Screening, Identification Of Chitinase-producing Bacteria And Cloning,Expressing Of Chitinase Gene

Chitin is a kind of straight chain macromolecule polymerized by N-acetylgcosamine group which is linked by beta-1, 4-glucosidic bonds. It is widely existed not only in crustaceans and the body case in insects, but also in peritrophic membrane of midgut. Chitinase, an important enzyme to catalyze bio-polymers, can efficiently inhibit the growth of fungi and kill insects, and it is environmentally unharmful. What's more, it is safe for plants and animals including human beings. It is therefore scientifically desirable in non-environmental pollution control of diseases and pests in plants, In our study, the screening, classification, disease resistance, and gene cloning as well as gene expressing of chintinase-producing bacteria were conducted and performed. The main results are summarized as below:I Study on screening and identification of chitinase-producing strains. One hundred and thirty two bacteria and actinomycetes were isolated from soils by using agar dilution method, and 32 strains able to produce chitinase were also screened by the appearance of clear zones in plates. Among the 32 strains, 20 were bacteria and 12 actinomycetes. Strains that were exhibited strong chitinase activity were numbered P6, C-10, 9, F2, J4, J7, 4-2 and X. Of them F2 was the strongest one, and the diameter of the clear zone to colony was 3.14. C-10 bacterium and P6 actinomyceto exhibited the strongest antagonism against pathogens of rice sheath blight, and the antagonistic zone was 7mm and 12mm respectively. P6, C-10 and F2 were determined as the advantageous strains by determining activity of chitinase and antagonism as well. They were Streptomyces globisporus, Bacillus pumilus and Aureobacterium flavesnes according to their morphological and growth features on plates, chemical composition of cell walls and physiological analysis. These 3 strains were all new chitinase-producing microbe that had not been reported.II Study on antagonism of chitinase-producing strains to pathogens. Eleven fungi were used as indicator pathogen in this study. They were Magnaporthe grisea, Rhizoctonia solani, Cephalosporium.sp, Gibberella zeae, Fusarium axysporum f. sp. Cucumerinum, Fusarium graminearum, Fusarium oxysporum f.sp niveum, Fusarium oxysporum f.sp. Melongenae, Colletotrichum gloeosporioide, Fusarium oxysporum f.sp. Coprinus comatus and Helminthosporinm.sp. Strain P6, F2 and C-10, were proved to be exhibited antagonism against these pathogens variously by detecting the degree of antagonism on solid plates. P6 showed the strongest antagonism to Rhizoctonia solani and Colletotrichum gloeosporioides, and its antagonistic zones were both 12mm . The...