The Spectral Analysis Of The Megaplamids From 30 Bacillus Thuringiensis Isolated Strains And The Toxic Activity Idetification On Crystal Proteins From These Strains To Necator Americanus

Bacillus thuringiens is(short for Bt) strains always contain sets of self-replicating, extrachromosomal DNA molecules, which are called plasmids. The plasmids vary in number and size in the different Bt strains. Up to now, almost all Cry proteins discovered were coded by the Bt plasmids DNA. The plasmid patterns obtained from gel electrophoresis have previously been used as a tool to characterize Bt strains.The megaplasmid bands of 30 Bt isolates from the Natural Reserves in Hainan, Guangxi and Heilongjiang were analyzed using the method of Pulsed Field Gel Electrophoresis (PFGE). The results showed that the megaplasmids of the tested Bt strains have good diversity in the number and the size.The megaplasmids can vary in number from 1 to 6 and in size from 45 to 582 kb. The Bt strains of S3119-2 and S3321-2, from Diaoluoshan Tropical rain forest reserves in Hainan province, had the same megaplasmid banding patterns, with only one band of 582 kb. The Bt strains of S3161-3 and S3208-3 had the same megaplasmid banding patterns, with 4 megaplasmids in size of 110,180,370,460 kb respectively. The Bt strain of S3322-2 and S3433-2 also had the same banding patterns (45,80,180 kb). While, the Bt strain of S3012-2/ S3136-1, along with S2685-1 and S2809-1 from Heilongjiang Liangshui Natural reserves had the same megaplasmid banding patterns with 3 megaplasmids(70,97,200 kb). Compared with the model strain Bti,30 Bt isolates emerged as different megaplasmid patterns, which was not associated with the ecological relationship or the geographical environment.In order to investigate the toxicity of Bt crystal proteins against hookworm larvae, we extract the crystal proteins of 30 Bt isolates using method of ultrasonic waves. The hookworm larvae were isolated from the infected hookworm human stool. The hookworm larvae were cultured in the Tissue Culture Plate with the Bt crystal proteins at 30℃in the dark. The results showed that only the crystal proteins of S2096-2 have some toxicity to the Necator americanusⅡrhabditiform larvae after 48 h. The death rate increased with increase in the concentration and the time of crystal proteins treatment and the adjusted death rate of S2096-2 crystal proteins againstⅡrhabditiform larvae were at a dose of 133.25μg/ml 30.78%,41.53% and 43.52% for 48 h,72 h and 96 h respectively.The Primers sets of 40Da-5/40Da-3 and pGEM-F/pGEM-R were designed to amplify the complete coding sequence of cry40Dal gene and the pGEM vector sequence containing complete promoter sequence of cry1Ac gene. The complete coding sequence of cry40Dal was inserted into pGEM. The positive recombinant was detected using the restricted enzyme DpnI. Expression products of the positive recombinant were detected by the analysis of SDS-PAGE and the result showed that there was the no expected product as same as our cooperator`s (Dr Neil Crickmore from Sussex University, UK). It is possible that cry40Dal is a pseudogene.