Team:Cornell/project/background/fungal genetic engineering

Recent years have shown increasing interest in the genetic engineering of filamentous fungi. This interest has been supplemented by many developments in transformation systems. Currently, there are three main methods commonly used to transform fungal species with recombinant DNA [1]. The primary method is polyethylene glycol (PEG) transformation, whereby the fungal species is protoplasted to rid the cells of their cell walls. By exposing the fragile protoplasts to PEG and recombinant DNA the cells uptake the DNA. Integration of the DNA into the fungal genome can then occur via homologous recombination, random insertion, or restriction enzyme mediated integration (REMI) [2]. Typically, homologous recombination is the most efficient, though this varies by species. Transformation markers are typically used in order to select for transformants. In fungal strains, these typically include resistances for hygromycin and geniticin.

Agrobacterium mediated transformation (AMT) is a method by which the fungus is co-cultivated with Agrobacterium [3]. The Agrobacterium is cloned to carry specific plasmids, and passes on these plasmids through horizontal gene transfer, allowing a path through the cell wall. The fungus then integrates the plasmids of the Agrobacterium.

Ganoderma lucidum and Cochliobolus heterostrophus were the fungal strains we used for genetic engineering. Protoplasting on both organisms was attempted but was only successful with Cochliobolus heterostrophus. The Cochliobolus protoplasts were then transformed with various constructs. The enzymes typically used for protoplasting Ganoderma lucidum are exclusively available in China, so we attempted to use alternate enzymes [4]. Future attempts at transforming Ganoderma lucidum will be done with agrobacterium mediated integration.

Reference

1. Weld, R. J., Plummer, K. M., Carpenter, M. A., & Ridgeway, H. J. (n.d.). Appraoches to functional genomics in filamentous fungi. N.p.: Nature.

2. Turgeon, G. B., Condon, B., Liu, J., & Zhang, N. (2010). Protoplast Transformation of Filamentous Fungi (Vol. 638). N.p.: Molecular and Cell Biology for Fungi.

3. Feldmann, K. A., & Marks, D. M. (1986). Agrobacterium-mediated transformation of germinating seeds of Arabidopsi thaliana: A non-tissue culture approach. N.p.: Zoecon Research Institute.

4. Sun, L., Cai, H., Xu, W., Hu, Y., Gao, Y., & Lin, Z. (2001). Efficient Transformation of The Medicinal Mushroom Ganoderma Lucidum (19th ed.). N.p.: Plant Molecular Biology Reporter.