Figure 1.
The modified OSCAR methodology for construction of gene disruption plasmids.
The donor plasmids contain the cassette of the herbicide-resistance genes Bar or Sur. The recipient binary plasmid pPk2-OSCAR-GFP was used for construction of gene disruption plasmids in E. coli. Primers B2r+5F/B1r+5R and B4+3F/B3+3R were used for cloning 5′ and 3′ flanking sequences of the gene to be deleted, respectively. B2r, B1r, B4 and B3 are recognition sites of Bp Clonase for recombination, which were added to 5′ end of their respective primers. Primer 5F (or B2r+5F) and Bar3 (Sur3 when pA-Sur-OSCAR used) and Bar5 (Sur5 when pA-Sur-OSCAR used) and 3R (or B3+3R) were used to confirm the construction of the gene disruption plasmids.
Figure 2.
The disruption of MrKu70 in M. robertsii using the herbicide-resistance gene Sur as the selection marker.
(A) The disruption plasmid of MrKu70 (bottom) and the relative position of MrKu70 in the wild-type strain. (B) Confirmation of the disruption of MrKu70 by PCR in the mutants with glufosinate ammonium resistance and without a GFP signal. 1 and 2 designate two different ΔMrKu70 mutants, and C is the wild-type strain. Top panel: PCR conducted with the primers Sur5 and MrKu70CF2, PCR products can be obtained only from MrKu70 disruption mutants; Bottom panel: PCR conducted using primers MrKu70CF1 and MrKu70CF2, and PCR products can be obtained in any strains other than MrKu70 disruption mutants. (C) Confirmation of the complementation of ΔMrKu70 by PCR using the primers MrKu70ORF-5 and MrKu70ORF-3 to amplify across the deleted region. 1 to 3: 3 different transformants with ΔMrKu70 complemented; C: the wild-type strain; M: ΔMrKu70; L: DNA ladder (DL 10004) from Generay (Shanghai, China).
Figure 3.
Disruption of Cag8 in ΔMrKu70 using the bar gene as the selection marker.
(A) the Cag8 disruption plasmid (pPK2-OSCAR-GFP-Cag8) and the relative position of Cag8 in ΔMrKu70. (B) The representative 74 of 301 glufosinate ammonium-resistant transformants randomly selected from fungal transformation plates for Cag8 disruption in ΔMrKu70. Arrows indicate colonies with conidiation, and the remaining 72 transformants show the fluffy phenotype without conidiation. (C) Two fluffy colonies were randomly selected from the transformation (B) (labeled as 1 and 2) for confirmation of Cag8 disruption in ΔMrKu70. Top panel: PCR conducted with the primers Bar5 and Cag8CF2, PCR products can be obtained only from ΔCag8:ΔMrKu70 mutants; Bottom panel: PCR conducted using primers Cag8CF1 and Cag8CF2, and PCR products can be obtained in any strain other than ΔCag8:ΔMrKu70 mutants. L: DNA ladder (DL 10004) from Generay (Shanghai, China).
Table 1.
Primers used in this study.