PNNL-13501 - Pacific Northwest National Laboratory

are shown in Figure 1(a) and 1(b), indicating that pgk1
promoter can regulate gene expression in all the
conditions tested while the glucoamylase promoter only
regulate gene expression in the conditions of glucose,
starch, xylose, and galactose, but not in mannose and
arabinose conditions. Therefore, pgk1 promoter is a
constitutive promoter while glucoamylase promoter is an
inducible promoter.
Figure 1. Genetic regulatory patterns of R. oryzae pgk1
promoter (a) and glucoamylase promoter (b). Lanes 1, 2, 3,
4, 5, and 6 are total RNA samples prepared from R. oryzae
cultures grown in the medium with glucose, potato starch,
xylose, mannose, galactose, and arabinose, respectively.
Design and Construction of Chromosomal Integrating
Vector System
A chromosomal integrating vector was designed as shown
in Figure 2, primarily containing two fungal expression
cassettes, one of which is an antibiotic resistance
(geneticin or sulfanilamide) gene and the other gfp marker
gene expression cassette. Both the expression cassettes
are under the control of the constitutive pgk1 promoter
and terminated by a fungal terminator Taox1. The gfp
gene expression cassette is flanked by a native R. oryzae
gene to serve as the chromosomal integrating element for
foreign gene insertion into the fungal host during
transformation. The antibiotic resistance gene expression
cassette is used to select transformant candidates after
transformation. The chromosomal integrating vectors
were constructed through a series of vector cloning
efforts, and used for the following transformation test.
Transformation Experiments
Germinated and enzyme-digested R. oryzae spores were
transformed with the constructed chromosomal
integrating vector using electroporation method. After
transformation, transformants were screened in selective
culture medium containing antibiotic geneticin. Potential
Figure 2. Plasmid map of a chromosomal integrating vector
for R. oryzae transformation. Amp, ampicillin resistance
gene; AR, antibiotic resistance gene; F5’and F3’,
chromosomal integrating element 5’ and 3’ end regions,
respectively; ori, plasmid replication origin; P pgk1,
phosphoglycerate kinase 1 promoter.
transformants were sub-cultured onto fresh selective
culture medium for further screening. Based on the
experiment, the transformation frequency was low, about
three to four transformants per µg DNA. After
sporulation, spores of transformed candidates were
collected and germinated in a culture medium containing
antibiotics. Genomic DNA samples were then prepared
for a gene insertion test using polymer chain reaction.
Results show that some of the transformed candidates
have the gfp marker gene insertion.
Feasibility of Converting Multi-Nucleate Fungal Spores
to Single-Nucleate Spores
Rhizopus was found to have an extremely large number of
nuclei for a given cell volume. In ungerminated spores,
individual nuclei could not be distinguished, probably
because of both close packing and inherent fluorescence
of the spore. In freshly germinated cells, 10 to 20 nuclei
are visible, and within a few hours of germination, a
growing mycelium usually contains nearly 100 nuclei as
shown in Figure 3. Because genetic manipulation of
Rhizopus is likely to affect only a fraction of the nuclei
present, the effects of such manipulations are likely to
become diluted in subsequent generations. If the nontransformed
nuclei can be identified and eliminated by
laser micromanipulation and microsurgery, then strains of
Rhizopus, which contain only transformed genomes,
might be obtained. Studies show that spores were
resistant to the cutting laser, while mycelia could readily
be ablated with the cutting laser on high power.
However, methods will need to be developed to
differentiate the transformed nuclei from untransformed
ones.
Biosciences and Biotechnology 77