An efficient transformation system for Trichoderma atroviride using the pyr4 gene as a selectable marker.
Braz J Microbiol
; 51(4): 1631-1643, 2020 Dec.
Article
em En
| MEDLINE
| ID: mdl-32627116
The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride. To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5 mM uracil restores their growth and conidia production, and 1 mg ml-1 is the lethal dose of 5-fluoroorotic acid in T. atroviride. Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura-1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆tmk3 and ∆pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆blr1 and ∆blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1, grg2, phr1, and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Orotidina-5'-Fosfato Descarboxilase
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Transformação Genética
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Proteínas Fúngicas
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Hypocreales
Idioma:
En
Revista:
Braz J Microbiol
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
México
País de publicação:
Brasil