基酸组成的蛋白质,通过SignalP4.1预测CSEP080含有其为1-23个氨基酸构成的分泌的信号肽。为了证实CSEP080的分泌功能,将CSEP080SP(CSEP 080的信号肽),CSEP080ΔSP(删除CSEP080的信号肽),Avr1bSP(Avr1b信号肽)转化至缺乏分泌转化酶的酵母菌株YTK12并观察其在CMD-W和YPRAA培养基上的生长状况。通过观察发现,表达CSEP080SP的酵母与阳性对照Avr1bSP的酵母一样可以在YPRAA培养基上生长,但表达CSEP118ΔSP和阴性对照的酵母不能生长。同时,用2,3,5-三苯基四唑基氯(TTC)确定表达CSEP080SP的酵母具有转化酶活性。这表明CSEP080是白粉菌En.NAFU1的分泌蛋白。
葡萄白粉菌是一种专性活体寄生真菌,在白粉菌侵染过程中抑制植物细胞坏死以获得其生长发育所需能量。因此,我们试着探索CSEP080是否抑制植物超敏反应,如程序性细胞死亡(PCD)。INF1作为引诱剂可诱导本氏烟草产生PCD。通过在烟草中共表达CSEP080和INF1,我们观察到CSEP080抑制了由INF1诱导产生的植物PCD并且减少了烟草的电导率。
CSEP080是一种具有特殊细胞器定位的葡萄白粉菌效应子且在侵染过程中诱导表达和抑制PCD。为了评价CSEP080对白粉菌致病性的影响,通过农杆菌介导的表达系统对CSEP080进行了过表达和沉默,并接种了白粉菌观察。通过在白粉菌侵染的1,3和5dpi的PI染色观察发现,在CSEP080沉默情况下,白粉病菌丝长度减少,吸器指数降低,白粉菌致病力降低。这些结果表明CSEP080是葡萄白粉菌致病过程中不可缺少效应因子。
实施例3:CSEP080与葡萄中VviB6f和VviPE互作且促进其积累
进一步探讨白粉菌效应因子CSEP080的分子机制,以CSEP080为诱饵,筛选葡萄cDNA酵母双杂交(Y2H)库。共筛选出葡萄中42个CSEP080候选的互作靶蛋白。由于CSEP080位于植物叶绿体和质膜,我们关注了预测定位于植物叶绿体中的VviB6f(葡萄树细胞色素b6-f复合铁硫亚基)和预测定位于质膜中的VviPE(葡萄树果胶酯酶)。为了确认CSEP080与VviB6f和VviPE之间的相互作用,根据NCBI中公布的CSEP080与VviB6f和VviPE基因序列,利用Vector NTI软件设计特异引物以白粉菌-葡萄互作cDNA为模板,用TAKARA公司的高保真酶PrimeSTAR HS DNA Polymerase进行PCR扩增,具体扩增体系见实施例1,切取单一的目的条带并用Genstar公司的胶回收试剂盒回收,然后分别连接至AD,BD载体。将重组后的载体转化酵母菌株Y2HGold,涂布至SD-LW培养基上,28℃,3天。待长出单克隆后,将其稀释分别点至SD-LW、SD-LWHA、SD-LWHA+X-a-gal和SD-LWHA+X-a-gal+AbA培养基上。通过观察发现,共转化CSEP080与VviB6f和VviPE的酵母同阳性对照一样,可以在SD-LWHA、SD-LWHA+X-a-gal和SD-LWHA+X-a-gal+AbA培养基上生长。为了进一步确认CSEP080与VviB6f和VviPE互作,将CSEP080重组载体nLUC,VviB6f和VviPE重组载体cLUC。然后,CSEP080-nluc、cluc-VviB6f和cluc-VviPE转化至农杆菌GV3101,通过农杆菌介导的基因表达在本氏烟草中共表达。通过荧光素酶互补成像,发现在本氏烟草中共表达CSEP080-nluc和cluc-VviB6f、CSEP080-nluc和cluc-VviPE有荧光产生。为了加强CSEP080与VviB6f和VviPE互作,将CSEP080重组载体pSPY-cYFP,VviB6fVviPE重组载体pSPY-nYFP。然后,CSEP080-cYFP、nYFP-VviB6f和nYFP-VviPE转化至农杆菌GV3101,通过农杆菌介导的基因表达在本氏烟草中共表达。在显微镜下观察发现,共表达CSEP080-cYFP和nYFP-VviB6f在叶绿体中富有荧光信号,CSEP080-cYFP和nYFP-VviPE富有质膜荧光信号。相比之下,在对照组中没有观察到荧光信号。为了确定CSEP080与VviB6f和VviPE的互作区域,我们通过SMART分析了VviB6f和VviPE的序列。VviB6f有两个结构域,transmembrane(TM)(67-88AA),Rieske(115-201AA)。VviPE有三个结构域signal peptide(SP)(1-30AA),plant pectin methylesterase inhibitor(PMI)(50-200AA)和pectinesterase(PE)(247-544AA)。将VviB6f划分为不同片段,并通过酵母双杂交系统,确定CSEP080与含有Rieske结构域的VviB6fC末端互作。但CSEP080不与VviPE的片段互作与完整蛋白互作。
由于CSEP080与VviB6f和VviPE互作,CSEP080促进VviB6f和VviPE积累或降解未知。基于这个问题,基于上述步骤构建CSEP080-mCherry、VviB6f-Luc和VviPE-Luc载体,转化至农杆菌GV3101,通过农杆菌介导的基因表达在本氏烟草中共表达。通过观察到荧光信号强度发现,共同表达CSEP080-mCherry和VviB6f-Luc比共同表达的mCherry和VviB6f-Luc强。共同表达CSEP080-mCherry和VviPE-Luc的荧光信号比共同表达mCherry和VviPE-Luc也要强。CSEP080-mCherry并没有影响Luc的LUC强度,同时对LUC的荧光强度进行了统计分析,与观测结果相同。这些数据表明CSEP080促进了VviB6f和VviPE积累。
实施例4:VviB6f调控葡萄抗白粉病分析
CSEP080与VviB6f在植物叶绿体中互作,且B6f是细胞色素b6-f复合铁硫亚基,在植物光合作用中起重要作用。针对此,首先分析了VviB6f在基因组中位置,并构建了系统发育树。VviB6f位于19号染色体(4.382.462-4.384.537bp)。同时,我们发现在构建的B6f系统发育树中,大多数同属的植物划分为同一族。同时,依据序列比对结果,我们发现B6F的C端包含Rieske结构域的序列是保守的,但包含TM结构域的N端则不保守。其次,我们发现VviB6f(XP_034679439.1)是由228个氨基酸的构成的蛋白质,通过LOCALIZER预测1-41个氨基酸是的叶绿体转移肽。为了验证VviB6f在植物中的位置,构建了载体VviB6f-GFP并转化至农杆菌GV3101中,通过烟草表达系统将其在烟草中表达。在共聚焦显微镜下观察到,VviB6f-GFP荧光信号富集在植物叶绿体中。同时,提取葡萄原生质体,在葡萄原生质体中表达VviB6f-GFP,在叶绿体中也观察到荧光信号。为了确定VviB6f是否影响植物的光合作用,在本氏烟草中表达了VviB6f,并测量植物的光合速率。其包括胞间CO2浓度,光合作用效率,气孔导度,蒸腾速率。最后结果显示植物表达VviB6f后胞间CO2浓度降低,光合作用速率提高。
为了研究VviB6f是否调控葡萄对白粉病抗性,通过农杆菌介导的表达系统对VviB6f进行了过表达和沉默,并接种了白粉菌观察。采用碘化丙染色1、3、5dpi法观察白粉病菌丝长度及菌丝长度。在VviB6f沉默情况下,白粉病菌丝长度减少,吸器指数降低,白粉菌致病力降低。这些结果表明VviB6f负调控葡萄对白粉病的抗性。
B6f连接光合电子传输链的PSII和PSI,影响CO2的同化速率,并将水分子分裂成光子和分子氧。因此,我们测量过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)的植物过氧化氢含量和酶活性。发现过氧化氢含量呈相反趋势,CAT、POD和SOD的酶活性随VviB6f表达水平的变化呈阳性趋势。这些数据表明VviB6f对葡萄过氧化氢的产生有负面的调节作用.
实施例5:VviPE调控葡萄抗白粉病分析
由于白粉菌效应因子CSEP080操纵宿主VviPE,我们想知道VviPE在葡萄树与E.necator的相互作用中起作用。基于这一奇观,我们分析了VviPE位置,并构建了系统发育树。VviPE位于17号染色体(3,365,207-3,368,241 bp)。系统发育树显示,VviPE与模式植物拟南芥较近。此外,为了研究VviPE是否调控葡萄对白粉病抗性,通过农杆菌介导的表达系统对VviPE进行了过表达和沉默,并接种了白粉菌观察。采用碘化丙染色1、3、5dpi法观察白粉病菌丝长度及菌丝长度。在VviPE沉默情况下,白粉病菌丝长度减少,吸器指数降低,白粉菌致病力降低。这些结果表明VviPE负调控葡萄对白粉病的抗性。
由于白粉菌效应因子CSEP080与VviPE互作,且VviPE对葡萄抗性有负面调节作用,那么白粉菌侵染葡萄时果胶含量是否降低。我们测量了未受白粉菌侵染和受白粉菌侵染的葡萄叶片中果胶含量,发现感染了白粉病的葡萄叶的果胶含量较未感染了白粉病的葡萄叶少。这意味着白粉菌在侵染葡萄过程中降低细胞果胶含量促进侵染。
参考文献
Gao YR,Han YT,Zhao FL,Li YJ,Cheng Y,Ding Q,Wang YJ,Wen YQ(2016)Identification and utilization of a new Erysiphe necator isolate NAFU 1toquickly evaluate powdery mildew resistance in wild Chinese grapevine speciesusing detached leaves.Plant Physiol Biochem 98:12-24.doi:10.1016/j.plaphy.2015.11.003
Wang W,Devoto A,Turner JG,Xiao S(2007)Expression of the membrane-associated resistance protein RPW8 enhances basal defense against biotrophicpathogens.Mol Plant Microbe Interact 20(8):966-976.doi:1 0.1094/MPMI-20-8-0966
Zhao FL,Li YJ,Hu Y,Gao YR,Zang XW,Ding Q,Wang YJ,Wen YQ(2016)A highlyefficient grapevine mesophyll protoplast system for transient gene expressionand the study of disease resistance proteins.Plant Cell Tiss Org 125(1):43-57.doi:10.1007/s11240-015-0928-7。
Claims (1)
1. 一种提高葡萄植株抗白粉病能力的方法,其特征在于将VviB6f和VviPE在葡萄中沉默,其中VviB6f的核苷酸序列为SEQ ID NO:2,VviPE的核苷酸序列为SEQ ID NO:3。
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