| Summary: | 碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 93 === Application of entomopathogenic fungi to control insect pests in the field was usually not effective as anticipated due to the stressed and harsh environmental conditions such as high temperature, desication and high sunlight UV radiation. To circumvent the obstacles encountered, we attempt to transform the prevalent entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae var. anisopliae, and Paecilomyces javanicus with melanin biosynthesis genes: polyketide synthase, scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase, melanin proved characterized with antistress and corelated with virulent capacity of some pathogenic fungi. Part of polyketide synthase, scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase gene fragments with length of 700, 250, 750 bp, respectively were amplified by polymerase chain reaction（PCR）using degenerate primers from Alternaria alternata, and being labeled with DIG as probes to screen the Fosmid library. The library was constructed based on the genomic DNA extracted from the mycelium of the dematiaceous A. alternata , which has been documented possessing three melanin biosynthesis genes clustered in a 30 Kb DNA contig. The Fosmid clones exhibiting positive signal against polyketide synthase and 1,3,8-trihydroxynaphthalene reductase were selected to construct shotgun library for sequencing and assembling. The assembled 41279 bp contig blastingX with nr NCBI revealed the encoded polyketide synthase, 1,3,8-trihydroxynaphthalene reductase plus four hypothetical proteins genes, flanked with function unknown DNA sequences. Unexpectedly, the scytalone dehydratase gene was not discovered within the contigs. However, all the full open reading frame of polyketide synthase, scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase genes were cloned and their full-length open reading frames accessed by rapid amplification of cDNA ends（RACE）. To further ascertain the existance of scytalone dehydratase gene, the specific Fosmid clones DNA will be restricted by an array of enzymes and verified by Southern blotting. We also constructed Ti-plasmid binary vectors, pCAM-GF-GT-Scy pCAM-GH-GT-Tri, which harboured GFP and scytalone dehydratase, hygromycinr and 1,3,8-trihydroxynaphthalene reductase gene insertions respectively, which has been cotransfered into the targeted entomopathogenic fungi B. beauveria, M. anisopliae var. anisopliae and P. javanicus by Agrobacterium tumefaciens Ti-plasmid mediated transformation using electroporation device. Preliminarly light and fluorescent microscopy showed the successful transformation as evident by the green fluorescent conidia and mycelium, or the darkened mycelium, which otherwise have not been observed in wild types. Experiments with respect to the phenotype、genotype and bioassay are undergoing and expected to come up with promising outcome.
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