鈍頂螺旋藻luxAB表達(dá)載體的構(gòu)建及電擊轉(zhuǎn)化研究.pdf

1、安徽農(nóng)業(yè)大學(xué)碩士學(xué)位論文鈍頂螺旋藻luxAB表達(dá)載體的構(gòu)建及電擊轉(zhuǎn)化研究姓名：朱一偉申請學(xué)位級(jí)別：碩士專業(yè)：微生物學(xué)指導(dǎo)教師：唐欣昀20090601AbstractTheaimofthisexperimentistosetupandtoperfectthegeneticsystemofSpirulinaWestudiedtheextentthattheSpirulinaA9strainissensitivetoAmp，andcloned

2、theluxABgenefragmentandgotthevectorfragment麗ⅡlpromoterUbilandampgeneWththetwofragments，weconstructedthenewplasmidvectorTheaimofthepretreatmentofSpirulinacellsistestraintheactivityofnuclease，andthentheelectropomtionwasuse

3、dtotransformatetheSpirulinacells111eSpirulinaA9strainisrelativelysensitivetoAmp朋1elninilnuminhibitoryconcentration(MIC)ofAmpis100肛g。ml—inliquidsituationand200pgml。1insolidsituationAmpmightbesuitableselectiongeneticmarker

4、sfortheSpirulina，andtheselectionconcentrationsisdesignedas200pgrni一InthisexperimenttheplasmidpUCf2GUSWasdigestedbytherestrictionenzymeSmalandEcoRIandthevectorfragmentwithpromoterUbilandampgeneWasrecoveredAccordingtothese

5、quenceofluxABgeneintheplasmidpRL1063aprimersweredesigned誦tllthedigestedpointsofSmalandE∞足jrintheupriveranddownrivenWi也thetemplateofpRLl063adigestedby&吐，theluxABgenefragmentwasclonedbyPC＆ThenthevictorfragmentsandluxABgene

6、wererecombined、衍thI“4DNAligaseThereconstructedplasmidisnamed鷦pUCD／uxAB、析ththeluxABandampgeneFurthermore，thecompetencecellofEcoliMC4100AstrainWasprepared謝mCaCl2andthereconstructedplasmidwastransformedintoEcoliMC4100AWecul

7、tivatedEcoliMC4100AcellstoreplicatethereconstructedplasmidDigestionidentifiedthereconstructedplasmidThereconstructedplasmidpUCD／uxABWastransformedandtheexpressionresultidentifiedwithfataldehydeInthisexperimentwegotthevec

8、torfragment、析thpromoterUbilandampgenewasobtainedandtheluxABgenefragmentwasamplifiedbyPCIL，nlevictorfragmentsandluxABgenewererecombined稍th“I4DNAligasenereconstructedplasmidpUCD／uxABWasprovedbyenzymedigestionWeculturedA9st

9、rainat24℃andtreatedwith2mmolL1EDTAfor24hbeforetransformationanduseedultrasonictreatmentwi廿lpowerof300wfor70sReconstructedplasmidWasusedtotransformateSpirulinaA9cellsusingelectropomtionInthisexperimentthetimeofelectropora

10、tionis50msandtheintensionofelectricfiledis4kvcm～melectroporationbufferis10mmolL以HEPES(pH75)05molL以sucrose，andtheeleetropomtionobjectisSpirulinaA9cellsthatareinmediumtermoflogarithmgrowthKeywords：Spirulinaplatens&vectoreo