Development of Gene Introduction and Vector System in Plant Pathogenic Fungus

在第一年，我们建立了一个有效的系统，直接基因转移到大孢子的尖孢镰刀菌f。sp.lycopersici（生理小种I）的突变体，该突变体在番茄植株中引起萎蔫。这项工作已发表在Journal of Phytopathology（1989）上。这种方法使我们能够将外源基因导入植物病原真菌，而不需要任何特殊的处理，如原生质体的形成和再生。因此，在去年，我们试图构建在这种真菌中起作用的载体系统。为了这个目的。我们首先从该真菌菌丝体中提取rRNA，通过合成cDNA的方法分离了真菌染色体上编码rRNA的基因。将这些cDNA与从染色体DNA制备的基因组DNA杂交，分离出28S rRNA特异性cDNA。用双脱氧法测定了该克隆的DNA序列，并将其切割成两部分片段。为了构建载体，将标记基因（潮霉素B）侧接在这两个级分之间。该质粒载体使我们能够确保载体和染色体之间的DNA序列的同源重组。利用该载体，通过电穿孔法成功地转化了大分生孢子。此外，我们还建立了用本研究中阐明的探针进行mRNA原位杂交的方法。将探针DNA通过显微注射导入该真菌的菌丝体中，以确保菌丝体细胞质中的细胞质杂交。这些结果已提交给《农业与生物化学》杂志。

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.