Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium.
The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (P gpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.
Institute of Biotechnology of León, INBIOTEC, Science Park, Av. Real 1, 24006 Leon, Spain.
R P
RP
Godio
R
R
Fouces
E J
EJ
Gudiña
J F
JF
Martín
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000378:metabolism; D000970:Antineoplastic Agents / Q000378:metabolism*; D001203:Ascomycota / Q000737:chemistry; D001487:Basidiomycota / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007810:Lanosterol / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D000970:Antineoplastic Agents; D004269:DNA, Bacterial; C115583:clavaric acid; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007810:Lanosterol; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase