CN105695467A - 基于麦长管蚜Sitobion avenae的保守型嗅觉受体Orco基因设计的siRNA及其应用 - Google Patents
基于麦长管蚜Sitobion avenae的保守型嗅觉受体Orco基因设计的siRNA及其应用 Download PDFInfo
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Abstract
本发明涉及基于麦长管蚜Sitobion avenae的保守型嗅觉受体Orco基因设计的siRNA及其应用。本发明通过饲喂siOrco8，干扰SaveOrco基因的转录。实时荧光定量PCR研究表明，SaveOrco基因的表达明显受到抑制。嗅觉行为反应检测结果显示，SaveOrco表达沉默后的蚜虫对顺-3-己烯醇,水杨酸甲酯和EBF的响应率均显著下降。EBF对麦长管蚜翅型分化的诱导实验，siRNA处理蚜虫停止了对EBF的诱导，蚜虫正常的化学通讯被干扰，同时逆境诱导的翅芽发生也被阻止。表明Orco基因的保守序列可应用于通过siRNA介导的RNAi技术，有效遏制蚜虫快速扩散为害、用于防治蚜虫。
Description
技术领域
本发明属于生物技术领域，涉及麦长管蚜Sitobionavenae嗅觉受体基因SaveOrco的siRNA序列及其在蚜害防控领域中的应用。
背景技术
蚜虫种类繁多、寄主范围广泛，许多是农业上的重要害虫。以刺吸取食寄主植物韧皮部汁液为生，通过掠夺寄主营养、携带和传播植物病毒、排泄蜜露等多种方式造成寄主农作物产量和品质的严重损失。昆虫主要通过嗅觉来认识周围的生境，蚜虫也不例外，诸如近距离的寄主植物定位[WebsterBEN(2012)Theroleofolfactioninaphidhostlocation.PhysiologicalEntomology37:10-18.]或外激素的识别[MustapartaH(1990)Chemicalinformationprocessingintheolfactorysystemofinsect.PhysiolRev70:199-245]等尤其依赖嗅觉。植物、植食性昆虫、肉食性天敌等多级营养水平间存在着复杂的化学通讯关系，引导这一关系的化学信号物质通常是一些挥发性小分子化合物，来源于植物挥发物(例如，绿叶气味和诱导植物挥发物)或昆虫信息素等，帮助昆虫实现趋利避害、生存繁衍，对昆虫具有重要的生态学意义。E-β-法尼烯(EBF)是蚜虫报警信息素的主要功能组分，是麦长管蚜报警信息素的唯一组分[FrancisF,/VandermotenS,VerheggenF,LognayG,HaubrugeE(2005)Isthe(E)-beta-farneseneonlyvolatileterpenoidinaphids？JournalofAppliedEntomology129:6-11.]。报警信息素的释放通常会引起周围蚜虫的防御反应或逃避行为[PickettJA,WadhamsLJ,WoodcockCM,HardieJ(1992)Thechemicalecologyofaphids.AnnualReviewofEntomology:67-90.]。另外，保持一定频率的EBF释放会引起豌豆蚜Acyrthosiphonpisum、麦长管蚜Sitiobionavenae成虫后代的有翅蚜(迁飞蚜)比例上升[FanJia,ZhangYong,FrancisFrédéric,ChengDengfa,SunJingrun,ChenJulian(2015)Orcomediatesolfactorybehaviorsandwingedmorphdifferentiationinducedbyalarmpheromoneinthegrainaphid,Sitobionavenae.InsectBiochemistryandMolecularBiology.9(64):16-24.PodjasekJO,BosnjakLM,BrookerDJ,MondorEB(2005)Alarmpheromoneinducesatransgenerationalwingpolyphenisminthepeaaphid,Acyrthosiphonpisum.CanadianJournalofZoology83:1138-1141.KunertG,OttoS,RoseUSR,GershenzonJ,WeisserWW(2005)Alarmpheromonemediatesproductionofwingeddispersalmorphsinaphids.EcologyLetters8:596-603.KunertG,TrautschJ,WeisserWW(2007)Densitydependenceofthealarmpheromoneeffectinpeaaphids,Acyrthosiphonpisum(Sternorrhyncha:Aphididae).EuropeanJournalofEntomology104:47-50.]。移除触角后的蚜虫不再响应EBF的诱导，提示该生理过程可能是通过嗅觉系统介导的[KunertG,WeisserWW(2005)Theimportanceofantennaeforpeaaphidwinginductioninthepresenceofnaturalenemies.BulletinofEntomologicalResearch95:125-131.]。进一步地，利用RNA干扰技术沉默麦长管蚜的嗅觉受体SaveOrco的表达，证实EBF诱导麦长管蚜发生迁飞蚜分化的诱导过程是由该受体介导的。迁飞蚜的发生有助于蚜虫种群的趋利避害、扩散增殖，进而实现种群的快速扩张。
嗅觉感受是昆虫获得外部环境化学信息的主要手段，广泛参与到昆虫生命活动的方方面面，例如：觅食、交尾、产卵、聚集、趋利避害等，并起着重要作用，蚜虫亦不例外。蚜虫发生转主寄主为害、意外坠落时，利用植物的挥发性“绿叶气味”对寄主植物进行探寻和再定位；或者对种内信息素，例如性信息素、报警信息素等的感受和响应均依赖于嗅觉。干扰或调控蚜虫的嗅觉行为，会直接影响蚜虫对寄主作物的识别和准确定位，破坏蚜虫间的信息交流，同时，不致死。符合农业有害生物综合治理策略的基本原则。
大多数蚜虫嗅觉信号的转导均是由G蛋白偶联受体(GPCR)完成的，昆虫却十分特殊，利用嗅觉受体(OR)来介导来自外界的化学信号。昆虫OR家族最初在果蝇中被鉴定[ClynePJ,WarrCG,FreemanMR,LessingD,KimJ,etal.(1999)Anovelfamilyofdivergentseven-transmembraneproteins:candidateodorantreceptorsinDrosophila.Neuron22:327-338.VosshallLB,AmreinH,MorozovPS,RzhetskyA,AxelR(1999)AspatialmapofolfactoryreceptorexpressionintheDrosophilaantenna.Cell96:725-736.GaoQ,ChessA(1999)IdentificationofCandidateDrosophilaOlfactoryReceptorsfromGenomicDNASequence.Genomics60:31-39.]，起初归类为GPCR超级蛋白家族。Westrand等[WistrandM,L,SonnhammerELL(2006)AgeneralmodelofGprotein-coupledreceptorsequencesanditsapplicationtodetectremotehomologs.ProteinScience15:509-521.WistrandM,L,SonnhammerELL(2006)AgeneralmodelofGprotein-coupledreceptorsequencesanditsapplicationtodetectremotehomologs.ProteinScience15:509-521.]利用GPCRHMM模型，即适合GPCR的隐马尔可夫模型(HiddenMarkovModel)分析不同物种的嗅觉受体家族时曾提出，昆虫OR的跨膜方向与GPCR相反，即N末端位于嗅觉神经细胞内，C末端则位于嗅觉神经膜外表面，这一推测很快通过一项利用β半乳糖苷酶标签来表达重组蛋白的技术被证实[WistrandM,L,SonnhammerELL(2006)AgeneralmodelofGprotein-coupledreceptorsequencesanditsapplicationtodetectremotehomologs.ProteinScience15:509-521.pologyandheteromericfunctionofDrosophilaodorantreceptorsinvivo.PLoSBiology4:e20.]。目前研究显示，昆虫OR区别于GPCR的特征主要有：虽然也是七次跨膜结构，但跨膜方向相反；实际上是由异源多聚体复合物行使嗅觉信号转导功能的，其中通常含有一个OR伴随受体(Orco)和至少一个OR，Orco是一个在昆虫中高度保守的直系同源蛋白，且为昆虫特异表达[WistrandM,L,SonnhammerELL(2006)AgeneralmodelofGprotein-coupledreceptorsequencesanditsapplicationtodetectremotehomologs.ProteinScience15:509-521.pologyandheteromericfunctionofDrosophilaodorantreceptorsinvivo.PLoSBiology4:e20.LarssonMC,DomingosAI,JonesWD,ChiappeME,AmreinH,etal.(2004)Or83bencodesabroadlyexpressedodorantreceptoressentialforDrosophilaolfaction.Neuron43:703-714.NakagawaT,SakuraiT,NishiokaT,TouharaK(2005)Insectsex-pheromonesignalsmediatedbyspecificcombinationsofolfactoryreceptors.Science307:1638-1642.NeuhausEM,GisselmannG,ZhangW,DooleyR,StortkuhlK,etal.(2005)OdorantreceptorheterodimerizationintheolfactorysystemofDrosophilamelanogaster.NatureNeuroscience8:15-17.JonesWD,NguyenTA,KlossB,LeeKJ,VosshallLB(2005)Functionalconservationofaninsectodorantreceptorgeneacross250millionyearsofevolution.CurrentBiology15:R119-121.]；昆虫嗅觉受体复合物具有配体依赖型离子通道的特征[SatoK,PellegrinoM,NakagawaT,NakagawaT,VosshallLB,etal.(2008)Insectolfactoryreceptorsareheteromericligand-gatedionchannels.Nature452:1002-1006.WicherD,SchaferR,BauernfeindR,StensmyrMC,HellerR,etal.(2008)Drosophilaodorantreceptorsarebothligand-gatedandcyclic-nucleotide-activatedcationchannels.Nature452:1007-1011.]，近来学者们发现，蚊子其中的Orco蛋白单体可以在特定条件下独立行使离子通道的功能[TaylorRW,RomaineIM,LiuC,MurthiP,JonesPL,etal.(2012)Structure-activityrelationshipofabroad-spectruminsectodorantreceptoragonist.ACSChemicalBiology7:1647-1652.JonesPL,PaskGM,RinkerDC,ZwiebelLJ(2011)Functionalagonismofinsectodorantreceptorionchannels.ProceedingsoftheNationalAcademyofSciences.JonesPL,PaskGM,RomaineIM,TaylorRW,ReidPR,etal.(2012)Allostericantagonismofinsectodorantreceptorionchannels.PLoSONE7:e30304.]，而OR则可能做为信号转导开关，当被特定气味激活即开启同Orco形成离子通道，实现嗅觉信号向生理电信号的转化[NicholsAS,ChenS,LuetjeCW(2011)Subunitcontributionstoinsectolfactoryreceptorfunction:channelblockandodorantrecognition.ChemicalSenses36:781-790.HallemEA,NicoleFoxA,ZwiebelLJ,CarlsonJR(2004)Olfaction:Mosquitoreceptorforhuman-sweatodorant.Nature427:212-213.HallemEA,CarlsonJR(2006)Codingofodorsbyareceptorrepertoire.Cell125:143-160.WangG,CareyAF,CarlsonJR,ZwiebelLJ(2010)MolecularbasisofodorcodinginthemalariavectormosquitoAnophelesgambiae.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica107:4418-4423.CareyAF,WangG,SuCY,ZwiebelLJ,CarlsonJR(2010)OdorantreceptioninthemalariamosquitoAnophelesgambiae.Nature464:66-71.]；昆虫嗅觉神经中普遍表达G蛋白[TalluriS,BhattA,SmithDP(1995)IdentificationofaDrosophilaGproteinalphasubunit(dGqalpha-3)expressedinchemosensorycellsandcentralneurons.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica92:11475-11479.LaueM,MaidaR,RedkozubovA(1997)G-proteinactivation,identificationandimmunolocalizationinpheromone-sensitivesensillatrichodeaofmoths.Cellandtissueresearch288:149-158.Jacquin-JolyE,FrancoisMC,BurnetM,LucasP,BourratF,etal.(2002)ExpressionpatternintheantennaeofanewlyisolatedlepidopteranGqproteinalphasubunitcDNA.Europeanjournalofbiochemistry/FEBS269:2133-2142.]，有其参与的代谢型嗅觉信号转导与昆虫ORs复合物的离子通道型信号转导同时进行[19]，并且有研究显示可能彼此间存在联系，例如：当Orco缺失表达时，过表达G蛋白可以帮助OR形成离子通道，实现不依赖于Orco的跨膜信号传递[WetzelCH,BehrendtHJ,GisselmannG,StortkuhlKF,HovemannB,etal.(2001)FunctionalexpressionandcharacterizationofaDrosophilaodorantreceptorinaheterologouscellsystem.ProceedingsofthenationalacademyofsciencesoftheUnitedStatesofAmerica98:9377-9380.SakuraiT,NakagawaT,MitsunoH,MoriH,EndoY,etal.(2004)IdentificationandfunctionalcharacterizationofasexpheromonereceptorinthesilkmothBombyxmori.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica101:16653-16658.KriegerJ,Grosse-WildeE,GohlT,BreerH(2005)CandidatepheromonereceptorsofthesilkmothBombyxmori.TheEuropeanjournalofneuroscience21:2167-2176.KielyA,AuthierA,KralicekAV,WarrCG,NewcombRD(2007)FunctionalanalysisofaDrosophilamelanogasterolfactoryreceptorexpressedinSf9cells.Journalofneurosciencemethods159:189-194.BentonR,VanniceKS,Gomez-DiazC,VosshallLB(2009)VariantionotropicglutamatereceptorsaschemosensoryreceptorsinDrosophila.Cell136:149-162.]。最近，果蝇中又发现了另一类嗅觉受体——离子转移受体(IRs)[SmadjaC,ShiP,ButlinRK,RobertsonHM(2009)Largegenefamilyexpansionsandadaptiveevolutionforodorantandgustatoryreceptorsinthepeaaphid,Acyrthosiphonpisum.MolecularBiologyandEvolution26:2073-2086.]。昆虫嗅觉受体在近10几年受到关注，相关功能研究在不同的昆虫特别是一些模式物种中取得很大进展。基于基因组信息获得的豌豆蚜OR家族是蚜虫中首个也是目前为止唯一被公布的OR家族，包含一个Orco蛋白[BruceTJA,BirkettMA,BlandeJ,HooperAM,MartinJL,etal.(2005)ResponseofeconomicallyimportantaphidstocomponentsofHemizygiapetiolataessentialoil.PestManagementScience61:1115-1121.]。
长期依赖化学农药防治蚜害带来诸多问题，例如，对非靶标生物(例如天敌昆虫、经济昆虫等非靶标昆虫和人畜等)的附带伤害、环境污染、食品安全等。近年来，大力发展的基于天然挥发物组分的绿色防控技术显示出较好发展潜力。田间释放蚜虫信息素(例如，EBF[BruceTJA,BirkettMA,BlandeJ,HooperAM,MartinJL,etal.(2005)ResponseofeconomicallyimportantaphidstocomponentsofHemizygiapetiolataessentialoil.PestManagementScience61:1115-1121.])或者通过基因工程改造的方式开发能够释放控蚜物质的作物(例如EBF[BealeMH,BirkettMA,BruceTJA,ChamberlainK,FieldLM,etal.(2006)Aphidalarmpheromoneproducedbytransgenicplantsaffectsaphidandparasitoidbehavior.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica103:10509-10513.YuXD,PickettJ,MaYZ,BruceT,NapierJ,JonesHD,XiaLQ(2012)MetabolicEngineeringofPlant-derived(E)-β-farneseneSynthaseGenesforaNovelTypeofAphid-resistantGeneticallyModifiedCropPlants.J.IntegrativePlantBiology54(5):282–299.])等均是利用蚜虫的嗅觉开展的绿色防控措施，可以扰乱蚜虫正常的化学通讯行为，即报警信息素驱避行为，同时引诱天敌、提高为害蚜虫后代中有翅蚜(迁飞蚜)的比例，从而抑制为害本地的蚜虫的种群增长，是安全、可靠代替化学农药防治蚜害的理想选择。但是上述措施的有效开展依赖于环境条件的配合，风、雨、低温等均会降低防治效果。
Orco是昆虫特有蛋白，以昆虫特有蛋白为治虫靶标是发展昆虫防治新途径的重要手段。在降低对非昆虫类生物产生的负面影响的同时，调节昆虫行为而不致死的防治效果符合有害生物综合防治策略(IPM)的宗旨。干扰蚜虫Orco蛋白的正常表达会影响蚜虫对嗅觉信号物质的嗅觉响应，利用本发明中的siRNA饲喂麦长管蚜，实验结果显示：被RNA干扰后的蚜虫对绿叶气味、取食诱导的植物次生挥发物质及信息素等的嗅觉反应显著降低；对蚜虫驱避剂E-β-法尼烯(EBF)的行为响应率下降的同时。与正常蚜虫相比，EBF处理前后，RNA干扰蚜虫后代中的有翅蚜比例并不显著上升。上述结果说明本发明的siRNA有效地干扰了蚜虫对寄主植物气味、中间信息素等的识别，同时后代迁飞蚜大幅度减少，将直接引起迁飞定殖率下降和定殖后扩散受阻。由于siRNA的靶标特异性，除蚜虫外的其它生物将不受影响，可以增加蚜虫天敌昆虫控制蚜虫的效率。
发明内容
针对上述领域中的缺陷，本发明提供与麦长管蚜Orco基因序列匹配的siRNA序列，通过经口饲喂siRNA的方法，抑制SaveOrco蛋白的转录水平，实现了RNA干扰(RNAi)，编码相应siRNA的DNA分子(包括麦长管蚜及其他昆虫Orco基因在内)也属于本发明的保护范围。干扰后的麦长管蚜对绿叶气味、取食诱导次生挥发物、信息素等的嗅觉响应显著降低，从而干扰迁飞蚜(有翅蚜)对寄主植物的选择、定位；同时降低感知周围环境气味信息的敏感性，例如无法及时识别其他同伴释放的报警信息素，导致蚜虫对天敌的捕食等危险“无动于衷”，因此有利于蚜虫天敌利用利它素(如报警信息素等)锁定蚜虫并捕食；另外，RNAi处理后，长时间、间断式的EBF诱导不再显著提高麦长管蚜的有翅后代比例，从而阻碍蚜虫种群及时响应环境信号进行迁飞扩散，破坏蚜虫群体的生活史对策(r对策)，使其无法迅速成灾。本发明为通过转基因手段开发具有上述生物学效应的作物品种提供了宝贵的基因资源，同时也可尝试利用喷施渗透siRNA的方式直接实现对害虫靶标基因的沉默。可应用于防治蚜虫或制备防治蚜虫的产品、抑制蚜虫有翅蚜发生或者制备抑制蚜虫有翅蚜发生的产品。
基于麦长管蚜Sitobionavenae的Orco基因设计的siRNA，命名为siOrco8，其序列如SEQIDNO：1所示。
所述的siRNA在制备防治蚜虫、干扰蚜虫化学通讯或降低蚜虫迁飞蚜比率的产品中应用。
所述的siRNA在防治麦蚜及其他具有相同基因靶标序列的害虫中的应用。
所述应用为将权利要求1所述的siRNA导入蚜虫，从而实现防治蚜虫、干扰蚜虫正常化学通讯或者降低蚜虫迁飞蚜比率。
所述导入的方式为饲喂蚜虫。
所述饲喂为将siOrco8溶解于人工饲料中，获得混合溶液，用所述混合溶液饲喂蚜虫。
所述siOrco8在人工饲料中的浓度为20ng/μl。
所述麦蚜为麦长管蚜Sitobionavenae。
本发明通过饲喂siRNA的方法，干扰SaveOrco基因的转录。实时荧光定量PCR研究表明，SaveOrco基因的表达明显受到抑制。嗅觉行为反应检测结果显示，SaveOrco表达沉默后的蚜虫对顺-3-己烯醇(绿叶气味物质),水杨酸甲酯(蚜害诱导植物挥发物)和EBF(麦长管蚜报警信息素)的响应率均显著下降。进一步开展EBF对麦长管蚜翅型分化的诱导实验，RNAi处理蚜虫停止了对EBF的诱导，沉默前后麦长管蚜及其后代群体迁飞蚜比例无显著变化。蚜虫正常的化学通讯被干扰，同时逆境诱导的翅芽发生也被阻止，表明Orco基因的保守序列可应用于通过siRNA介导的RNAi技术，有效遏制蚜虫快速扩散为害、用于防治蚜虫。
Orco蛋白为昆虫特有蛋白，直系同源Orco的氨基酸序列间具有较高的序列一致性(通常超过50﹪)。尽管如此，不同昆虫的Orco基因仍包含不少变异区序列。这类变异区段是理想的siRNA靶标区域，可以实现RNA干扰效应的针对性、甚至实现“种”特异性，适合筛选用于转基因开发材料；以变异区为潜在作用靶标，设计并筛选目标受体的兴奋剂或者拮抗剂等，可进一步通过对嗅觉功能蛋白表达的调控干预靶标昆虫的行为，达到防控目的，是开发直接施用药剂、进行有针对性地害虫防治的宝贵资源。总之，针对昆虫特有蛋白开展害虫防治，有望降低对非靶标生物甚至非靶标昆虫的伤害，同时提高防治效果。
附图说明
图1.RNAi干扰处理后，麦长管蚜SaveOrco转录水平被抑制情况(荧光定量PCR)，
其中(120+72)h–RNAi干扰处理停止后，第72hSaveOrco的表达情况。0h–以RNAi干扰处理起始时间的SaveOrco表达水平为外参标准化实验数据，比较不同处理时间目的基因的相对表达情况；120h–RNAi干扰处理满120hSaveOrco的表达情况；120+72h–RNAi干扰处理满120h后，第72小时SaveOrco的表达情况
图2.RNAi干扰麦长管蚜嗅觉行为反应测定，
图3.EBF诱导处理后RNAi干扰麦长管蚜的有翅蚜分化情况。
具体实施方式
下面结合实例对本发明做进一步的详细说明。
1.材料与方法
1.1供试昆虫
麦长管蚜单性系实验种群最初来源于廊坊野外种群，经单头扩繁，饲喂于小麦植株，小麦品种是中麦175。选择低密度(10株麦苗/盆、平均1头/株麦苗)饲喂3代以上的麦长管蚜成虫及其后代做为试虫。饲养温度保持在22±1℃，相对湿度75％，光周期16h︰8h。
1.2siRNA序列设计
以SaveOrco的cDNA(Genbank登录号：GQ275379)为模板，根据SiRNA序列设计原则设计并合成siRNA，命名为SiOrco8，分别以打乱顺序的siRNA作为阴性对照，命名为NegOrco8。序列内容见表1。
表1.siRNA序列内容
1.3饲喂法RNAi干扰实验
麦长管蚜人工饲料经0.22μm滤膜过滤处理后，用于溶解siRNA，浓度为20ng/μl。饲喂法RNAi干扰实验取高6cm、直径2.5cm的通透玻璃管，一端附两层充分拉伸的石蜡膜，在膜间注入含siRNA的100μl人工饲料，朝上放置。另一端以纱布包裹，保持通气；设纯人工饲料作为空白对照，同时设20ng/μl的NegOrco为阴性对照。每玻璃管接入10头成蚜，饲喂时间持续120h，进行三次平行实验。人工饲料饲喂满120h后，收集蚜虫准备下一步实验。其中，第72h时更换新鲜人工饲料。
1.4荧光定量PCR
利用SYBRGreenqPCR技术检测RNAi干扰处理0h、24h、48h、72h、96h和120h的麦长管蚜成虫(30头)SaveOrco表达量，引物序列见表2。同时，检测RNAi干扰处理结束转接麦苗后第72h麦长管蚜SaveOrco的表达量。
表2.引物列表(名称、序列内容、扩增区域、扩增片段长度、退火温度)
快速切取并收集麦长管蚜触角于1.5ml离心管。电动组织研磨器研磨处理，提取总RNA并合成第一链cDNA。具体步骤见总RNA提取试剂盒(PureLinkRNAMiniKit,Ambion,德国)及cDNA第一链合成试剂盒(SuperScriptIIIFirstStrandSynthesisSystem,Invitrogen,美国)说明书。SYBRGreen(SYBRGreenReal-TimePCRMasterMixes，Lifetechnologies，美国)PCR体系为20μl，含5μM上下游引物各1μl、第一链cDNA4μl。引物及PCR反应条件参见表2。内参选择Actin基因，利用2-ΔΔCT法分析、计算目的基因相对于内参的表达量，结果进一步以RNAi干扰处理起始时间即正常蚜虫SaveOrco表达量为外参进行数据标准化处理。每个处理的qPCR检测设3个重复，平行实验3次。
1.5风洞法嗅觉行为反应检测
风洞测试区长30cm，内径3cm。两端各依次连接装载测试气味的玻璃气室(50ml)、装有亲水硅胶和活性炭的空气净化干燥塔(500ml)、空气流量计、大气采集仪等。管中央的出口用于放置测试蚜虫。将载有测试气味物质的滤纸条置入玻璃气室密封后，接通抽气泵电源，通气1min使气味充满管道即可开始测试。测试时，保持两边气流流速相同,均为100ml/min。释放单头蚜虫到风洞嗅觉仪的正中位置，观察其行为。蚜虫1min内离开释放点进入气味源一侧超过5cm，定义蚜虫对该气味物质有趋性；远离气味源而进入对照一侧超过5cm，定义该气味物质对蚜虫具有驱避性。规定时间内未离开中央区域，则定义为无选择。每个气味物质测试20头蚜虫。气味物质分别选择对蚜虫具有驱避性的水杨酸甲酯(虫害诱导挥发物)、EBF(报警信息素)和吸引蚜虫的顺-3-己烯醇(绿叶气味物质)。所有测试均在相对封闭、空气洁净、光线均匀的专用嗅觉测试室内进行。以干净空气为对照。
1.6EBF对麦长管蚜翅型分化的诱导作用
麦长管蚜翅型分化存在2个敏感期[FanJia,ZhangYong,FrancisFrédéric,ChengDengfa,SunJingrun,ChenJulian*.Orcomediatesolfactorybehaviorsandwingedmorphdifferentiationinducedbyalarmpheromoneinthegrainaphid,Sitobionavenae.InsectBiochemistryandMolecularBiology.2015,64:16-24]：伪胚胎时期(也称“出生前期”，pre-natal)和1-2龄期(也称“初生期”，post-natal)，在此期间，拥挤、营养恶化等逆境信号可刺激群体有翅蚜的发生率上升[MüllerCB,WilliamsIS,HardieJ(2001)Theroleofnutrition,crowdingandinterspecificinteractionsinthedevelopmentofwingedaphids.EcologicalEntomology26:330-340.]。本实验主要参考Kunert发表的处理及检测方法[KunertG,OttoS,RoseUSR,GershenzonJ,WeisserWW(2005)Alarmpheromonemediatesproductionofwingeddispersalmorphsinaphids.EcologyLetters8:596-603.]。针对伪胚胎期即出生前期，以RNAi干扰处理后的成蚜为实验对象，进行EBF诱导处理。同时，设正常成蚜的EBF处理为阳性对照，统计后代有翅蚜比例，比较处理结果与对照间差异。
将RNAi处理后的蚜虫、阳性对照和空白对照成蚜分别定殖到处于二叶期的麦苗上(10头蚜虫/盆)，外扣圆筒形塑料罩子，取10ul浓度为100ng/ulEBF矿物油溶液，点滴在1cm见方的小滤纸片，将滤纸片固定在饲喂供试蚜虫的容器基部。每天从上午9点到下午5点，共释放5次，每两小时1次，处理5天。以正常无翅成蚜的EBF处理为阳性对照，同时设立无EBF处理的空白对照。移除在EBF处理期间出生的所有若蚜，但保留EBF处理结束后24h内出生若蚜，此若蚜即是在伪胚胎时期(出生前期)接受EBF诱导的成蚜后代。保持饲喂条件至若蚜进入成蚜阶段，统计有翅蚜和无翅蚜数量。
1.7统计分析方法
实验数据以平均值±标准误(means±MSE)表示。1.5中RNAi处理及其空白对照蚜虫经EBF诱导，其后代有翅蚜比例差异采用单方面分类的方差分析(One-WayANOVA,Turkey，p＝0.01)；采用SAS的单因素方差分析(One-WayANOVA,Turkey)对1.6中RNAi处理及其空白对照蚜虫经EBF诱导，其后代有翅蚜比例差异进行分析(p＝0.01)。
2、结果分析与讨论
2.1针对SaveOrco的RNAi处理
很多刺吸式昆虫可以通过饲喂法实现对特定基因表达的RNAi干扰，例如，麦长管蚜、豌豆蚜、烟粉虱、长红锥蝽等。将siRNA溶解在人工饲料中饲喂麦长管蚜至120h，为保持人工饲料的洁净及siRNA的活性，在第72h时更换新鲜饲料。设立阴性对照和空白对照。
2.2SaveOrco表达的荧光定量PCR检测
成蚜SaveOrco的表达均在第72h检测到下降，分别是正常表达量(0h)的74.77％，之后持续下降，96h下降为正常表达量的41.92％；120h下降为31.98％(图1)。停止RNAi处理后的第72h((120+72)h)，SaveOrco的表达量仍然保持在较低水平，为正常表达量的32.10％(图1)。阴性对照蚜虫与空白对照蚜虫SaveOrco的表达量无显著差异。
上述结果说明饲喂siRNA可以通过干扰基因转录引起麦长管蚜成、若虫的基因表达沉默。RNAi干扰的瞬时效应可持续至少72h。
2.3风洞法嗅觉行为反应检测
RNAi处理满120h的麦长管蚜对三种小分子气味物质水杨酸甲酯、EBF和顺-3-己烯醇的嗅觉行为反应数据分析结果显示，SaveOrco表达被干扰后，麦长管蚜对水杨酸甲酯、EBF和顺3己烯醇的嗅觉反应阳性率(图2)分别由干扰前的87.6％、73.4％和68.2％下降为44.4％、41.7％和52.8％。与对照相比，RNAi干扰处理蚜虫对三种气味分子的嗅觉行为响应率均显著下降。上述结果表明SaveOrco蛋白表达被干扰后，蚜虫对包括绿叶气味、诱导植物挥发性气味和蚜虫信息素在内的多种气味信号物质的嗅觉响应均大幅减弱，证实SaveOrco蛋白功能的缺失会引起麦蚜嗅觉障碍。
2.4EBF对麦长管蚜翅型分化的诱导作用
开展RNAi干扰蚜虫的EBF诱导翅型分化调查。与未经EBF处理的蚜虫后代(空白对照，10.2％)相比，EBF诱导处理蚜虫其后代(阳性对照，79.7％)有翅蚜比例显著提高；而RNAi干扰后的蚜虫，经EBF诱导处理(RNAi+EBF，8.9％)其后代有翅蚜比例则不再出现显著上升(图3，F＝321.53，P＜0.0001)。
嗅觉功能损伤蚜虫不响应EBF的翅型分化诱导，说明SaveOrco或者麦长管蚜嗅觉信号转导系统与EBF诱导有翅蚜分化这一生理过程有关。
不同种蚜虫翅型分化的敏感时期有所不同，主要分为三种类型：出生前期、初生期或者二者兼顾。麦长管蚜上述两个时期均为翅型分化的敏感期。本实验结果说明，被RNAi干扰后的蚜虫不再响应EBF的翅型分化诱导，其后代即使在EBF这一典型逆境信号存在情况下，依旧保持较低迁飞蚜比例，不利于蚜虫扩散为害。
Claims (8)
1.基于麦长管蚜Sitobionavenae的Orco基因设计的siRNA，命名为siOrco8，其序列如SEQIDNO：1所示。
2.权利要求1所述的siRNA在制备防治蚜虫、干扰蚜虫化学通讯或降低蚜虫迁飞蚜比率的产品中应用。
3.权利要求1所述的siRNA在防治麦蚜及其他具有相同基因靶标序列的害虫中的应用。
4.根据权利要求3所述的应用，其特征在于：所述应用为将权利要求1所述的siRNA导入蚜虫，从而实现防治蚜虫、干扰蚜虫正常化学通讯或者降低蚜虫迁飞蚜比率。
5.根据权利要求4所述的应用，其特征在于：所述导入的方式为饲喂蚜虫。
6.根据权利要求5所述的应用，其特征在于：所述饲喂为将siOrco8溶解于人工饲料中，获得混合溶液，用所述混合溶液饲喂蚜虫。
7.根据权利要求5所述的应用，所述siOrco8在人工饲料中的浓度为20ng/μl。
8.根据权利要求3-7任一所述的应用，所述麦蚜为麦长管蚜Sitobionavenae。
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