Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Poster: Microbial Genome Technology
            

EXPRESSION OF HYGROMYCIN B RESISTANCE AND PIGMENTATION GENES IN TRANSFORMANTS OF MONASCUS PURPUREUS

¹ Department of Microbiology, Faculty of Science, Kasetsart University, Bangkhen, Bangkok Thailand 10900

² USDA/ARS Department of Crop and Soil Science, 209 Johnson Hall, Washington State University, Pullman, WA. 99164-6420

Two different transformation procedures have been studied. Microprojectile bombardment of conidia and mycelial fragments with cosmid pMOcosX containing the E. coli Hygromycin B phosphotransferase gene (HPH) resulted in two mitotically stable transformants. The efficiency of transformation to Hygromycin resistance was 0.1/ug DNA. Southern blot analysis indicated that a single copy of the cosmid DNA was integrated into the fungal genome. Electroporation-mediated transformation was attempted with fungal protoplasts and Hygromycin B resistance genes in two plasmids. Abortive transformants that grew beneath an agarose overlay containing Hygromycin B were obtained with frequencies of 4.7 and 5.4/ug DNA of plasmids pCB1003 and pCB1004, respectively. These transformants did not survive after subculturing to selective media. Electroporation of fungal protoplasts from an albino strain in the presence of total genomic DNA from a red strain resulted in eight cultures that transiently, but consistently, produce red or yellow pigments in culture. The frequency of these putative transformants was 1.67/ug DNA. The pigmentation was observed under a compound light microscope and was determined to be produced within the mycelia. PCR analysis using primers designed for a polyketide synthase gene, and RAPD primers, revealed novel DNA fragments in these cultures, further suggesting they were transformed. Southern analysis has suggested that homologous gene replacement may have occurred in these transformants.