RNi技術(shù)原理及應用課件

RNAi技術(shù)原理及其應用2013.10.15什么是RNA干擾？RNA干擾(RNAinterference,RNAi)是指由與靶基因序列同源的雙鏈RNA(double-strandRNA，dsRNA)所誘導的mRNA特異性降解，從而使基因轉(zhuǎn)錄后沉默的一種現(xiàn)象(FireA,2002)。

2002年美國《科學》雜志將其評為全球年度十大科學進展之首1RNAi現(xiàn)象的發(fā)現(xiàn)及發(fā)展轉(zhuǎn)錄水平轉(zhuǎn)錄后水平基因沉默DNA甲基化異染色質(zhì)形成DNA分子的消融siRNA介導miRNA介導2RNAi的作用機制2.1轉(zhuǎn)錄水平的基因沉默RNA分子介導的轉(zhuǎn)錄水平的基因沉默（transicriptionalgenesilencing，TGS）是指基因信息從DNA到mRNA的轉(zhuǎn)錄過程尚未啟動時，siRNA分子通過修飾染色體DNA分子或與其結(jié)合的組蛋白分子阻礙轉(zhuǎn)錄的發(fā)生2.2轉(zhuǎn)錄后水平的基因沉默RNA分子介導的轉(zhuǎn)錄后水平的基因沉默（posttranscriptionalgenesilencing，PTGS）PTGS是指基因信息從DNA到mRNA的轉(zhuǎn)錄過程啟動后，siRNA分子通過RNA干擾機制降解mRNA或抑制mRNA翻譯，也包括在轉(zhuǎn)錄啟動后siRNA介導的基因第一外顯子序列的DNA甲基化。2.3RNAi的主要過程起始階段dsRNA(500nt左右最佳)被Dicer酶特異性識別，以一種ATP依賴的方式逐步切割成siRNASmallinterferingRNA(siRNA)長度：21-25ntDicer是一種ATP依賴性RNaseⅢ型核酸內(nèi)切酶，對單鏈RNA沒有活性，對200~500nt的dsRNA作用效果最好2.效應階段siRNA誘導沉默復合體（RNA-inducedsilencingcomplex，RISC）的形成RISC中解旋酶可依靠ATP供能，解開siRNA雙鏈，激活RISC在siRNA反義鏈的指導下(靶序列的識別），RISC中核酸內(nèi)切酶特異性切割、降解靶mRNA，導致基因表達失活dsRNA外源dsRNA病毒dsRNA其他dsRNADicer雙鏈siRNAP特異性蛋白結(jié)合形成RISCsiRNA解鏈靶序列識別RDRP以siRNA為引物，RDRP催化合成新的dsRNA核酸酶降解mRNAmRNAsiRNARNAi作用機制Table1.miRNA和siRNA介導RNAi的區(qū)別名稱miRNAsiRNA來源內(nèi)源轉(zhuǎn)錄本轉(zhuǎn)基因病毒RNA前體莖環(huán)狀的pre-miRNA雙鏈結(jié)構(gòu)dsRNA催化酶Dicer或者類似于Dicer的酶復合物Dicer匹配方式不完全互補（動物）或完全互補（植物）完全互補作用目標的專一性（特異性）相對較低高作用點蛋白質(zhì)合成水平轉(zhuǎn)錄后水平大小約22nt約22nt功能發(fā)育過程中調(diào)節(jié)內(nèi)源基因表達抑制轉(zhuǎn)錄活性，病毒感染，表現(xiàn)遺傳靶基因的命運抑制轉(zhuǎn)錄或者被降解被降解確定目的基因

根據(jù)相對應的核酸序列確定dsRNA模板區(qū)域

獲得dsRNA

用dsRNA轉(zhuǎn)染細胞

分析RNA干擾的效果3昆蟲RNAi技術(shù)的實驗方法RNA干擾研究的一般流程RNAi實驗對照設置對照組設置原理普通陰性對照

與目的基因的序列無同源性和目的靶細胞中其它基因同源性很低熒光標記陰性對照

方便地在熒光顯微鏡下觀察轉(zhuǎn)染情況可用于優(yōu)化轉(zhuǎn)染條件和評價轉(zhuǎn)染效率陽性對照確認RNAi實驗中轉(zhuǎn)染、RNA提取和基因表達檢測方法是否可靠

LaminA/C、GFP22、

LuciferaseGL2、MAPK1、Beta-Actin、Vimentin、P53、GAPDH、CyclophilinB轉(zhuǎn)染試劑對照檢測轉(zhuǎn)染試劑對細胞的毒性、細胞的成活率等細胞轉(zhuǎn)染的各個因素影響避免Off-target對照針對同一個基因的不同區(qū)域的多個siRNA進行實驗Table2.RNAi實驗對照設置1.dsRNA模板的選擇選擇的dsRNA模板區(qū)域不要針對5’和3’端的非編碼區(qū)通常選擇的模板長度約為500bp在合成dsRNA之前，應該先進行dsRNA和其他mRNA的同源性比對3.1dsRNA的獲得3.2dsRNA的合成方法1.體外轉(zhuǎn)錄法原理：采用5’-末端含有T7、Sp6或T3RNA多聚酶識別序列的寡核苷酸引物進行PCR擴增目的片段，然后在T7、Sp6或T3RNA多聚酶的作用下轉(zhuǎn)錄成RNA，通過簡單的退火、核酸酶消化和離心等步驟后即可獲得dsRNA2.構(gòu)建RNAi表達載體制備dsRNA

表達載體可分為兩個類型：單啟動子載體，需要插入反向重復DNA（由正義鏈、間隔序列和反義鏈組成），在轉(zhuǎn)錄的過程中，反向重復序列可產(chǎn)生由內(nèi)部堿基互補而回折形成的發(fā)夾RNA分子，然后加工形成dsRNA；雙驅(qū)動子載體，將靶DNA置入克隆位點后，雙啟動子分別表達正義鏈和反義鏈，然后在胞內(nèi)結(jié)合形成dsRNA單啟動子載體3.3昆蟲RNAi的導入方法dsRNA微量注射法InjectionAdultPupaEmbryoLarvalNanojectIIAuto-NanoliterInjectorTable3.昆蟲RNAi的導入方法注射法飼喂法組織培養(yǎng)法操作時期卵、幼蟲、蛹、成蟲幼蟲、成蟲培養(yǎng)細胞、胚胎部分幼蟲操作方法微量注射器dsRNA人工飼料菌液、轉(zhuǎn)基因dsRNA培養(yǎng)基優(yōu)點dsRNA迅速到達組織精確地導入dsRNA操作容易、省時、經(jīng)濟不造成機械損傷成活率高大量昆蟲同時連續(xù)攝取dsRNA適合用于害蟲控制適用于長期研究操作簡單適用于大規(guī)劃的基因功能篩選缺點操作難度大、耗時成活率較低難于用于大規(guī)模分析基因沉默效率可能較低不同昆蟲由于腸道環(huán)境不同導致dsRNA攝取效率差異較大昆蟲攝取的dsRNA量難于確定基因沉默效率較低需要較高濃度的dsRNATable4.RNAiresearchonfunctionalgenesininsectsInsectsGenesandReferencesMethodsEffectsSilkwormBombyxmori家蠶Circadianclockgeneper(Sandrellietal.,2007)

Transgenics

Disruptionofegg-atchingEcdysis-triggeringhormonegeneETH(Daietal.,2008)TransgenicsLethalatpharatesecond-instarlarvalstage

EgyptiancottonleafwormSpodopteralittoralisβ-actingene(Gvakhariaetal.,2003)InjectionDisruptionofspermreleaseCircadianclockgeneper(Kotwicaetal.,2009)InjectionDelayedspermreleaseLightbrownapplemothEpiphyasPostvittana蘋果淺褐卷蛾CarboxylesterasegeneEposCXE1andpheromonebindingproteingeneEposPBP1(Turneretal.,2006)FeedingInhibitionofgeneexpressionCottonbollwormHelicoverpaarmigeraCytochromeP450geneCYP6AE14(Maoetal.,2007)FeedingInhibitionoflarvalgrowthGlutathione-S-transferasegeneGST1(Maoetal.,2007)FeedingSuccessfulinhibitionofgeneexpressionInsectsGenesandReferencesMethodsEffectsBeetarmywormSpodopteraexiguaChitinsynthasegene(Chenetal.,2008)InjectionDisorderintheinsectcuticle,noexpansionofthelarvaltracheaepithelialwall,andotherlarvalabnormalitiesJapanesepinesawyerMonochamusAlternatus松墨天牛LaccasegeneMaLac2(Niuetal.,2008)InjectionPupalandadultcuticlesclerotisation,deathatahighdoseRedflourbeetleTriboliumcastaneumChitinsynthasegenesTcCHS1andTcCHS2(Arakaneetal.,2005)InjectionDisruptioninalltypesofmoulting(larva-larva,larva-pupa,andpupa-adult),cessationofingestion,decreaseinlarvalsize,andreductionofchitincontentinthemidgutChitinase-likeproteinsTcCHT5,TcCHT10,TcCHT7,andTcIDGF4(Zhuetal.,2008)InjectionEffectsonpupal-adultmoultingEffectsonegghatching,larvalmoulting,pupation,andadultmetamorphosis.Effectsonabdominalcontractionandwing/elytraextension.Effectsonadulteclosion續(xù)Table4.RNAiresearchonfunctionalgenesininsects續(xù)Table4.RNAiresearchonfunctionalgenesininsectsInsectsGenesandReferencesMethodsEffectsWesterncornrootwormDiabroticavirgiferavirgiferaLeConte玉米根螢葉甲

VacuolarATPase(v-ATP)(Baumetal.,2007)FeedingDelayedlarvaldevelopmentandincreasedmortalityStripedfleabeetlePhyllotretastriolataArgininekinasegeneAK(Zhaoetal.,2008)FeedingDelayeddevelopment,increasedmortality,andreducedfertilityMediterraneanfieldcricketGryllusBimaculatus蟋蟀Circadianclockgeneper(Moriyamaetal.,2008)InjectionCompletelossofcircadiancontroloflocomotoractivityandelectricalactivityintheopticlobeNitricoxidesynthasegeneNOS(Takahashietal.,2009)InjectionDestructionoflong-termmemory續(xù)表Table4.RNAiresearchonfunctionalgenesininsectsInsectsGenesandReferencesMethodsEffectsEuropeanhoneybeeApismelliferaTranscriptionfactorgeneRelish(SchlünsandCrozier,2007)InjectionInhibitionofRelishgeneexpressionandreductionintheexpressionoftwootherimmunegenes,abaecinandhymenoptaecinTriatomidbugRhodniusprolixus長紅獵蝽Salivarynitrophorin2geneNP2(Araujoetal.,2006)InjectionandfeedingShortenedplasmacoagulationtimeSavannahtsetseflyGlossinamorsitansMorsitans刺舌蠅TsetseEPgeneandtransferringene2A192(Walsheetal.,2009)feedingInhibitionofTsetseEPgeneexpression,butnoinhibitionof2A192geneexpressionEasternsubterraneanTermiteReticulitermesFlavipes東方散白蟻CellulaseenzymegeneCell-1andcasteregulatoryhexamerinstorageproteingeneHex-2(Zhouetal.,2008)feedingReductioningroupfitnessandincreasedmortalityTitleTitleTitleInfeedingexperimentsmostsequencesrangebetween300and520bp，ButInthecaseofS2cells,itshouldbeminimally211bp.Thesequenceusedwilldeterminepossibleoff-targeteffectsinthetargetorganism,butalsoinotherinsects.Thepersistenceofthesilencingeffectdependonthedeliverywaysandtheorganisms.Foreverytargetgeneandorganismanoptimalconcentrationhastobedeterminedtoinduceoptimalsilencing.Althougholderlifestagesaremoreefficientforhandling,theyoungerstagesoftenshowlargersilencingeffects.ConcentrationNucleotidesequenceLengthofthefragmentPersistenceOftheeffectLifestageoftheorganism4.3RNAi技術(shù)在害蟲控制的應用5.1Fiveimportantfactorslargelyinfluencingthesilencingeffect1.靶標基因沉默導致害蟲死亡Betten