RED同源重组法敲除发光光状杆菌晶体蛋白基因cipA和cipB

Knockout of the Crystalline Inclusion Protein Gene cipA and cipB in Photorhabdus luminescens by RED Homologous Recombination

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。

Study on the knockout of the crystalline inclusion protein gene cipA and cipB in Photorhabdus luminescens subsp. A khurstii by RED homologous recombination provide a gene knockout method for P. lurninescens subsp. A khurstii, 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-GentarlnCipA or pTA-AprarlnCipB was gained via the gentamycine resistant gene （Gentd） or apramycin resistant gene （Aprd） with two homology arms was amplified and exchanged with a part ofcipA or cipB ofpTA-cipA or pTA-cipB by ET reconbination; GentarlnCipA DNA fragment was purificated from 0.7% agarose gel after pTA-GentaIncipA was digested by BamH I, tans-formed into P. lurninescens subsp. A khurstii horbaring pASK-αβγA and exchanged with the cipA gene from P. luminescens subsp. A khurstii by ET recombination; AprarIncipB DNA fragment was purificated from 0.7% agarose gel after pTA-AprarInCipB digested by EcoR I, tansformed into P. luminescens subsp. Akhurstii without cipA horbaring pASK-αβγA was exchanged with the cipB gene from P. luminescens subsp. Akhurstii by ET recombination; P. luminescens TZR001 was acquired after pASK-αβγA was removed from P. luminescens subsp. A khurstii mutant. The results of coloning PCR and SDS-PAGE analysis were shown that the crystalline inclusion protein cipA and cipB in P. luminescens subsp. A khurstii was knocked out by RED homologous recombination.Conclion the crystalline inclusion protein cipA and cipB in P. luminescens subsp. A khurstii