| Bacillus thuringiensis is an important insecticidal microorganism. It has not yet showed toxicity to people , domestic animals and natural enemies of pests and can keep the ecological balance. It is less likely than chemical pesticides to cause field resistance in target insects. So, to study on the screening, identification, molecular biology and fermentation of Bacillus thuringiensis is of importance. Resource of Bacillus thuringiensis was studied in Sichuan in this paper. The research lays the foundation for transferring gene engineering about anti-pest breeding, the construction of the new engineering strains against pests and the application and production of Bacillus thuringiensis pesticide.Nine Bacillus thuringiensis strains were isolated from Wenjiang in Sichuan by Sodium acetate-antibiotic method. Their classification status in microbiology was deduced by studying the physiological and biochemical character. Each strain is toxic to Pieris rapae, Parnara guttata, Chilo suppressalis et al in vitro and field trial . Insecticidal crystal protein produced from 39 strain was isolated and purified, and the crylAb gene of 39 strain was cloned through PCR method. The genes were located on the plasmids of the strains through dot-blot hybridization. In addition, the potential promoting in rice was studied.The main results are summarized as below:1. Two hundred and seventy-nine Bacillus were isolated from ninety-four soils samples collected from Wenjiang in Sichuan by sodium acetate-antibiotic method. Nine Bacillus thuringiensis 39, D3, Hi2, Dj, LI, 2, 4-2, 4-14 and A\ were obtainded according to their physiological and biochemical character.2. Based on PCR method, two-step multiplex Polymerase Chain Reaction with primers designed by the cry genes sequences were used to detect the cry genes from nine strains of Bacillus thuringiensis . cry I and cryV genes were found in all of them ; LI and 39 strains have cry II genes; 39, 4-14, 2, AI and D3 strains have cryIII. genes. Each strain not only has different cry gene type, but also different cry-subclass. For example, 39, 2 and 4-14 contain crylAb and crylAc genes. The others have crylAb, cryl Ac and crylC genes.3. The bioassays against Pieris rapae, Parnara guttata, Chilo suppressalis et al in vitro showed that Bacillus thuringiensis had the best biocontrol effects to Pieris rapa, and the averageeffect is 90%. The highest mortality to Parnara guttat is 85.68%, and to Chilo suppressalis is 83.67%. The total tests showed that 39 and Da are higher toxin Bacillus thuringiensis.4. The controlling experiments in field showed that the biocontrol effect against pests would change by the influence of external environment. Howevert, the control result was consistent with the result in vitro. The best control effect against Pieris rapae was 100%. 39 and D3 strains against Parnara guttata was 85.06%, 83.67% respectively, and against Chilo suppressalis was excellent.5. Study on the biological characteristics of 39 strain indicated that its growth curve was bacteria typical growth curve, including four phases: lag phase, exponential phase, stationary phase and decline phase. The best cultural condition was: pH7, 35 . The Biocontrol effect could be affected by the ultraviolet ray.6. The growth-promoting effect of Bacillus thuringiensis on dry weight of root and step was tested in vitro. The field trail showed that the treatments of Bacillus thuringiensis made the number of efficient seeds, the weight of 1000 seeds, the rate of seeding, and length of spike increased respectively.7. SDS-PAG electrophoresis indicated the toxic protein of 39 strain was consisted of three protein :90kDa, 60kDa and 46kDa.8. With the cryl Ab gene primer, PCR was performed using DNA from 39 as the template and 1330bp fragment was obtained. The recovered fragment was inserted into T-vector, and then the recombinant DNA was transformed into the competent cells of DH5 .Sequence analysis showed that the fragment contains 1330bp, coding a protein of 443 amino acids. Compa...
|
PDF Full Text Request