Identification And Function Research Of Novel Bacillus Thuringiensis Strains And Its Insecticidal Gene

Bacillus thuringiensis has a special insecticidal activity,has been widely used in agriculture,forestry,health and medical aspects,and is currently the world's most widely used,the highest security microorganism.Because of the enormous potential value of Bt,many countries in the world have carried out extensive research on it.The isolation and identification of new Bt strains and the excavation and utilization of new genes have been the focus of research,and a lot of new resources have been obtained.In this study,the biological characteristics of ST7,ST8 and YWC2-8 strains isolated from the Sichuan basin were studied,including morphological characteristics,physiological and biochemical characteristics,serum type;The novel cry62Aal,cry8Kb1,cry8Pa1 and cry8Qa1 genes were cloned from the above strains,And cyt3Aa1 was cloned from TD516 strain.And the genes were deeply studied.The results are as follows:1.This paper analyzed the biological characteristics of ST7,ST8 and YWC2-8 strains.Optical microscope observation results show that three strains with week flagella,have similar morphological characteristics and growth curve;Electron microscopic observation indicated that ST7 and YWC2-8 could prouduce spherical and square parasporal crystal,and ST8 can only produce spherical crystals.SDS-PAGE results showed that YWC2-8 mainly produced molecular weight of 70 kDa and 130 kDa protein,ST7 and ST8 strains produce molecular weight of 70 kDa and 130 kDa protein,respectively.Physiological and biochemical characteristics and H serotype identification results showed that the ST7 strain belong to serotype H20a,20b Yunnan subspecies,ST8 strain belong to serum H10a,10C BT serovar Londrina subspecies,and YWC2-8 is a new subspecies named Bt serovar sichuansis;Biological activity test results indicated that ST8 had high insecticidal activity to Anomala corpulenta larvae(LC50=8.835 ?g toxin g-1 soil),YWC2-8 had high insecticidal activity to Helicoverpa armigera(LC50=1.54 ?g/mL)and Ostrinia furnacalis(LC50=14.03 ?g/mL).2.The virulence test of ST7 showed that the ST7 had no toxicity to Lepidoptera,Coleoptera and Diptera pests,and had slight toxicity to breast cancer cells with mild toxicity of breast cancer cells.Therefore,according to the conserved sequence of the PS anticancer protein gene,the specific primer were designed to amplify the genomic DNA,and the full-length gene primers were designed to clone the cry62Aa1 gene,The analysis of SDS-PAGE showed that the gene produced 92 kDa protein,including the Trx carrier label with 19 kDa.The protein was purified and activated with protease K,which is highly toxic to breast cancer cells(IC50=30?g/mL).The discovery of the cry62Aa1 gene will be important significance for the study of its anticancer mechanism and the production of specific anticancer drugs.3.PCR-restriction fragment length polymorphism(PCR-RFLP)analysis showed that the ST8 strain contained three different cry8 genes.These full-length genes were obtained by SON-PCR(single oligo nucleotide nested-PCR)technology,which were named as cry8Kb1,cry8Pa1 and cry8Qa1,respectively.Only Cry8Pa1 is highly toxic to Anomala corpulenta larvae(LC50=3.219 ?g/mL).Bacillus thuringiensis strain ST8 has special biological characteristics and rich insecticidal crystal protein gene,indicateed that ST8 and its toxin may be widely used as a new insecticide.4.Bioassay results have shown that Cyt3Aa1 cloned from TD516 strain has weak hemolytic activity against human red blood cells,and only have teratogenic effects on Aedes aegypti.Therefore,the relationship between Cyt3Aa1 and the Cyt family members was analyzed in this study.The initial three-dimensional structure of Cyt3Aa1 was constructed using homologous modeling.The results showed that the model molecules of Cyt3Aa1 have a typical cytolysin fold,two ?-helical layers surround a ?-sheet.Moreover,the biological activity of Cyt3Aa1 was implied based on structure analysis and comparison of Cyt3 Aa with structurally related proteins.The results in this study present a strong foundation for understanding the biological characterization of Cyt3Aa1 and will aid in the development of strategies to cope with the potential problem of insect resistance and the control of various pests.