Figure 1.
Morphologies of U. muehlenbergii.
Images of the hyphae phase (A) and the yeast phase (B) were obtained using a stereoscope (left) and a scanning electron microscope (right).
Figure 2.
pBHt2 contains the hygromycin B resistance gene under the control of the Aspergillus nidulans trpC promoter [8] was used to construct pSK1044 by insertion of the eGFP gene cassette between the EcoRI and HindIII sites in the multi-cloning site of pBHt2.
Figure 3.
Growth, transformation efficiency, and Southern analysis.
(A) Sensitivity of a wild-type U. muehlenbergii strain to hygromycin B was evaluated by culturing it on PDA amended with 0, 5, 10 and 20 µg/ml of hygromycin B. (B) Membranes overlaid with only fungal cells (1st on the left), and a mix of A. tumefaciens and fungal cells (2nd, 3rd and 4th) were placed on PDA containing 20 µg/ml hygromycin B after culturing on co-cultivation medium for 24, 36 and 48 hrs (2nd, 3rd and 4th). The pictures were taken after 28 days of culture on this selection medium. (C) Effect on the transformation efficiency by increasing the co-cultivation time. Data presented as the average of eight plates per treatments. Error bars indicates standard error. (D) Distribution of T-DNA copy number among transformants of different time interval of co-cultivation. Genomic DNAs were digested with HindIII, a restriction enzyme that does not cut the hph cassette (see Figure 2). The digested DNAs, fractionated using 0.7% agarose gel and transferred to a nylon membrane, were probed with a labeled hph cassette. (E) Representative result of Southern blot analysis from 15 randomly selected transformants and a wild-type strain. (F) Distribution of T-DNA copy numbers among 784 transformants.
Figure 4.
Expression of eGFP in 11 transformants. DIC, differential interference contrast images; GFP, fluorescence image.
Table 1.
Results from BlastX searches of the NCBI database with U. muehlenbergii genomic sequences flanking inserted T-DNA as queries.
Figure 5.
Agarose gel analysis of products resulted from TAIL-PCR of eight randomly selected transformants.
The gels from the top to the bottom show products from the primary, secondary, and tertiary reactions, respectively.
Figure 6.
Genomic sequences of U. muehlenbergii flanking the right border of inserted T-DNA.
All sequences are shown 5′→3′. Bold letters correspond to the 30 bp border sequences.
Figure 7.
Colonies of four putative mutants of U. muehlenbergii generated via ATMT.
From left to right, colonies of a wild-type strain and four mutants are shown. These strains were grown for 28 days on PDA.
Table 2.
Primers used in this study.