hwtx-Ⅰ基因四价体表达载体构建及苏云金芽孢杆菌工程菌株的研究.pdf

示，转化子Bh4一经超声波破碎并稀释304倍后72h对小菜蛾CPlutella xylostella)的毒杀效果是转化菌B4-304(转化pHT304后的Bt4.0718) 的141.17%0 关键词:苏云金芽抱杆菌，crylAc基因启动子，Hwtx-I，基因四价 体，工程菌株 ABSTRACT Thepromoterofinsecticidalcrystalproteins(ICPs)genecry]wasa sporulation-dependantandactivatedinthephaseofsporulation. Huwentoxin-I(Hwtx-I)isapeptideneurotoxinpurifiedrfomthecrude vemonsecretedbytheChinesebirdspiderSeleocosmiahuwena.Itisone ofaninhibitorofacetylcholinereceptorandcancutthesignalexpress betweennerveandmuscle.ItcanactasoneofaN-typeCa2+ionchannel inhibitors.Syntheticgenemonomerencodingrecombinanthwtx一Iin rfagmentsusingBtspreferencecode,basedontheknownprotein sequence.Phosphorylatedtherfagmentsandlinkedthem.Alinkerwith withnucleateendonucleaseSpeIand XbaIsitesandtermination codon(TAG)wasdesigned.Thesitessequencesselectedinthelinker wereBtpreferencecodeandtheycould互inkhwtx-Igenemonomerinto multimerbyheadtotaillinking. ThevectorpUA19eliminatedtheORFofinstecticidalcrystalproteins crylAcgeneafterdoubledigestedbyBsaBIandMunI,andthe promoterandtheterminatorofcrylAcgenewereremained.Thelinker wasinsertedinthissite,andgotanewvectorpUKHl.pUKHIdouble digestedwithSalIandSpeI,andreclaimedthelargerrfagmentand linkedtothesmallerrfagmentwhichpUKHldoubledigestedwithSalI andXbaI,thengotpUKH2.RepeatthedigestandlinksteponpUKH2as pUKHl,wewouldgetpUKH4.Doubledigesting plasmidpUKH4with SalIandBamHI,reclaimingthesmallerfragmentandinsertintothe E.coliandBtshuttlevectorpHT304betweenSalIandBamHIsites, gotpXH4,thehwtx-Igenequadrivalentexpressionvector.pXH4was transformatedintoBacillusthuringiensisacrystalliferousstrainXBUOOI andwildtypestrainBt4.0718.Theinterestprotein expressed successfullyinrecombinatestrains.Theproductionofthequadrivalentin shakeflaskis14.0mg/l.Theinterestproteinaccountsfor11.48%oftotal solubleproteinsinrecombinaneUH04and4.51% inBh4-4through analysisbygelimagingsystemandproteinanalysissoft.Theprotein formhwtx-Iquadrivalentwasproduced.Bh4-4,are