Screening Of B. Thuringiensis Strains Against Coleopteran Pest And Cloning, Expression And Insecticidal Activity Of Cry8Ea2 Gene

Bacillus thuringiensis(Bt) is the most in-depth study of microbial pathogens, and its insecticidal protein gene is the most widely and potentially used against pest insect, Therefore, exploring new Bt strains and isolating novel insecticidal gene to control pest are of great significance.In recent years, the damagement of coleopteran pest is serious increasingly especially Scarabaeoid beetle and it is very difficult to control it because of its larvae living in the soil . This study mainly includes isolation of Bt stains from soil in Shandong Province, identification of cry gene-type of Bt strains through PCR-RFLP system and cloning, expression and insecticidal activity assay of new protein gene that is toxic to Scarabaeoid beetle in order to provide new genetic resources for constructing high Bt engineered strain and cultivating genetically modified plant. The main contents of this study are as follows:1 57 Bt strains were isolated from 750 soil samples in Shandong Province by sel ective medium (NGKG) method. Crystal shapes could be observed, such as bipyramid al, spherical and so on.2 Making use of two pair of primers S5un8/S3un8 and S5un3/S3un3 were designed by'using conserved block to ampify gene, there are B-DLL and B-JJX strain that have positive fragment from the identification strains. Alignment results showed that B-DLL and B-JJX strain contain cry8 gene which is toxic against coleopteran pest. SDS-PAGE analysis showed that protein of 130 kD were in strain B-DLL and B-JJX.3 Iecticidal activity assay results showed that B-DLL strain is toxic to the larvae of Popillia flavosellata, Holotrichia parallela and Plagiodera versicolora, with the corrected mortality of 26.67%, 33.33% and 33.33%, respectively. B-JJX strain is toxic to the larvae of Plagiodera versicolora, with the corrected mortality of 30.80%, but it has no insecticidal activity against other kinds of insects.4 The 3.5 kb gene was amplified using a pair of special primers, JJX5 and JJX3, designed according to cry8-type genes sequences by PCR, and inserted into cloning vector pBluescriptSK (+) to obtain the recombinant plasmid. Nucleic acid sequence analysis showed that this gene was 3495 base pairs encoding 1164 amino acids, which were homolog of 99.31% compared with Cry8Eal with eight different amino acids which are in 452,526,527,537,542,752,907 and 975 position respectively, the molecular weight of the protein was 131.8 kD with isoelectric point pH 4.71. This gene sequence had been registered in GenBank (Accession number was EU047597), and named cry8Ea2 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee.5 The full length cry8Ea2 gene were inserted into E.coli expression vector pET-21b to construct the recombinant expression plasmid pET21b-cry8Ea by control of T7 promotor. The result of SDS-PAGE indicated that cry8Ea2 gene could be expressed as a 130 kD protein in E. coli BL21 (DE3) strain induced by IPTG6 The bioassay of Cry8Ea2 expression product showed that it is toxic to the larvae of Holotrichia parallela, Yopillia flavosellata and Plagiodera versicolora, with the corrected mortality of 46.67%, 55.56% and 33.33%, respectively. But it has no insecticidal activity to Holotrichia oblita, Henosepilachna vigintioctopunctata, Tenebrio molitor, Helicoverpa armigera, Spodoptera exigua, Mythimna separata, Trichoplusia ni.