Development of Gene Introduction and Vector System in Plant Pathogenic Fungus

植物病原真菌基因导入及载体系统的开发

基本信息

批准号：
01560057
负责人：
TOYODA Hideyoshi
金额：
$ 1.73万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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.

在第一年，我们建立了一个有效的系统，用于直接基因转移至氧气孢子菌的大孢子。 sp。 Lycopersici（种族I），通过电穿孔导致番茄植物的枯萎。这项工作已发表在《植物病理学杂志》（1989年）中。这种方法使我们能够将外源基因引入植物病原真菌中，而没有任何特殊治疗（例如原生质体形成及其再生）。因此，在去年，我们试图在这种真菌中构建矢量系统。为此目的。我们首先通过从该真菌的菌丝体中提取的rRNA合成cDNA来分离真菌染色体中编码rRNA的基因。这些cDNA与已经从染色体DNA制备的基因组DNA杂交，并分离了针对28S rRNA的cDNA。该克隆的DNA序列通过二氧化法确定，并裂解成片段的两个部分。为了构建载体，标记基因（湿霉素B）在这两个部分之间是两侧。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。使用该载体，通过电穿孔成功地转化了大胶质细胞。此外，我们开发了MRNA的原位杂交方法，并在本研究中阐明了探针。通过显微注射将探针DNA引入这种真菌的菌丝体中，以确保菌丝细胞质中的细胞质杂交。这些结果已提交给杂志，农业和生物化学杂志。