根癌农杆菌介导的CryⅠA(b)基因在毛壳菌中的转化

Transformation of Gene CryⅠA(b)Mediated by Agrobacterium Tumefaciens in Chaetomium Globosum

利用根癌农杆菌介导的遗传转化方法,将CryⅠA(b)基因转入生物防治真菌毛壳菌中,转化率约为40～180个转化子·10-7个孢子。通过对转化子的RT-PCR检测和Southern杂交分析表明,CryⅠA(b)基因已整合进毛壳菌基因组中,而且在转录水平上得到表达,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有高转化率、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具。

Agrobacterium tumefacines-mediated transformation system is routinely used for genetic transformation in a wide range of plant species. Not restricted to plant species, A. tumefaciens is able to transfer T-DNA to yeasts, filamentous fungi and human cells. Using this transformation system, we successfully transferred CryⅠA(b) gene to biological control fungus C. globosum with anefficiency of 40～180 transformants per 107 spores, and constructed genetically engineered strains against plant pathogens and pest insects. Compared with the transformation mediated by PEG-CaCl2, the transformation efficiency was increased 20～50 folds. Putative transformants were analysised for the presence of CryⅠA(b) gene by PCR and southern analysis. The latter analysis proved that the gene was integrated into the genome, and most transformants contained a single copy. RT-PCR analysis showed that the CryⅠA(b) gene was transcribed intoC. globosum transformants. The CryⅠA(b) gene used here was artificially modified from its wild gene according to plant codon bias without changing amino acid sequence, which maybe contributed to the expression of the gene in the fungusC. globosum. Genetic stability of the transformants through mitotic cell division were also observed. Good performance of the tranformation system mediated by A. tumefaciens in C. globosum may further prove to be a powerful tool for the filamentous fungal transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability oftransformants.