Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea.
This study describes the development of an efficient and reliable activation tagging system for the medicinal fungus Antrodia cinnamomea. For successful Agrobacterium tumefaciens-mediated transformation, different parameters were considered. The Agrobacterium concentration of 5 x 10(8) cfu ml(-1), 1mm acetosyringone, 25-d-old mycelia at 0.2 g ml(-1), and co-culture period of 6d were found to be the most optimal conditions for enhancing the transformation efficiency. The mitotic stability of transferred DNA (T-DNA) was demonstrated by growing eight randomly selected putative transformants in malt extract agar medium for five subcultures. Insertion of T-DNA into the genome of transformants was confirmed by PCR and Southern hybridization. Results showed that 88% of the mutants contained a single T-DNA insertion. Two of the mutants were observed with different triterpenoid profiles compared with the untransformed cultures. Our results suggest a new functional genomics approach to tag the triterpenoid biosynthesis genes in A. cinnamomea.
Chen
Emily Chin-Fun
EC
Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, 168 Gifeng E Road, Wufeng, Taichung County 41349, Taiwan.
Su
Yu-Hsiu
YH
Kanagarajan
Selvaraju
S
Agrawal
Dinesh Chandra
DC
Tsay
Hsin-Sheng
HS
D016960:Agrobacterium tumefaciens / Q000235:genetics; D055491:Antrodia / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004271:DNA, Fungal / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation; D014170:Transformation, Genetic; D014315:Triterpenes / Q000032:analysis / Q000378:metabolism
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D014315:Triterpenes