A highly e⁄cient polyethylene glycol-mediated transformation

method for mushrooms

Gang Li1, Ruixue Li1, Qiuyun Liu1, Qiang Wang1, Min Chen2 & Baojian Li1
1
The Key Laboratory of Gene Engineering of Ministry of Education, Sun Yat-sen University, Guangzhou, China; and 2Department of Biotechnology
Engineering, Zhejiang Gongshang University, Hangzhou, China

Correspondence: Baojian Li, The Key Abstract

Laboratory of Gene Engineering of Ministry of
Education, Sun Yat-sen University,
Guangzhou 510275, China. Tel.: 186 20
84110296; fax: 186 20 84036551; e-mail:
lsslbj@zsu.edu.cn
Received 8 July 2005; revised 13 December
2005; accepted 13 December 2005.
First published online 2 February 2006.
doi:10.1111/j.1574-6968.2006.00110.x
Editor: Bernard Prior
Keywords
PEG-mediated transformation; Pleurotus
ostreatus; hph; gfp.
A highly efficient transformation system mediated by polyethylene glycol was
developed for the cultivated mushroom Pleurotus ostreatus. Eighty to 180
integrative and stable-resistant colonies appeared per mg of DNA per 107 viable
protoplasts in a transformation experiment with the hygromycin B phosphotrans-
ferase gene (hph), which is about 40–1800 times higher than that previously
reported in P. ostreatus. One hundred to 150 transformants emitting green
fluorescence were observed per mg of DNA per 107 viable protoplasts in a
transformation with the green fluorescent protein gene, but green fluorescence
disappeared 30 h after transformation, suggesting that the green fluorescent
protein gene was only transiently expressed in P. ostreatus. Plasmid pAN7-1 was
also transferred into two important cultivated mushrooms, Ganoderma lucidum
and Lentinus edodes, and 120–150 and 85–100 transformants per mg of DNA per
107 viable protoplasts were obtained, respectively, which is seven to 38 times and
24–28 times greater than previously reported. These data indicate that this new
polyethylene glycol-mediated transformation procedure is highly efficient for
mushrooms, and could be a useful tool in mushroom improvement by gene
engineering.

of DNA per 107 viable protoplasts (Sunkyung et al., 2004),

Introduction
Pleurotus ostreatus is one of the most important edible
mushrooms in many countries, especially in China and
Japan. Cellular and genetic engineering techniques for this
mushroom are well developed, and polyethylene glycol
(PEG)-mediated transformation systems have been de-
scribed (Ming et al., 1992; Koji et al., 1996; Irie et al., 2001).
However, only 2 transformants per mg plasmid DNA (Koji
et al., 1996) or 0.1–0.8 transformants per mg plasmid (Irie
et al., 2001) have been reported, which is very low and not
up to the demands of commercial strain improvement. In
another transformation experiment mediated by PEG,
transformation efficiency reached 5–46 transformants per
mg plasmid DNA, but the majority of the transformants lost
the resistance phenotype after 3–6 weeks of growth in the
absence of selection, and no integrated transformants were
found (Ming et al., 1992). Ganoderma lucidum and Lentinus
edodes are well known in Asia for their curative effects
against cancers. Transformation systems for these two
mushrooms have been established, but the transformation
efficiency of G. lucidum was only 4–17 transformants per mg
and for L. edodes only 3.6 transformants per mg of DNA per
107 viable protoplasts (Irie et al., 2003). Therefore, the
development of a reliable and highly efficient transforma-
tion system for mushrooms that may ultimately prove to be
useful in gene engineering towards producing mushrooms
of improved nutritional value is needed.
As a dominant selectable marker for antibiotic resistance,
the hygromycin B phosphotransferase gene (hph) has been
used successfully in transforming at least seven basidiomy-
cetes, two plant pathogenic smut fungi, Ustilago maydis
(Wang et al., 1988) and U. violacea (Bej & Perlin, 1989), an
ectomycorrhizal fungus, Laccaria laccata (Barrett et al.,
1990), and four edible fungi, P. ostreatus, Agaricus bisporus,
L. edodes and G. lucidum (Ming et al., 1992; Rhee et al., 1996;
Hirano et al., 2000; Li et al., 2004). However, in these
studies, transformation efficiencies were very low.
Green fluorescent protein (GFP) of the jellyfish Aequorea
victorea, which can be directly visualized as a result of the
emission of green light when excited with long UV or blue
light (Chalfie et al., 1994), fulfills the prerequisite of a
versatile reporter gene (Cubitt et al., 1995). The gfp gene

FEMS Microbiol Lett 256 (2006) 203–208

c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
204
has been developed as a tool to monitor gene expression in
situ and in vivo for many reasons. The fluorescence results
from its intrinsic chromophore structure and does not
require any substrate or cofactor. Moreover, because of the
fluorescence properties of GFP, it has been widely used in the
gene engineering of plants, mammalian cells, viruses and
filamentous fungi. Despite these advances, to date GFP has
been successfully expressed in only four homobasidiomy-
cetes, Schizophyllum commune (Lugones et al., 1999), Pha-
nerochaete chrysosporium (Ma et al., 2001), A. bisporus and
Coprinus cinereus (Burns et al., 2005). Transgene expression
of GFP in basidiomycetes appears to be hampered by a
number of factors. In the model species, S. commune,
transforming DNA was inactivated by preferential methyla-
tion (Mooibroek et al., 1990). AT-rich sequences inactivated
gene expression (Schuren & Wessels, 1998; Scholtmeijer
et al., 2001), and introns were needed for mRNA accumula-
tion to occur (Lugones et al., 1999; Scholtmeijer et al., 2001).
Results from Foster et al. showed that efficient GFP expres-
sion in A. bisporus and C. cinereus required introns (Burns
et al., 2005).
In this study, we report a highly efficient transformation
protocol mediated by PEG. We transferred the hph gene into
P. ostreatus and into two other mushrooms, G. lucidum and
L. edodes. We also explored the potential of the gfp gene as a
reporter in the transformation of P. ostreatus.
Materials and methods
Strains and plasmids
Pleurotus ostreatus strain Life No. 1 was purchased from the
Fungal Institute of Jinxiang (Shandong Province, China).
Lentinus edodes strain GIM5.15 was purchased from Guang-
dong Institute of Microbiology (Guangzhou, China). Gano-
derma lucidum strain AM21 is our laboratory strain.
Plasmid PRset-S65TGFP was kindly provided by Professor
R. Y. Tsien from HHMI, and contains a gfp gene modified
for plant transformation. Plasmid pAN7-1 was a gift of Dr P.
J. Punt from the Department of Applied Microbiology and
Gene Technology, TNO Voeding, the Netherlands. This
vector contains the Escherichia coli hph gene fused to the 5 0
expression signals from the Aspergillus nidulans glyceralde-
hyde-3-phosphate dehydrogenase gene (gpd) and the tran-
scription-termination sequence from the A. nidulans
tryptophan synthetase gene (trpC) (Punt et al., 1987).
Expression of the hph gene confers resistance to the amino-
cyclitol antibiotic hygromycin B (HmB).
Plasmid construction
Plasmid pAN7-1 was digested to completion with BamHI,
followed by a partial EcoRI digest. A 5.8 kb fragment was
recovered after agarose gel electrophoresis. Plasmid pRset-


c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Li et al.
S65GFP was double digested with EcoRI and BamHI and a
0.7 kb gfp gene fragment was isolated. Ligation between the
5.8 kb vector fragment and the 0.7 kb gfp gene gave rise to
a new expression vector named pAN-GFP.
Preparation of protoplasts from Pleurotus
ostreatus
Mycelium grown in 100 mL of MYG (1% maltose, 0.4%
glucose, 0.4% yeast extract) was collected by filtration
through gauze and rinsed three times with 0.6 M mannitol.
Then the mycelia were incubated for 2.5–3.0 h in 15 mL of
2.5% lywallzyme (Guangdong Institute of Microbiology) in
0.6 M mannitol at 30 1C. Protoplasts were purified (proto-
plasts were separated from hyphal debris by filtration
through a G-2 glass filter, collected by centrifugation at
1621 g for 10 min at 4 1C, washed once with 0.6 M mannitol
and 15 mL MTC buffer (0.6 M mannitol, 100 mM Tris-HCl,
pH 8.0, 100 mM CaCl2) and finally resuspended in MTC
buffer and diluted to 108 mL1 for storage at 4 1C until use.
Highly efficient PEG transformation of
protoplasts
Protoplasts (about 1  108 in 160 mL MTC buffer) were
mixed thoroughly with pAN7-1 (10 mg), 10 mL 20 mM
aurintricarboxylic acid (ATA, a nuclease inhibitor; Sigma,
St Louis, MO), 5 mL 50 mM spermidine, 100 mg heparin and
50 mL of PTC buffer (40% PEG3350, 100 mM CaCl2, 10 mM
Tris-HCl (pH7.4)), then incubated on ice for 45 min. One
milliliter of MTC buffer was added and the mixture incu-
bated for an additional 25 min at room temperature. Proto-
plasts were recovered by centrifugation (4 1C, 5 min at 4150
g) and resuspended in 500 mL MTC buffer. Then protoplasts
were centrifuged (4 1C, 5 min at 1621 g) and resuspended in
1 mL MYG regeneration medium (1% maltose, 0.4% glu-
cose, 0.4% yeast extract and 0.6 M mannitol). Subsequently,
protoplasts were allowed to regenerate for 18–24 h in 1 mL
MYG regeneration medium without hygromycin B, then
mixed with 5 mL MYG-selective medium (37 1C) containing
0.6 M mannitol, 1% low-melting point (LMP) agarose and
100 mg mL1 hygromycin B. The mixture was poured onto
an MYG plate containing 0.6 M mannitol, 1.5% agar and
100 mg mL1 hygromycin B. The plate was incubated at 28 1C
for 5–7 days. Colonies were subcultured individually onto
fresh MYG plates containing 100 mg mL1 hygromycin B.
PCR amplification of the transformants of
Pleurotus ostreatus
Polymerase chain reaction primers were designed by DNA-
SIS (V7.12, Hitachi Software Engineering Corp., Tokyo,
Japan) and were synthesized by Shanghai Sangon Biological
Engineering Technology & Services Co., Ltd. (Shanghai,
FEMS Microbiol Lett 256 (2006) 203–208
Polyethylene glycol-mediated transformation method
China). The sequences of the primers are P1: 5 0 -GCA GCT
TGA CTA ACA GCT AC-3 0 and P2: 5 0 -CGG TCG GCA TCT
ACT CTA TT-3 0 . The amplified fragment spans
2261–3336 bp of pAN7-1 plasmid, and is 1076 bp in length.
Genomic DNA of the transformants was extracted (Hirano
et al., 1999) and used as template. Thirty-five cycles of PCR
amplification were carried out as follows: 94 1C for 30 s,
58 1C for 30 s and 72 1C for 1 min.
Southern blot analysis of transgenic Pleurotus
ostreatus
The hph gene fragment used as a probe was labeled with
(a-32P)-dCTP, and the Southern blot was performed as
previously described (Sambrook et al., 1989).
Observation by fluorescence microscopy
For the formation of the GFP chromophore, transgenic
protoplasts were incubated under aerobic conditions at
4 1C for various times (Cody et al., 1993). Fluorescence was
detected using excitation wavelengths of 480 nm in an
Olympus Fluo epifluorescence microscope (Japan). Images
were documented using Kodak Colorsharp ISO 400 film
(Kodak, Rochester, NY).
Results and discussion
Susceptibility of Pleurotus ostreatus to
hygromycin B
Mycelia and protoplasts of Pleurotus ostreatus were inocu-
lated onto MYG plates containing various concentrations
of hygromycin B. Their growth was markedly inhibited at
50 mg mL1 hygromycin B. In order to reduce the possibility
of false positives, 100 mg mL1 hygromycin B was used for
selection in the transformation experiments, and no growth
of controls was detected under these conditions.
Transformation efficiency of our improved PEG
method in the transformation of Pleurotus
ostreatus, Ganoderma lucidum and Lentinus
edodes
The efficiency of previously reported transformation meth-
ods for P. ostreatus is not high enough for commercial strain
improvement. In order to increase the transformation
efficiency of P. ostreatus, we included several chemical
substances in the improved PEG-mediated transformation
method. These substances are heparin, ATA and spermidine.
Heparin is an anticoagulant, which can prevent aggregation
of protoplasts and is beneficial to the uptake of foreign
DNA. ATA is a nuclease inhibitor, which prevents degrada-
tion of foreign DNA in vivo. Spermidine carries positive
charges, which can bind the negatively charged foreign DNA
FEMS Microbiol Lett 256 (2006) 203–208
205
Table 1. Comparison of the transformation efficiency of our improved
polyethylene glycol method with previously reported results for Pleurotus
ostreatus
Transformation efficiency
(transformants per mg
PEG transformation methods DNA per 107 protoplasts)
Method 1: Ming et al. (1992) 5–46
Method 2: Koji et al. (1996) 2
Method 3: Irie et al. (2001) 0.1–0.8
Our improved PEG method 80–180
PEG, polyethylene glycol.
to the surface of protoplasts and increase the likelihood of
entrance. In addition, we also washed and resuspended
protoplasts in MTC buffer instead of 0.6 M mannitol so as
to increase their competence.
We compared the transformation efficiency of our im-
proved PEG method with previous results reported in the
literature for the transformation of P. ostreatus. Transforma-
tion experiments were repeated at least five times, and the
results are shown in Table 1. Our transformation method
resulted in 80–180 resistant colonies per mg DNA per 107
viable protoplasts. Further investigation revealed that these
transformants were mostly stable and integrative transfor-
mants, whereas the other three methods only resulted in
0.1–46 resistant colonies per mg DNA per 107 viable proto-
plasts. The transformation efficiency of our method is about
100–1800 times higher than that of Irie et al. (2001), and
40–90 times higher than that of Koji et al. (1996). Further-
more, although the transformation efficiency of the PEG
method of Ming et al. reached 5–46 transformants per mg
DNA per 107 viable protoplasts, much more than the other
two methods, most of these were unstable transformants
that would lose the resistance phenotype after 3–6 weeks of
growth in the absence of selection (Ming et al., 1992). As
only method 2, method 3 and our new method could
produce stable and integrative transformants, our novel
method represents a marked improvement in transforma-
tion efficiency in terms of stable and integrative transfor-
mants over other PEG methods.
In addition to P. ostreatus, this new PEG transformation
procedure was used to transform other mushroom species.
We transferred plasmid pAN7-1 into G. lucidum and L.
edodes and high transformation frequencies were obtained.
We compared the transformation efficiency of our new PEG
method with the transformation results of other researchers
for G. lucidum and L. edodes as shown in Table 2. Our new
transformation method resulted in 120–150 resistant colo-
nies per mg DNA per 107 viable protoplasts for G. lucidum,
which is seven to 38 times greater than that reported by
other researchers, and resulted in 85–100 transformants per
mg DNA per 107 viable protoplasts for L. edodes, which is
24–28 times greater than figures reported in the literature.


c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
206
Table 2. Comparison of the transformation efficiency of our improved polyethylene glycol method with previous transformation methods for
Ganoderma lucidum and Lentinus edodes
Transformation method
Method 1: Sunkyung et al. (2004)
Method 2: Sun et al. (2001)
Method 3: Irie et al. (2003)
Our improved PEG method
NA, not available; PEG, polyethylene glycol.
Fig. 1. Growth of subcultures of Pleurotus ostreatus transformants on
an MYG plate containing 100 mg mL1 hygromycin B.
Moreover, hygromycin B resistance persisted in nearly all
transformants throughout many subcultures.
Test for hygromycin B resistance of subcultures
of transformants
Six primary transformants were picked randomly from a
transformation plate of P. ostreatus and were transferred
onto an MYG plate without hygromycin B and cultivated for
five subculturings at 28 1C; then, they were inoculated onto
an MYG plate with 100 mg mL1 hygromycin B. All these
transformants were capable of vigorous growth (Fig. 1),
confirming that our transformation method resulted in
stable transformants.
PCR amplification of hph gene from
transformants of Pleurotus ostreatus
The genomic DNAs of the above six transformants were
used as the template for PCR with hph-specific primers in
order to confirm transformation of plasmid pAN7-1 into P.
ostreatus. The expected 1076 bp amplified band appeared in
the PCR products of transformants on agarose gels, and the
band was absent in the case of the untransformed control


c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Li et al.
Transformation efficiency Transformation efficiency
of G. lucidum (transformants of L. edodes (transformants
per mg DNA per 107 protoplasts) per mg DNA per 107 protoplasts)
4–17 NA
15 NA
NA 3.6
120–150 85–100
(data not shown). This confirmed that plasmid pAN7-1 had
been transferred into P. ostreatus.
Southern blot analysis of transgenic Pleurotus
ostreatus
To ascertain the presence of plasmid pAN7-1 in the trans-
genic P. ostreatus, Southern blot analysis was performed
using the hph gene as a probe. No specific hybridization
signal was found in the DNA samples of the untransformed
control (data not shown), while specific strong hybridiza-
tion signals were clearly visible in the DNA samples of the
transformants at the high-molecular weight DNA region
corresponding to the migrating chromosomal DNA (data
not shown). These results indicated that the transformants
had acquired the introduced DNA, which was integrated
into the chromosomal DNA. In addition, the total DNA
samples were prepared from mycelia cultivated in liquid
media without hygromycin B, suggesting that the transfor-
mants obtained with this transformation procedure were
stably inherited.
Expression of gfp gene in transgenic Pleurotus
ostreatus
As expression of the gfp transgene in basidiomycetes has
been found to be very difficult and modifications of the gfp
gene have proved necessary, an exploratory experiment was
performed to examine if the gfp gene modified for plant
transformation could be expressed in P. ostreatus. A gfp gene
from vector pRset-GFP, a Pgpd promotor and a TtrpC
terminator from plasmid pAN7-1 were fused together to
produce a new expression vector pAN-GFP, which was then
transformed into protoplasts of P. ostreatus by our PEG
transformation method. The transformed protoplasts were
analyzed by fluorescence microscopy using an excitation
wavelength of 480 nm. Observation of green fluorescence
revealed that the transformation efficiency was 100–150
transformants per mg vector pAN-GFP per 107 viable proto-
plasts, and no green fluorescence was detected in the control.
A weak green fluorescence was first visible in transformed
protoplasts after 4–6 h. Bright fluorescence could be mon-
itored after 16 h posttransformation, reaching a maximum
FEMS Microbiol Lett 256 (2006) 203–208
Polyethylene glycol-mediated transformation method
intensity after about 19 h. The fluorescence then began to
fade, finally disappearing beyond 30 h after transformation
(data not shown). One reason why the gfp gene was not
persistently expressed in P. ostreatus could be that the GFP
protein was degraded or inactivated by modification beyond
30 h posttransformation. Another reason could be that the
transforming gfp gene was inactivated by preferential
methylation. Because the gfp gene was modified for plant
expression, it might not be the most suitable form for fungal
expression. In our opinion, some changes to the gfp gene
need to be made in order to acquire persistent expression of
GFP in P. ostreatus; these would include base alterations for
preferential codon usage (Chiu et al., 1996), and the addi-
tion of an intron in the upstream region of gfp (Burns et al.,
2005).
In this study, we developed a reliable, integrative and
highly efficient transformation system for the white-rot
basidiomycete P. ostreatus. We transferred hph and gfp genes
into P. ostreatus using hygromycin B resistance and green
fluorescence as dominant markers. In the transformation
experiment with hph, a transformation efficiency of 80–180
transformants per mg of DNA per 107 viable protoplasts was
obtained and transformants still showed hygromycin B
resistance after five subculturings. In the transformation
experiment with gfp, a transformation efficiency of about
100–150 transformants per mg of DNA per 107 viable
protoplasts was obtained, but gfp expression in transfor-
mants was transient, and green fluorescence disappeared
after 30 h. This constitutes an initial investigation of GFP
expression in P. ostreatus, and further research is needed to
resolve the transient nature of GFP expression in this
important cultivated mushroom.
Acknowledgements
We are grateful to Professor R. Y. Tsien and Dr P. J. Punt for
supplying Plasmids PRset-S65TGFP and pAN7-1. This work
was supported by the Natural Science Foundation of
Guangdong Province (No. 4203112), the National Natural
Science Foundation of China (No. 30300055), and a key
grant from the Science and Technology Program of Guangz-
hou city (No. 99-Z-021-01, No. 2001-Z-006-01).
Gang Li and Ruixue Li contributed equally to this work.
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