肉桂地鏈霉菌接合轉(zhuǎn)移體系的構(gòu)建及nsdA基因中斷對(duì)莫能菌素產(chǎn)量的影響.pdf

1、華中農(nóng)業(yè)大學(xué)碩士學(xué)位論文肉桂地鏈霉菌接合轉(zhuǎn)移體系的構(gòu)建及nsdA基因中斷對(duì)莫能菌素產(chǎn)量的影響姓名：陳芬申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：微生物學(xué)指導(dǎo)教師：鄭應(yīng)華20070601華中農(nóng)業(yè)大學(xué)2007屆碩士學(xué)位論文ConstructionoftheconjugaltransfersystemofStreptomycescinnamonensisandeffectofPCRmediatednsdAgenedisruptiononyieldofmonen

2、sinABSTRACTConjugaltransferapowerfultransgenetool，hasbeensuccessfullyappliedinvariousStreptomycesstainsforgeneticmanipulationThismethodprotectsforeignDNAfromnucleaseinduceddegradationtherebyavoidingrestrictionofhostcells

3、andlargelyenhancingthegenetransferefficiencyAtpresentthegeneticmanipulationofStreptomycescinnamonensiswasmainlyaccomplishedbyprotoplasttransformationandapplicationofconjugationmethodologyhasnotbeenreportedInthisstudywein

4、vestigatedtheconjugationbetweenEcoliandStreptomycescinnamonensisforthefirsttimeUsingEcoliStreptomycesshuttleplasmidtotransformEcolistudlnETl2567(pUZS002)followedbyconjugaltransferintoStreptomycescinnamonensis，theoptimize

5、dconditionwasobtainednsdAgene(negativeregulatorofstreptomycesdifferentiation)isaglobalnegativeregulatorforantibioticproductionpresentingconservedsequenceinthestreptomycesgenomeHowevertheapplicationofnsdAinstreptomycesind

6、ustrialstrainhasnotbeenreported曲nowInpresentworkthedismptionofnsdAinproductionstrainwasaccomplishedatfirsttimeTheprocessisasfollowing：AnalyzingthebioinformationofnsdAencodinggeneandflankingsequence，universalprimerP1，P2wa

7、sdesignedtosuccessfullyamplifiedandverified48kblengthcorrespondingfragmentsfromStreptomycescinnamonensisBIB2005Firstlyapairoflong(5859nt)primerswerepreparedwhichhaveat5’end39ntmatchingtheflankingsequenceofndsA，andthe3seq

8、uencematchingtherightorleftendoftheapramycinresistantcassettes(aac’例]VofiT)ThelinearDNAPCRamplifiedbytheseprimerswaselectrotransformedtothestrainBW25113／pU790／pBIBll8whichexpresses7、一Redenzymeandcontainstargetplasmidthen