Team:Cornell/project/background/fungal genetic engineering

Recent years have shown increasing interest in the genetic engineering of filamentous fungus. This interest has been supplemented by many developments of transformation systems. Currently, there are 3 main methods commonly used to transform fungal species with recombinant DNA[4]. The primary method is PEG transformation, whereby the fungal species is protoplasted to rid the cells of their cell wall. By exposing the fragile protoplasts to polyethylene glycol 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)[3]. Typically homologous recombination is the most efficient. This does vary by species however. 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[1]. The agrobacterium is cloned to carry specific plasmids and uptakes through horizontal gene transfer, the fungus integrates the plasmids of the agrobacterium.

Ganoderma lucidum and Cochliobolus heterostrophus were used as the fungal strains used for genetic engineering. Protoplasting on both organisms was attempted but was only successful with Cochliobolus heterostrophus. The protoplasts were then transformed with various constructs. The enzymes typically used for protoplasting Ganoderma lucidum are exclusively available in china and thus alternate enzymes were attempted[2]. Future attempts at transforming Ganoderma lucidum will be done with agrobacterium mediated integration.

Reference

1. 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.

2. 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.

3. 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.

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