Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: cloning, characterization and application as a dominant selectable marker for transformation.
A pyrithiamine (PT) resistance gene (ptrA) was cloned from a genomic DNA library prepared from a PT resistant mutant of Aspergillus oryzae. It conferred high resistance to PT on an A. oryzae industrial strain as well as A. nidulans. Nucleotide sequence analysis showed that the ptrA gene contained one intron (58-bp) and encodes 327 amino acid (aa) residues. Additionally, the deduced aa sequence has 72% and 63% identity to Fusarium solani sti35 encoding a stress-inducible protein and Saccharomyces cerevisiae THI4 encoding an enzyme involved in thiamine biosynthesis, respectively, indicating that ptrA is a mutated allele of a gene belonging to the THI4 family. The mutation point was identified in the conserved motif in 5'-flanking region of these three THI4 homologous genes (ptrA, sti35, and THI4). The introduction of the ptrA gene allowed an A. oryzae industrial strain to grow on the minimum medium containing PT (0.1 mg/l) on which an untransformed strain did not grow. This result indicates that the ptrA is applicable as a dominant selectable marker for transformation of A. oryzae.
Kubodera
T
T
Research & Development Department, Hakutsuru Sake Brewing Co. Ltd., Kobe, Japan. kubodera@hakutsuru.co.jp
Yamashita
N
N
Nishimura
A
A
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001236:Aspergillus oryzae / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D015415:Biomarkers; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004352:Drug Resistance, Microbial / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D011745:Pyrithiamine / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013831:Thiamine / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D000935:Antifungal Agents; D015415:Biomarkers; D004271:DNA, Fungal; D005656:Fungal Proteins; C416685:THIA protein, Aspergillus oryzae; D011745:Pyrithiamine; D013831:Thiamine