| Beauveria bassiana is one of the popular entomopathogenic fungi and had been widely used for agricultural and forest pest control all over the world. Due to the advantage of safety and sound to environment hi its application, as well as the character of invading host actively and infecting pest circularly, compared with chemical pesticide, B.bassiana has been attracting more and more attention in recent years. However, the slow and unstable virulent effect to pest makes great handicap on the widespread and acceptance of fungal insecticides. The main course of such phenomena is unclear and ambiguous about the molecular basis of fungal entomopathogenicity.Since the penetration to host s cuticle is a critical step and chitinase play an important role in it, which involves both mechanical pressure and enzymatic degradation, and the facts reported in recent years showed that there are several chitinases produced in the process of penetration and the general roles of them in pathogenesis remain to be further studied. So, it is urgent and necessary for us to explore the infection mechanism of B.bassiana on the molecular level. Upon these problems and facts mentioned above, this dissertation focuses on the purification of chitinase from B.bassiana.In addition, based on the special reaction between antigen and antibody, which is very sensitive, simple, fast and credible, immunoassay is widely used in the expression of a particular gene or in the study and certification of protein about its characteristics, such as function, structure, homologue, relationship in evolution, etc. This paper also carries out thepreparation work of chitinase antiserum. Then, the main results are summarized as following:1. Comparating of B.bassiana strains producing chitinaseThree strains of B.bassiana, Bbl-1, Bb02, Bb05, preserved in our laboratory, were induced respectively in the minimum medium containing colloidal chitin as sole carbon and nitrogen source. The chitinase activity was detected every 6 hours. According to the curve of chitinase activity, Bbl-1 was selected out for following studies.2. Timecourse of chitinase produced by B.bassianaIn order to find the time of chitinase s tiptop activity and investigate the effect of glucose on production of chitinase, Bbl-1 mycelia were transferred into the induction medium with or without 2% glucose after incubated in SDY for 2-3 days. The chitinase activity in both cultures was assayed every 12 hours. The result showed that the highest activity of chitinase came at the point of 168 hours after the mycelia was transferred into the induction medium, which would be used for chitinase purification, and the expression of chitinase was inhibited in the initial stage in the induced medium supplemented with glucose, which indicated that glucose repressed the expression of chitinase.3. Purification of chitinase from B.bassianaFiltrated from the induction culture, the liquid was subjected to 75% Ammonium Sulfate for precipitation. Then, the pellet, after centrifugation, was resolved in 0.02mol/L pH8.0 Tris-HCl and the crude extract was obtained. The next step was Ultrgel AcA54 gel filtration chromatography, which was carried out by 0.02 mol/L pHS.O Tris-HCl. The unique peak of endochitinase activity, ranged in 67-76 fractions, was pooled and dialyzed completely against 0.02 mol/L pH7.8 Tris-HCl. After concentrated for a while by PEG20 000, the sample from Ultragel AcAs4 was chromatographed on the DEAE-Cellulose anion exchange column, which was also preequilibrated by 0.02 mol/L pH7.8 Tris-HCl fully. Then, the column was washed with the same buffer before it was eluted with 100ml of linear salt gradient from 0 to lmol/1 NaCl in 0.02 mol/L pH7.8 Tris-HCl. There was also one single peak of endochitinase activity and it was in column-through fraction.4. Identification and characterization of purified chitinaseSubjected to SDS-PAGE, the pooled activity fraction from DEAE-Cellulose migrated as a single band, indicated that the purified chitinase from B.bassiana...
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