Expression And Cloning Of Cry Gene Of Bacillus Thuringiensis Isolated From Liaoning Province

Bacillus thuringiensis(Bt)is a gram-positive rod-shaped bacterium and insecticidal proteins which show a wide range of specificity for different insect orders are produced during metabolism.Due to the fact that Bt has high specificity and great safety to environment,Bt has been one of the most widely applied microbial pesticides.Recently,as the amount of Bt preparation increases year by year and the planting area of Bt transgenic plants expands,the problem of pest resistance to Bt insecticidal proteins is becoming more serious.Simultaneously,the problem that Bt biological pesticide have a limited range of insecticides and short duration of action and are susceptible to environmental influences such as temperature and ultraviolet light are gradually exposed.Therefore,the isolation and screening of highly virulent strains and the discovery of novel Bt genes have important research value and practical application significance.In this study 524 samples were collected from the soil of hills,farmland and urban erosion in Liaoning Province,and 66 wild-type strains of Bt were isolated.The shape of parasporal crystal is spherical,elliptical,short biconical,rhomboid,and irregular.The study has screened genotypes which includes cry1Ac?cry2Aa?cry8E and cry8 F using the PCR-RFLP identification system.There are many gene combinations presented,for instance,cry1 Ac was combinated with cry8 E,and cry2 Ab was combinated with cry8 E,in addition,the combination of cry1Ac?cry2Aa and cry8 E types was also presented,which fully reflects the diversity of Bt resources in Liaoning Province.Bt proteins were tested against the lepidopteran insects Helicoverpa armigera,Plutella xylostella,Spodoptera litura,Ostrinia furnacalis and Mythimna separate.The bioassay showed that strains DD214-2?DD229?YK524 and YK540 had high insecticidal activity.According to the analysis of genome sequencing and sequence similarity analysis of Bt strain LTS290,this strain was found might contain novel cry genes.The novel cry gene was successfully cloned from the strain LTS290.It has a full length of 2187 bp,encodes 729 amino acid residues,42% similarity to known Cry19Bb1 protein and it was identified as the first-rank novel gene.The gene sequence was submitted to Gen Bank and got the accession number which was MK131319 and it was designated as cry79Aa1 by International Nomenclature Committee of Bt.It was successfully expressed in E.coli Rosetta(DE3)by p ET-21 b expression vector and named p ET-79 Aa.The peptide about 85 k Da that was in accord with expected was detected by SDS-PAGE.The bioassay was done against the Lepidoptera insects Leguminivora glycinivorella,Helicoverpa armigera,Spodoptera exigua and Plutella xylostella.The bioassay showed that p ET-79 Aa had no insecticidal activity against Leguminivora glycinivorella but had insecticidal activity against Helicoverpa armigera,Plutellaxylostella and Spodoptera exigua,with 50% lethal concentration(LC50)of 26.037 ?g/ml?37.579 ?g/ml and 34.582 ?g/ml.According to the PCR-RFLP identification system,strain BJH500 was found might contain novel cry8 genes.The novel cry8Ca?cry8Ea gene was successfully cloned from the strain BJH500 and the cry8 Ca gene was identified as the fourth-rank novel gene.The gene sequence was submitted to Gen Bank and got the accession number which was MK167020 and it was designated as cry8Ca5 by International Nomenclature Committee of Bt.They were successfully expressed in E.coli Rosetta(DE3)by p ET-21 b expression vector and named p ET-8Ca and p ET-8Ea,with peptides of 130 k Da were detected by SDS-PAGE.Both p ET-8Ca and p ET-8Ea showed no insecticidal activity against the Lepidoptera insects,but with some weight inhibitory against Leguminivora glycinivorella.