［植物科學(xué)領(lǐng)域］利用農(nóng)桿菌侵染煙草進(jìn)行體內(nèi)瞬時(shí)表達(dá)的方法MethodfortransientexpressioninvivobyinfectingtobaccowithAgrobacterium一、 原理(Principle)煙草葉片的瞬時(shí)表達(dá)系統(tǒng)常用來(lái)進(jìn)行基因的亞細(xì)胞定位監(jiān)測(cè)，監(jiān)測(cè)蛋白在細(xì)胞中分布的位置，從而對(duì)其功能進(jìn)行預(yù)測(cè)。通過(guò)基因翻譯的蛋白與綠色熒光蛋白構(gòu)成融合蛋白，在激光共聚焦儀器下觀測(cè)綠色熒光的分布，判斷蛋白表達(dá)的部位。同時(shí)，還可根據(jù)熒光的光強(qiáng)度檢測(cè)蛋白的表達(dá)量。該過(guò)程是經(jīng)根癌農(nóng)桿菌介導(dǎo)的，進(jìn)而將目的基因整合到到煙草的細(xì)胞內(nèi)的。Tobaccoleaftransientexpressionsystemsareoftenusedtomonitorthesubcellularlocalizationofgenes,monitorthedistributionofproteinsincells,andpredicttheirfunction.Thegenetranslatedproteinandgreenfluorescentproteinconstituteafusionprotein.Thedistributionofgreenfluorescenceisobservedunderalaserconfocalinstrumenttodeterminetheproteinexpressionsite.Atthesametime,theexpressionleveloftheproteincanalsobedetectedbasedonthelightintensityofthefluorescence.ThisprocessismediatedbyAgrobacteriumtumefaciens,whichintegratesthegeneofinterestintotobaccocells.二、 材料與試劑攜帶表達(dá)載體的農(nóng)桿菌菌株(通常表達(dá)載體由35S啟動(dòng)子驅(qū)動(dòng))2-4周的煙草植株LB培養(yǎng)基乙酰丁香酮MES:2-(N-嗎琳代)乙磺酸抗生素注射器MaterialsandreagentsAgrobacteriumstraincarryinganexpressionvector(usuallytheexpressionvectorisdrivenbythe35Spromoter)Tobaccoplantsfor2-4weeksLBmediumAcetosyringoneMES:2-(N-morpholino)ethanesulfonicacidAntibioticssyringe三、 儀器50ml離心管光譜儀紫外燈熒光顯微鏡Third,theinstrument50mlcentrifugetubespectrometerUVlampfluorescencemicroscope

四、 步驟挑取單克隆于5mlLB液體培養(yǎng)中，28?30°C震蕩培養(yǎng)。通常，LB中加入100gg/ml慶大霉素(農(nóng)桿菌株GV3101攜帶抗性)，50gg/ml大觀霉素(載體攜帶)。將1ml過(guò)夜培養(yǎng)的農(nóng)桿菌轉(zhuǎn)接到25mlLB液體培養(yǎng)基中(加有與1相同的抗生素，另外加入高壓滅菌的乙酰丁香酮)。檢測(cè)過(guò)夜培養(yǎng)的菌液OD600的值。5000g，15分鐘集菌，用重懸液重懸菌體，最終OD600為0.4。室溫放置2~3h后注射煙草。將侵染液裝入5ml注射器內(nèi)，用拇指按壓注射器反板將液體從葉片下表皮注射到煙草葉片內(nèi)(勿使用子葉)。注射后，煙草葉片會(huì)出現(xiàn)濕潤(rùn)的現(xiàn)象。注射后2-5天，在便攜式長(zhǎng)波長(zhǎng)紫外燈下檢測(cè)GFP熒光信號(hào)(只適用于熒光很強(qiáng)的葉片)。通過(guò)熒光顯微鏡或者激光共聚交熒光顯微鏡檢測(cè)GFP信號(hào)。同時(shí)，可以提取蛋白，檢測(cè)蛋白的含量。StepsPickasingleclonein5mlLBliquidcultureandshakecultureat28?30°C.Generally,LBisaddedwith100gg/mlgentamicin(agrobacteriumstrainGV3101carriesresistance)and50gg/mlspectinomycin(carriedbycarrier).Transfer1mlofAgrobacteriumculturedovernightto25mlofLBliquidmedium(addthesameantibioticas1andaddautoclavedacetylsyringone).ChecktheOD600valueofthebacterialsolutionculturedovernight.5000g,15minutestocollectbacteria,resuspendthebacteriawitharesuspension,thefinalOD600is0.4.Injecttobaccoafterplacingatroomtemperaturefor2?3hours.Filltheinfectioussolutionintoa5mlsyringe,andpressthebackofthesyringewithyourthumbtoinjecttheliquidfromthelowerepidermisoftheleafintothetobaccoleaf(donotusecotyledons).Afterinjection,thetobaccoleaveswillappearmoist.2-5daysafterinjection,detecttheGFPfluorescencesignalunderaportablelong-wavelengthUVlamp(onlyapplicabletohighlyfluorescentleaves).DetecttheGFPsignalusingafluorescencemicroscopeoralasercopolymerizedfluorescencemicroscope.Atthesametime,proteincanbeextractedandthecontentofproteincanbedetected.五、 配方加有相應(yīng)抗生素的LB液體培養(yǎng)基(一種抗生素是菌株攜帶，一種為載體攜帶)。乙酰丁香酮(100mM溶于乙醇，-20°C儲(chǔ)存)。1MMgCl2重懸液(10mMMgCl2，10mM2-(N-嗎琳代)乙磺酸(pH5.6)高溫高壓滅菌15分鐘，100uM乙酰丁香酮，高溫高壓滅菌)。Five,formulaLBliquidmediumsupplementedwithcorrespondingantibiotics(oneantibioticiscarriedbythestrainandoneiscarriedbythecarrier).Acetylsyringone(100mMinethan