| The indoor and field toxicity assay of Bacillus thuringiensis strain 4.0718 were studied, the parasporal crystals were separated and the observation was made under elecronmicroscope, the solubility and properties of trypsinisation were studied, too. protoxin and toxin of parasporal crystals recovered from SDS-PAGE, and the method of analysis by mass spectrometry of recovered protein from SDS-PAGE were explored, two-dimensional gel electrophoresis were emploied to analyse the ICPs of Bt for its patterns of subunits composition, molecular weight and isoelectric point of each subunits.1. Toxicity assayIn indoor bioassay the corrected mortality of strain 4.0718 preparation diluted 250 times to 3 instar plutella xylostella larvae was 100% within 72hr, the corresponding results to 3 to 4 instar Heliothis armigera larvae was 79.3%. The results showed that the control effect of strain 4.0718 is higher than other strains.In field plots strain 4.0718 preparation diluted 100, 200 and 300 times to control third generation Heliothis armigera, the rate of insect drop (RID) were 76.2%, 66.7% and 47.6 at 3rd day, 81.4%, 65.1% and 62.8% at 7th days respectively. The control effect is obviously higher than similar insecticide Bt preparation "qiansheng" diluted 200 times, whose RID were 21.4% and 34.2% at 3rd or 7th day respectively, and chemical insecticide acephatemet diluted 670 times, whose RID were 28.5% and30.1% at 3rd or 7th day respectively.In field regions strain 4.0718 preparation diluted 200 times to control 3rd generation Heliothis armigera, RID were 59.71% and 75.59% at 3rd day, wheras the control biological insecticide "big hero" diluted 330 times were 59.40% and 63.80% respectively, the control effect at 3rd day is similar to "big hero", and is 11.79% higher than "big hero" at 7th day.In comparison fields demonstration strain 4.0718 preparation diluted 250 times to control 4th generation Heliothis armigera, RID were 79.39% at 3rd day, the control effect of chemical insecticide high permeability monocrotophos diluted 250 times and acephatemet diluted 500 times added esfenvalerate diluted 1000 times was similar.2. Separation of parasporal crystals and observation under electronmicroscopeThe separation of crystals from strain 4.0718 and its morphology under electronmicroscope were studied, its insecticidal crystal proteins were analyzed by SDS-PAGE, too. Based on shape, there are two major types of crystals, those with shape of bipyramidal, composed of 130 kD Cryl protoxin, and those with shape of cubic, made up of 65 kD Cryll protoxin .The strain is toxic to insects of Lepidoptera and Diptera.3. Comparison of character of SDS-PAGE and content of insecticidal crystal proteins of different Bt strainsCharacter of electrophoresis and content of parasporal crystal proteins of strain 4.0718 and those of other four strains were compared, the results showed that the protein content of parasporal crystals of strain 4.0718 is higher than other four strains.4. Solubility and properties of trypsinisation of parasporal crystals The solubility of strain 4.0718 parasporal crystals handled bydistilled water, alaki and surface active reagent were studied, and theeffect of different buffer pH values and reaction time on trypsinisation were compared, Primary purification way of toxin solubilized in alaki buffer contain B - mercaptoethanol, precipitated again with acid, followed by trypsinisation and ammonium sulfate precipitation were made.5. Purification of protoxin and toxin and analysis by mass spectrometry The way of recovering proteins from SDS-PAGE gel to use 65 kD protoxin from Bt strain 4.0718 as a model for mass spectrometry was explored. The effect of different staining methods to recovering potein was compared. The purifmg steps includes SDS-PAGE, electroelution, desalting, and SDS removal. Entrapment of the eluted protein with semipermeable membrane for eletroelution, centrifugation ultrafiltration for desalting and cold acetone method was used for SDS removal. Th... |
