【摘要】 RED同源重组法敲除发光光状杆菌晶体蛋白基因cipA和cipB,为发光光状杆菌基因敲除提供方法。采用TOPO克隆方法将基因cipA和cipB克隆到质粒pTA上,获得质粒pTA-cipA和pTA-cipB;采用Red/ET方法分别将Gentar基因和Aprar基因分别插入pTA-cipA和pTA-cipB的cipA和cipB基因中,并替换cipA和cipB基因部分序列,获得pTA-GentaIncipA和pTA-ApraIncipB;pTA-GentaIncipA通过BamHⅠ酶切,从0.7%琼脂糖胶上回收获得GentaIncipA,将GentaIncipA导入带有质粒pASK-αβγA的P.luminescenssubsp.Akhurstii中,通过的RED同源重组敲除发光光状杆菌晶体蛋白cipA;pTA-ApraIncipB用EcoRⅠ酶切,从0.7%琼脂糖胶上回收获得ApraIncipB,将ApraIncipB导入带有质粒pASK-αβγA的P.luminescenssubsp.Akhurstii中,通过的RED同源重组敲除发光光状杆菌晶体蛋白cipB;42℃下热激去除突变菌中的质粒pASK-αβγA。克隆PCR和SDS-PAGE电泳结果表明发光光状杆菌晶体蛋白基因cipA和cipB均被敲除。采用RED同源重组法成功地敲除了发光光状杆菌晶体蛋白基因cipA和cipB。更多还原

【Abstract】 Study on the knockout of the crystalline inclusion protein gene cipA and cipB in Photorhabdus lumi-nescens subsp.Akhurstii by RED homologous recombination provide a gene knockout method for P.luminescens subsp.Akhurstii .pTA-cipA and pTA-cipB were acquired via cipA and cipB amplified from P.luminescens subsp.Akhurstii genome DNA and cloned on pTA by TOPO Cloning;pTA-GentarInCipA or pTA-AprarInCipB was gained via the gentamycine resistant gene(Gentar) or apramycin resistant gene(Aprar) with two homology arms was amplified and exchanged with a part of cipA or cipB of pTA-cipA or pTA-cipB by ET reconbination;GentarInCi-pA DNA fragment was purificated from 0.7% agarose gel after pTA-GentaIncipA was digested by BamHⅠ, tans-formed into P.luminescens subsp.Akhurstii horbaring pASK-αβγA and exchanged with the cipA gene from P.luminescens subsp.Akhurstii by ET recombination;AprarIncipB DNA fragment was purificated from 0.7% a-garose gel after pTA-AprarInCipB digested by EcoRⅠ, tansformed into P.luminescens subsp.Akhurstii without cipA horbaring pASK-αβγA was exchanged with the cipB gene from P.luminescens subsp.Akhurstii by ET re-combination;P.luminescens TZR001 was acquired after pASK-αβγA was removed from P.luminescens subsp.Akhurstii mutant.The results of coloning PCR and SDS-PAGE analysis were shown that the crystalline inclusion protein cipA and cipB in P.luminescens subsp.Akhurstii was knocked out by RED homologous recombination.Conclion the crystalline inclusion protein cipA and cipB in P.luminescens subsp.Akhurstii更多还原