Identification, Expression And Bioactivity Of Cry1Genes From Bacillus Thuringiensis

Many years ago people began to control the pests by microorganism. Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt) is a kind of special bacteria, it used to control pests with many other compounds. Bt has been commercialized already and makes an important contribution to agriculture and the environment. Although other bacteria including Bacillus Popilliae and Bacillus sphaericus also used as a microbial insecticide, their spectrum insecticidal activity compared with Bt are quite limited. Importantly Bt is not only safe for humans but also the most widely used environmentally compatible biopesticide worldwide. Furthermore, Bt insecticidal gene has been already transformed into several major crops, rendering them insect resistant, and thus providing a model for genetic engineering in agriculture. The main insecticidal components are insecticidal crystal proteins (ICPs) that encoded by cry and cyt genes. The Cry1proteins which have strong insecticidal activity against many kinds of Lepidoptera pests is considered the deepest fields in the study of Bt insecticidal crystal proteins. In order to fully exploit the strains of Bt cry1-type genes resources,in this study we use PCR-RFLP method for screening cryl-type genes.Genomes of300strains of Bacillus thuringiensis separated from laboratory were identify by PCR using cry1-type universal primers, the analysis of the PCR-RFLP results suggest45strains of them contain cry1-type genes. Bt V4strain contains both crylEa and crylla genes. Bt FH21strain contain six cry1-type genes, as crylAa,crylAc,crylBb,crylDa,crylJa and cry1ld.Further analysis of these Bt strains revealed some other genes also exist, such as Bt V4strain contains cry2Aa gene. we selected crylEa,crylBb,crylDa,cry1Id,cry1Ia and cry2Aa for cloning and expression.According to full-length gene sequences, we designed corresponding primers and successfully cloned the genes above. CrylEa,crylBb,crylDa,crylld,cry1Ia and cry2Aa genes were registered in GenBank with the respective accession numbers of KF601559,KJ619659,KJ619660,KJ619661,KJ619662and KF667522..They were named crylEa12、cry1Bb3、cry1Da4、cryld3、cry1la35and cry2Aal6respectively by International Nomenclature Committee of Bt.. The genes were connected with PEB plasmid and transformed into Escherichia coli Rosetta (DE3) strain. And expression products weight from60kDa to130kDa. The protein of CrylEa12was used for purification and Western blot.We used the expression proteins of CrylEal2,CrylBb3,Cry1Da4,Cry1Id3,Cry2Aa16and the proteins of Bt V4strain and Bt FH21strain to do bioassay experiment. The selected insects are Helicoverpa armigera, Ostrinia furnacalis, Spodoptera exigua Hiibner and Plutella xylostella. The bioassay results indicated that CrylEa12and Cry2Aal6had low insecticidal activity against Plutella xylostella, Ostrinia furnacalis and Helicoverpa armigera, but had growth inhibition. The proteins of Bt V4strain had a higher insecticidal activity against Helicoverpa armigera with LC50was7.5466μg/ml and Ostrinia furnacalis with LC50was2.939μg/ml μg/ml. The proteins of Bt FH21strain had a higher insecticidal activity against Helicoverpa armigera with LC50was2.858μg/ml and Ostrinia furnacalis with LC5Owas0.502μg/ml. The proteins of Cry1Bb3had a higher insecticidal activity against Ostrinia furnacalis with LC50was1.697μg/ml. The proteins of Cry1Id3had a higher insecticidal activity against Ostrinia furnacalis with LC50was1.800μg/ml.