Insecticidal Screening And Genetic Recombination Of Marine Microorganisms No.050101

In contrast to the terrestrial environment, little pharmaceutical information is available to guide current marine research. Many structurally unprecedented, and often highly bioactive metabolites have now been isolated from marine plants and marine animals. Here marine microorganisms from eastern and southern China Sea were evaluated for anti-insect activities. The results are briefly summarized as below:1 .Two methods of common larva-dipping and micro-plate were employed for screening marine anti-insect microorganism against Heliothis armigera. 917 strains were screened, and 1.7448% had insecticidal activity, including 12 strains bacterium, 2 strains actinomycetes and 2 strains fungus. With 54.17% mortality against neonate larvae of H. armigera, 050101 was identified to be Bacillus subtilis. Fermentation broth of 050101 had above 50% activity against neonate larvae of H. armigera after it was cultivated seven generations.2. A medium was optimized by single factor and orthogonal experiments. The results showed that the best medium was consisted of 3.5% starch, 2.5% tryptone,0.2% CaCO3, 75% seawater and 25% distilled water and the value of pH was 7.5. Fermentation broth of 050101 had 67.33% activity after it was cultivated at 28 and 200 r/min in rotary vibrator for 3d.3.With DES and 60Co- radiation, a mutant strain 050101D was obtained by anti-insect screening. Fermentation broth of 050101D had 77.78% activity on neonate larvae of H. armigera. Continues culture exhibited that the mutant strain had high genetic stability.4.Experiment of cellular membrane broken showed that insecticidal toxin/toxins of 050101D was/were excreted the outside of cells. The extractions with several organic solvents had not any anti-insect activity against H. armigera. Experiments of ammonium sulfate sediment exhibited that toxins had high 78.9% activity with 80% ammonium sulfate saturation. Its activity kept stability with 50 癈 for 1h and under pH 6~10. Experiment of dialysis showed that its molecule weight was above 8000Da. It had the biggest absorption in 280nm wavelength with scanning UV spectrum. According to all the facts above, we inferred that the toxins were proteins or peptides. Rudiment purified sample J was obtained through sediment with ammonium sulfate, dialysis and purification with HW-50S column chromatography. Assaying by HPLC and FPLC exhibited that Sample J had three compounds with 47.22% activity. Methods of isolation and purification needed to be studied further.4. The full length sequence of the promoter and Cry1Ac gene were obtained respectively by PCR with two pairs of unique primers Cry1AcyF/R and Pxy/F/R, which were designed according to the CrylAc gene and promoter sequence ofxylase operon from Bacillus subtilis 168. The fused translational expression cassette PxylR-CrylAc was constructed using overlapping PCR technique with the primers pair PxylF/CrylAcR. and the mixture of above PCR production. After being digested by Sph I and Bamh I, PxylR-CrylAc expression cassette was inserted into E.coli-Bthuringiesnsis shuttle vector pHT315, and the recombinant plasmids was named as pCrylAc. Furthermore, it was transferred into B. subtilis lab strain 050101D. Available express of Cry1Ac gene was proved with restriction electrophoresis analysis and SDS-PAGE electrophoresis analysis. The engineered 050101D-1AC had high activity on P. xylostella, S. exigua and O. Jurnacalis, mortality for three insects was 71.1%, 56.7% and 88.9% respectively.