1990 Fiscal Year Final Research Report Summary
Development of Gene Introduction and Vector System in Plant Pathogenic Fungus
| Project/Area Number |
01560057
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
植物保護
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| Research Institution | Kinki University |
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Principal Investigator |
TOYODA Hideyoshi Kinki University, Agriculture, Associate Professor, 農学部 (00150805)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Motonobu Kinki University, Research Insti, Food Science,, 講師 (80192425)
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| Project Period (FY) |
1989 – 1990
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| Keywords | electroporation / rRNA gene / homologous recombination / in situ hybridization / microinjection |
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| Research Abstract |
In a first year, we established an efficient system for direct gene transfer to macrospores of Fusarium oxysporum f. sp. lycopersici (race I), which causes the wilting in tomato plant, by electroporation. This work has been published in Journal of Phytopathology (1989). This method enabled us to introduce foreign genes into plant pathogic fungus without any special treatments such as protoplast formation and its regeneration. In a last year, therefore, we attempted to construct the vector system functional in this fungus. For this purpose. We first isolated the genes encoding rRNA in fungal chromosome by synthesizing cDNAs from rRNAs extracted from mycelia of this fungus. These cDNAs were hybridized with genomic DNAs which had been prepared from chromosomal DNA, and the cDNA specific to 28S rRNA was isolated. The DNA sequence of this clone was determined by dideoxy method and cleaved into two parts of the fragment. In order to construct the vector, the marker gene (hygromycin B) was flanked between these two fractions. This plasmid vector allowed us to ensure the homologous recombination of DNA sequences between the vector and chromosome. With this vector, macroconidia were successfully transformed by electroporation. In addition, we developed the method for in situ hybridization of mRNA with the probe clarified in this study. The probe DNA was introduced into mycelia of this fungus by microinjection in order to ensure cytoplasmic hybridization in mycelial cytoplasm. These results were submitted to the Journal, Agricultural and Biological Chemistry.
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