Cloning And Expression Of The Main Mosquitocidol Protein Of Bacillus Thuringiensis And Bacillus Sphaericus

Cry4Aa and Cry4Ba are two kinds crystal protein of Bacillus thuringiensissubsp.iraelensis. The molecular weight of the protein Cry4Aa is134Kda, The molecular weightof the protein Cry4Ba is128Kda; Mtx1、Mtx2and Mtx3are toxins synthesized in vegetativegrowth stage of Bacillus sphaericus SSII-1. The molecular weight of the protein Mtx1is100Kda,The molecular weight of the protein Mtx2is31.8Kda, The molecular weight of the protein Mtx3is35.8Kda.With the aim of understanding the characteristics of proteins,referring to thepublished NCBI GenBank, we used the Primer5to design the gene amplification primersequence and add the restriction enzyme cutting site.The PCR template is the plasmid of theBacillus thuringiensis subsp.iraelensis and chromosome of the Bacillus sphaericus SSII-1.ThePCR products was connected to the cloning vectorPMD18-T.We obtained the target genes byPCR and restrition enzyme analysis.The Cry4Aa、Cry4Ba、 Mtx1、Mtx2and Mtx3genes were clonedto a expression vector PHT315. Five recombinant plasmids were obtained.The recombinantplasmids were transfered to E.coli and Bacillus thuringiensis.With the IPTG inducing the E.coliand Bacillus thuringiensis recombinants which contained recombinant plasmids could expressthe toxins.The bioassay results that the recombinants Cry4Aa and Cry4Ba had almost identicaltoxicities to Culex pipiens pallens larvae but their toxicities were lower than Bacillusthuringiensis subsp.iraelensis.This is because that Bacillus thuringiensis subsp.iraelensiscontains kinds of toxins which have synergy. The toxicity of recombinants Mtx1was similar toBacillus sphaericus SSII-1but Mtx2and Mtx3didn’t have toxicity.