21396477
not annotated - annotated - LINNAEUS only
Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii.
Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.
Ann file
T1 Species 0 26 Agrobacterium tumefasciens
N1 Reference T1 Taxonomy:358
T2 Species 73 98 Blastocladiella emersonii
N2 Reference T2 Taxonomy:4808
T3 Species 102 127 Agrobacterium tumefaciens
N3 Reference T3 Taxonomy:358
T4 Out-of-scope 223 228 yeast
T5 Species 257 262 human
N5 Reference T5 Taxonomy:9606
T6 Species 371 396 Blastocladiella emersonii
N6 Reference T6 Taxonomy:4808
T7 Species 735 755 Aspergillus nidulans
N7 Reference T7 Taxonomy:162425
T8 Species 926 951 Agrobacterium tumefsciens
N8 Reference T8 Taxonomy:358
N4 Reference T4 Taxonomy:147537 Saccharomycotina