Genetic Transformation Systems for Characterization of Gene Promoters in MarineAlgae

摘要

This report summarizes the results of ONR funded research focused on thedevelopment of a general method for directed genetic manipulation of marine algae based on DNA transfection. The research established the use of electroporation for DNA transfection and macromolecular loading of walled diatom cells through the development of an osmotically compatible electroporation buffer, Seapore Buffer. This technique was extended to several other diverse groups of marine phytoplankton. Expression of transfected genes was demonstrated and stability of transfection assessed. Kanamycin and formaldehyde were identified as two useful selective agents for enrichment of transformed cell lines and enhancement of expression from plasmids bearing the resistance markers. A variety of protein encoding genes where characterized from the diatom Skeletonema costatum and used to identify potential requirements for efficient translation of heterospecific genes in diatoms. Flanking regulatory sequences of highly expressed genes were targeted for future development of diatom specific transformation vectors. The protocols developed through this research provide a foundation for biotechnological utilization of marine chromophyte algae.