Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea .

Autor: Dörnte B; Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, University of Goettingen, Büsgenweg 2, 37077 Goettingen, Germany., Peng C; School of Life Sciences, Anhui University, Hefei, 230601 China.; Anhui Key Laboratory of Modern Biomanufacturing, Hefei, 230601 China., Fang Z; School of Life Sciences, Anhui University, Hefei, 230601 China.; Anhui Key Laboratory of Modern Biomanufacturing, Hefei, 230601 China., Kamran A; Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, University of Goettingen, Büsgenweg 2, 37077 Goettingen, Germany.; Present Address: Institute for Microbiology and Genetics, University of Goettingen, 37077 Goettingen, Germany., Yulvizar C; Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, University of Goettingen, Büsgenweg 2, 37077 Goettingen, Germany., Kües U; Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, University of Goettingen, Büsgenweg 2, 37077 Goettingen, Germany.; Goettingen Center for Molecular Biosciences (GZMB), University of Goettingen, Goettingen, Germany.
Jazyk: angličtina
Zdroj: Fungal biology and biotechnology [Fungal Biol Biotechnol] 2020 Oct 12; Vol. 7, pp. 15. Date of Electronic Publication: 2020 Oct 12 (Print Publication: 2020).
DOI: 10.1186/s40694-020-00105-0
Abstrakt: Background: Two reference strains have been sequenced from the mushroom Coprinopsis cinerea , monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 ( ade8-1 ) and a para -aminobenzoic acid (PABA)-auxotrophy in AmutBmut ( pab1-1 ) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph , respectively.
Results: Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8 + vector p Cc Ade8 complements the auxotrophy of OK130 in transformations. Transformation rates with p Cc Ade8 in single-vector and co-transformations with ade8 + -selection were similarly high, unlike for trp1 + plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected p Cc Ade8 helped in co-transformations of trp1 strains with a trp1 + -selection vector to overcome suicidal effects by transferred trp1 + . Co-transformation rates of p Cc Ade8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26-42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase.
Conclusions: ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. p Cc Ade8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8 + successive rounds of transformation possible.
Competing Interests: Competing interestsThe authors declare no competing interests.
(© The Author(s) 2020.)
Databáze: MEDLINE
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