| Summary: | 碩士 === 國立臺灣大學 === 植物病理學研究所 === 89 === Chitin, a -1,4-linked polymer of N-acetylglucosamine, is widely existed in many organisms, including crustances, fungi, nematodes and insect, and is able to protect and support the body of organisms. Many kinds of organisms produce chitinase to cleave -1,4-linkage between N-acetylglucosamines and hydrolyzes the chitin polymer to release N-acetylglucosamine oligomers or monomer. The goal of this study is to screen fluorescent Pseudomonas antagonists from rhizosphere and construct Bacillus chitinase gene-containing Pseudomonas strains. Based on the synergism of chitinase and antibioticts, the biocontrol activity can be enhanced. In order to make fluorescent Pseudomonas translocate Bacillus chitinase to the bacterial cell surface, firstly, the DNA sequence encoding signal peptide and transmembrane domain of OmpA (ST-DNA) was amplified from the genomic DNA of Escherichia coli and ligated with the vector pCR2.1. A Bacillus chiA-containing DNA was amplified from pNTU111 and placed downstream of ST-DNA. The chitinase translocated to the outer membrane of E. coli TOP10F' was assumed. Then, ST-chiA DNA was ligated with the shuttle vector, pMMB6, in the position downstream of tac promoter. In Pseudomonas putida, chitinase activity was apparently detected in membrane fraction, supporting that ompA ST-DNA could drive Bacillus chitinase to outer membrane. In addition, we have isolated thirty-six strains of fluorescent pseudomonads from plant rhizosphere. In the dual culture with Sclerotium rolfsii, different strains of fluorescent pseudomonads showed different antifungal activities. Among these antagonistic Pseudomonas strains, seven was performed biocontrol assay. Three Pseudomonas strains could decrease the diseased incidence on bean after seed-coating with Pseudomonas antagonist. Then, plasmid pMMST2000 was transferred into one of Pseudomonas strain, FP-16, the biocontrol activity of FP-16(pMMST2000) was higher than wild strain, FP-16, however, the biocontrol activity between wild and genetic engineered strain did not show significant difference.
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