Isolation Of Bacillus Thuringiensis In Heilongjiang Province And Cloning And Expression Of Novel Cry Gene

Noctuidae had a wide variety of insects and had be harmful for a long time and a wide range,which had brought great losses to agricultural production.The treatment of pest insects was still dominated by chemical control,which's long-term large-scale application would bring the "3R" problem and the destruction of the ecological environment.Therefore the green control of pests had become more and more popular.Bacillus thuringiensis(Bt)was widely used in pest biological control because of its insect-specific,environment-friendly and other characteristics.However,with the large-scale application of Bt,the field resistance population was also produced.Therefore,it was particularly important to continuously excavate new Bt strains and obtain both efficient and broad-spectrum cry gene.In this study,Bacillus thuringiensis was isolated by selection of sodium acetate from different types of soils in Heilongjiang Province and its genotypes were identified.The bioactivity of the strain against Spodoptera litura,Mamestra brassicae Linnaeus and Helicoverpa armigera Hubner was studied.The new genes of the highly virulent strains were also cloned and expressed.The following experimental results were obtained:Bt strains were isolated from 352 soil samples from different areas in Heilongjiang Province,and the strain rate was 13.6%.Among them,the Bt strains isolated from the dark brown soils in the areas such as Harbin and Yichun had the largest number and the rate of bacteria was 18.81% and 18.83%,respectively.The bacteria rate of Jiagedaqi and other areas' brown coniferous forest soil and Qiqihar and other areas' meadow soil was 7.89% and 9.52%,respectively.The sandy soil of bacteria rate of Jiamusi was 2.38%.In addition,Daqing and other areas of saline soil did not separate Bt strains.Through the observation of the microscope,the phylogenetic morphology of the isolates was different.The main crystal type is diamond-shaped,divided into long rhombic and blunt rhombic.There were also some irregular type,spherical,mosaic shape and other crystal types.Some isolates were also observed the coexistence of several forms of crystals.According to the PCR-RFLP identification system,4 genotypes including cry1,cry2,cry8 and cry9 were screened by 10 pairs of universal primers.Among them,34 strains with cry1 gene were detected and the detection rate was 73.91% which was the highest one.There were 18 strains contained cry2 gene,4 samples contained cry8 gene and 6 samples contained cry9 gene.16 Bt strains had only a single cry1 or cry2 gene.Others presented a variety of gene combinations,containing the combination of cry1 and cry2 types,the combination of cry1,cry2 and cry9 types and so on.And 9 isolates did not detect the genotype,accounting for 19.57% of the total isolate,but expressed the protein.The reason might be that the number of identified primers was so small that some genotypes were not detected or some new insecticidal genes that were not found.The amount of insecticidal crystals expressed by Cry protein of Bt isolates was analyzed by SDS-PAGE.It was found that the molecular weight of 130 kDa,90 kDa,70 k Da and 60 k Da protein had the most expression.The protein expression of 13 strains containing only cry1 gene was about 130 kDa.The protein expression of 3 strains containing cry2 gene was about 60kDa-70 kDa.The strains containing both cry1 gene and cry2 genes showed rhombic crystals and its ICPs' molecular was 130 kDa and 60 kDa.The strains presented multiple genotypes had different protein expressions,which was mostly 130 kDa and 60 k Da.The strains did not recognize the cry genotype,but detected the expressed protein and the protein expression was different.cry1Ab36 and cry2Ab35 genes were derived from strain HT2,which had a high insecticidal activity against Noctuidae larvae and their gene accession numbers were KY440260 and KY501107,respectively.cry1Ab36 and cry2Ab35 genes had been cloned and analyzed.The cry1Ab36 gene had an open reading frame of 3546 bp,encoding 1181 amino acids with a molecular weight of 133.52 kDa and isoelectric point of 4.98.The cry2Ab35 gene had an open reading frame of 1902 bp,encoding 633 amino acids with a molecular weight of 70.72 kDa and isoelectric point of 8.73.The proteins encoded by these two genes having typical three domains and do not containing signal peptides.SDS-PAGE analysis and Western Blot verification showed that cry2Ab35 gene was successfully expressed in Escherichia coli,and its supernatant and precipitate were both expressed in E.coli,indicating that the protein was partially dissolved.