Pustaka Ilmiah Universitas Padjadjaran

Abstrak

Agrobacterium Tumefaciens-Mediated Genetic Transformation Of Monascus Purpureus Albino Mutant

Monascus purpureus ITBCC-HD-F002, an EMS-induced albino strain, was transformed into hygromycin B resistance using the hygromycin B phosphotransferase (hph) of Escherichia coli as the selective trait, governed by gdp promoter of Aspergillus nidulans in pUR5750 plasmid or CaMV35S promoter in pCAMBIA1304 plasmid. These plasmids were transformed into protoplasts and spores from this fungus mediated by A. tumefaciens LBA1100 and A. tumefaciens AGL1. The transformation efficiency achieved with pUR5750 mediated by A. tumefaciens LBA11 was 346 ± 4.00 transformants/107 protoplast, with pUR5750 mediated by A. tumefaciens AGL1 was 635.7 ± 13.32 transformants/107 spores and with pCAMBIA1304 mediated by A. tumefaciens AGL1 was 650.3 ± 10.02 transformants/107 spores. This result indicating the highly virulent strain A. tumefaciens AGL1 was found to be more efficient in DNA transfer than A. tumefaciens LBA1100 and the CaMV35S promoter from pCAMBIA1304 worked well in this fungus. The majority of transformants were mitotically stable up to five generations (92-96 %). The presence of the hph genes were detected by PCR. In four randomly chosen transformants, single-copy integration of the marker gene at different chromosomal site were proven by Southern Blot analysis. It favoured single-copy integration in genome, which facilitate genomic study using this method and as a tool for insertional mutagenesis for a mycotoxin gene disruption.. The additional marker in pCAMBIA1304 might serve for physiological study on another metabolite-targeting in this fungus, like natural pigments, antihypercholesterolemia, inflammatory and antitumour agents.