Bt Strains Cry7、cry8 Gene Cloning And Expression

Bacillus thuringiensis,Bacillus species,a gram-positive bacteria are widely distributed around the world as an environmentally friendly insecticide is widely used in pest management constraints,which produces a protein for each pests have effective insecticidal activity. Bacillus thuringiensis is considered chemical pesticides in agriculture,forestry pest control administering an effective substitude.With the development of transgenic technology, Bacillus thuringiensis gene has become genetically modified crops,in order to provide the main source of insect resistance.Therefore,to search new insecticidal activity of genes for pest control is very important.Since Coleoptera increasingly grave threat,and most insects living in the underground environment,the use of chemical methods can not achieve the desired results.Therefore,screening for strains pests Chrysomelidae high activity is important.The main contents of this study are as follows(1)In this study,423 Bacillus thuringiensis strains were isolated from Hainan area,Using polymerase chain reaction(PCR) and polyacrylamide gel electrophorsis(SDS-PAGE) method to identify the strain of genotype and insecticidal crystal protein composition.The PCR amplification results showed that 23 strains contained cry7 Ab gene, The fragment size is 3417 bp.To contain Cry7 Ab BJH705 strains for further protein expression and bioassay(Gen Bank accession number is KX010669).SDS-PAGE showed that this strain can express molecular weight is about 130 kDa insecticidal crystal protein.For Colaphellus bowringi Baly bioassay,the rescreening results showed that,this protein have higher insecticidal activity on Colaphellus bowringi Baly and LC50 is 53.386 μg/m L.(2)To identify 3-1-a strain in the laboratory saved,and found it contain cry8 gene,Further full-length primers of amplification,the sequencing results showed that the strain contain cry8 Ax and cry8 Ea genes at the same time(Genbank accession number are KU986725.KU955850).Connected to the p EB expression vector,and expressed in E.coli BL21,SDS-PAGE showed, cry8 Ax and cry8 Ea genes can be expressed as the molecular weight of 80 k Da,130 k Da insecticidal protein.Corrected mortalities are 56.89 %,77.87%.The 80 kDa protein were rescreening and LC50 is 83.329 μg/m L.In this study,we conducted the strain screening and gene cloning research in Hainan area and laboratory strains,and obtained new BJH705 and 3-1-a strain,clond 3 Bt gene.The results have important implications for large ape beetle has insecticidal activity of the sustainability of the new clone prevention and Coleptera pets.