Hygromycin B

A simple and efficient genetic transformation method
of Ganoderma weberianum
Yu-Ping Zhou & Min-Hua Chen & Jun-Jie Lu & Xun Kang &
Qiong-Hua Chen & Xiao-Ling Huang & Chang-En Tian
Received: 28 May 2014 /Accepted: 23 January 2015
# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015
Abstract In this study, the Agrobacterium tumefaciens￾mediated transformation method for Ganderma
weberianum has been established. Driven by the cauli￾flower mosaic virus (CaMV) 35S promoter, the
hygromycin phosphotransferase (hpt), β-glucuronidase
(uidA), and enhanced green fluorescent protein (egfp)
genes have been efficiently expressed in transgenic
mycelia and spores. The transformation system was
composed of the growing mycelia, A. tumefaciens strain
GV3101, and the expression vector pBI-H1, harboring
the CaMV 35S promoter and selective hpt marker. The
genetic transformation of G. weberianum was achieved
through co-cultivation of Agrobacterium lawn and fun￾gal mycelia at 28 °C on yeast extract agar (YEA) me￾dium. Stable genetic transformants were obtained
through successive hygromycin B selections and single
spore isolation. Over 80 % of transformants showed
genetic stability even after ten rounds of subculturing.
The simple and efficient genetic transformation method
is a useful tool for molecular genetics analyses and gene
manipulation of G. weberianum.
Similar to Ganoderma lucidum, Ganoderma weberianum, a
tropical white-rot fungus, has been used as a traditional
Chinese medicine with anticancer and antivirus properties
and that promotes immunity (Gao and Zhou 2003).
G. weberianum TZC-1 was able to produce a high yield of
laccase, which exhibited the strong ability to decolorize dif￾ferent dyes (Chen et al. 2010a, b). Furthermore, its mycelia
pellets could also decolorize indigo carmine, indigo dye,
and its effluents efficiently (Zhou et al. 2011; Tian et al.
2013). Although G. weberianum is of great economic
and environmental importance, its genetic studies are
very limited, which impedes the strain development
and the improvement of production efficiency through
advanced molecular tools. Therefore, there is an urgent
need for fundamental and applied research based on
genetic manipulation in G. weberianum.
G. lucidum, a widely cultivated medicinal fungus in
China, has been widely studied for its biological and
pharmacological activities (Cao and Lin 2004; Wang
and Ng 2006). Despite its complete genomic sequences
(Chen et al. 2012) and several methods to deliver exog￾enous DNA into it, such as the restriction enzyme￾mediated integration method (Kim et al. 2004),
protoplast-polyethylene glycol (PEG) transformation
method (Li et al. 2004) and Agrobacterium tumefaciens￾mediated transformation (ATMT) method (Shi et al.
2012), were available, all mentioned methods need com￾plex protoplast separation processes and have merely
low transformation efficiency.
ATMT has been widely used in gene transfer and gene
tagging of plants and fungi (Bundock et al. 1995; de Groot
et al. 1998; Jiang et al. 2013; Michielse et al. 2005; Vain
2007). However, this method was not available for
G. weberianum. For the first time, we provided a simple and
efficient genetic transformation method for the fungus in this
paper. Driven by the cauliflower mosaic virus (CaMV) 35S
promoter, the hygromycin phosphotransferase (hpt) gene, β-
glucuronidase (uidA) gene, and the enhanced green fluores￾cent protein (egfp) gene harbored in the vector pBI-H1
(Kimura et al. 1993) can be efficiently and stably expressed
in the transgenic mycelia and spores.
Y. X. Guangzhou Key Laboratory for Functional Study on Stress-Resistant
Genes in Plants, School of Life Sciences, Guangzhou University,
Guangzhou 510006, China
e-mail: [email protected]
Folia Microbiol
DOI 10.1007/s12223-015-0377-z
Materials and methods
Strains and culture conditions
G. weberianum TZC-1 was isolated by our research group
(Chen et al. 2010a) and maintained on potato dextrose agar
(PDA) slants at 4 °C. Escherichia coli DH5a was used for
plasmid amplification and grown on Luria-Bertani (LB) me￾dium containing 100 mg/L ampicillin or 50 mg/L kanamycin
as required at 37 °C. A. tumefaciens strain GV3101 (Koncz
and Schell 1986) was used as the T-DNA donor for
G. weberianum and cultured on LB medium containing
50 mg/L streptomycin at 28 °C.
Construction of binary vectors
The binary vector pBI-35S:uidA (Fig. 1a) was constructed by
inserting the CaMV 35S promoter into the HindIII and SalI
sites of the multiple cloning site of plasmid pBI-H1 (Kimura
et al. 1993), which harbored a bacterial hpt gene. To construct
the binary vector pBI-35S:egfp (Fig. 1b), the uidA gene in
plasmid pBI-35S:uidA was replaced with the egfp reporter
gene from plasmid pEGFP (Clontech, USA) using a XbaI
and SacI double restriction enzyme digestion.
Selection of media for the mycelial growth of G. weberianum
G. weberianum TZC-1 was recovered on PDA plates at 28 °C
for 4 days. Thereafter, the mycelia disk, each 7 mm in diam￾eter, were placed in the center of culture plates containing
20 mL of LB, malt extract agar (MEA), yeast extract agar
(YEA), and potato dextrose agar (PDA) media. The diameter
of the mycelia colonies was measured after the mycelia had
grown at 28 °C for 96 h, respectively. The experiment was
repeated three times.
Determination of the sensitivity of G. weberianum
to hygromycin B
To determine the sensitivity of G. weberianum mycelia to
hygromycin B, mycelial disks were placed onto PDA plates
containing different concentrations of hygromycin B (75, 100,
125, and 150 mg/L) and cultivated at 28 °C for 5 days. The
growth rate was monitored. Each experiment included a neg￾ative control and was repeated three times.
Genetic transformation facilitated by A. tumefaciens strain
According to the method described by Sharma and
Kuhad (2010), an improved ATMT method was per￾formed. The PDA medium in the inner dish (6 cm in
diameter) was chosen to cultivate the fungal mycelia.
The YEA medium, supplemented with 50 mg/L kanamy￾cin and 50 mg/L streptomycin in the outer dish (12.5 cm
in diameter), was chosen to co-cultivate both bacteria
and fungi (Fig. 2a). Three mycelial disks was placed in
the center of the PDA medium and cultivated at 28 °C to
let the daughter hyphae grow to fill the inner plate and
move on to the YEA medium. Then, A. tumefaciens
strain GV3101, harboring the plasmid pBI-35S:uidA or
pBI-35S:egfp, was streaked onto the YEA medium and
transferred to the constant temperature incubator at dif￾ferent temperatures (24, 26, 28, and 30 °C) to form a
confluent layer of cells. The bacteria was grown in LB
broth containing 50 mg/L kanamycin and 50 mg/L strep￾tomycin to an A600=0.5–0.6 prior to inoculation. After
the co-cultivation of bacteria and mycelia, mycelial disks
were randomly picked from the periphery of the growing
mycelia in the outer plate, and washed five times with
MEA broth supplemented with 250 mg/L augmentin to
kill the bacteria. Thereafter, three mycelia disks were
dried with sterile papers, along with a negative control,
transferred onto the screening PDA plate containing
125 mg/L hygromycin B, and incubated at 28 °C. After
a 4-day incubation, daughter hyphae developed from the
co-cultivated mycelial disks were subcultured and the
subculturing was conducted three times. Thus, a number
of hygromycin B-resistant transformants were obtained
and the transformation efficiency was calculated in each
experiment (transformation efficiency = no. of hygromycin￾resistant mycelial disks/no. of mycelial disks inoculated×
100 %). Each experiment included an untransformed negative
control. The transformation experiment and the subculturing
were performed in three repeated experiments.
Fig. 1 Diagram of binary vectors harboring both the hygromycin
phosphotransferase (hpt) gene and the β-glucuronidase (uidA)/enhanced
green fluorescent protein (egfp) gene driven by the cauliflower mosaic
virus (CaMV) 35S promoter, hpt, uidA, and egfp. a Diagram of pBI-
35S:uidA. b Diagram of pBI-35S:egfp
Folia Microbiol
Single spore isolation and PCR identification of transformants
A hygromycin B-resistant transformant with abundant
sporulation was mixed with 0.7 % sterile saline and
diluted to 105 CFU/mL as determined by the plate
count method. The spore suspension (100 μL) was plat￾ed on a PDA plate containing 125 mg/L hygromycin B.
After incubating for 3 days at 28 °C, individual germi￾nated spores were picked out with a sterilized scalpel
blade under a dissecting microscope in a laminar flow
cabinet, and transferred to a PDA plate containing
125 mg/L hygromycin B. The hyphae derived from a
spore were identified as a homozygous transformant.
The mycelia of 20 homozygous transformants were har￾vested and ground to a fine powder in liquid nitrogen.
Genomic DNA was isolated by the cetyltrimethyl ammonium
bromide (CTAB) method (Kuhad et al. 2004). A PCR analysis
was performed with the primer pair 5′-GCTGTTATGCGGCC
the hpt gene, amplifying a 600-bp fragment. A 1.5-kb fusion
fragment containing CaMV 35S promoter and uidA gene was
amplified using the primer pair 5′-AGATTAGCCTTTTCAA
and another 1.5-kb fusion fragment containing the CaMV 35S
promoter and egfp gene was detected using the primer pair 5′-
Detection of GUS and GFP in transformants
To detect the GUS activity, mycelia were incubated at 37 °C
for 10 h in detection buffer containing 0.1 % (v/v) Triton
X-100, 5 mmol/L potassium ferrocyanide, 5 mmol/L potassi￾um ferricyanide, 50 mmol/L sodium phosphate (pH 7), and
2 mmol/L 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X￾Gluc) (Sigma, USA). Microscopy was performed using a dis￾secting microscope (SMZ800, Nikon). The green fluores￾cence emission from eGFP was observed by an epifluorescent
microscope (TE2000-U, Nikon, Japan). Images were recorded
and processed using the NIS-Elements F package.
Genetic stability of transformants
To determine the genetic stability of the transformants,
18 positive transformants identified by PCR were indi￾vidually cultured on the PDA medium at 28 °C. Five
days later, daughter mycelia disks of each plate were
transferred onto fresh PDA medium. Ten rounds of
subculturing were performed. Then, transformants tested
were again transferred onto the screening PDA medium
containing 125 mg/L hygromycin B to analyze the
hygromycin B resistance and identify the hpt gene by
PCR. In addition, the GUS and GFP levels in the
transformants were determined again.
Fig. 3 Co-cultivation of Agrobacterium tumefaciens and Ganoderma
weberianum mycelia. a G. weberianum mycelia grown on the PDA
medium in the inner dish and A. tumefaciens grown on the YEA
medium in the outer dish. Bacteria were streaked on the outer region of
the white cycle. b Co-cultivation of A. tumefaciens and G. weberianum
mycelia on the YEA medium in the outer dish at 24 °C for 3 days. c Co￾cultivation of A. tumefaciens and G. weberianum mycelia on the YEA
medium in the outer dish at 28 °C for 3 days. Size bar, 1 cm
Fig. 2 Sensitivity test of Ganoderma weberianum mycelia to hygromycin B. PDA medium without hygromycin B (a) and with 75 (b), 100 (c) , 125 (d),
150 mg/L (e) hygromycin B. Size bar, 1 cm
Folia Microbiol
Results and discussion
Selection of media for mycelia growth of G. weberianum
and co-cultivation of G. weberianum and Agrobacterium
Given that PDA and MEA media are widely used as mycelia
growth media, LB and YEA media were used as the
Agrobacterium culture media. To select the optimal medium
for the mycelia growth of G. weberianum TZC-1 and the co￾cultivation medium for G. weberianum TZC-1 and
Agrobacterium GV3101, four kinds of media (PDA, MEA,
LB, and YEA) were tested. The results showed that the PDA
medium was optimal for mycelia growth, with the mycelia
grown on the PDA medium producing more aerial hyphae
and spores than on the MEA medium. Thus, the PDA medium
was chosen to cultivate G. weberianum mycelia. Meanwhile,
Agrobacterium GV3101 grew well in LB and YEA medium,
and G. weberianum mycelia grew better in YEA medium than
in LB medium. So, the YEA medium was chosen as the co￾cultivation medium for both G. weberianum and
Agrobacterium GV3101.
Sensitivity analysis of G. weberianum to hygromycin B
To determine the sensitivity of G. weberianum mycelia to
hygromycin B, mycelial disks were inoculated on PDA sup￾plemented with different concentrations of hygromycin B.
After a 5-day inoculation period, hygromycin B was found
to partially inhibit mycelial growth at 75 and 100 mg/L
(Fig. 2b, c), whereas it completely inhibited growth at 125
and 150 mg/L (Fig. 2d, e). Thus, hygromycin B was used at
a concentration of 125 mg/L.
Genetic transformation facilitated by A. tumefaciens
To optimize the experimental protocol, different inoculation
sites for the bacteria and co-cultivation temperatures were
tested. When streaked adjacent to the outer edge of the inner
dish, Agrobacteria proliferated rapidly and formed the lawn.
This would restrain the hyphal growth and resulted in the
failure of co-cultivation. So, bacteria were streaked ~1.5 cm
away from the edge of the inner plate on the surface of medi￾um in the outer dish (Fig. 3a). The co-cultivation temperature
was found to seriously affect the transformation efficiency.
When the co-cultivation temperature was 24 °C, both the fun￾gal mycelia and bacterial lawns grew slowly (Fig. 3b), and
Fig. 5 PCR identification of both
the hygromycin
phosphotransferase (hpt) gene
and the β-glucuronidase (uidA)/
enhanced green fluorescent
protein (egfp) gene in putative
Ganoderma weberianum
transformants. a The hpt band in
transformants of pBI-35S:egfp. b
The fusion fragment of CaMV
35S and egfp in transformants of
pBI-35S:egfp. c The hpt band in
transformants of pBI-35S:uidA. d
The fusion fragment of CaMV
35S and uidA in transformants of
pBI-35S:uidA. Lane M, DNA
marker; lanes 1–9, transformed
mycelia; lane 10, untransformed
Fig. 4 Effect of co-cultivation temperature on the transformation
efficiency of Ganoderma weberianum
Folia Microbiol
only 61 % of the co-cultivated mycelia disks had the resistance
to hygromycin B (Fig. 4). When the co-cultivation tempera￾ture was 28 °C, fungal mycelia quickly reached to the bacterial
lawn (Fig. 3c), and 100 % of the co-cultivated mycelia disks
were resistant to hygromycin B (Fig. 4). However, when the
co-cultivation temperature was 30 °C, the transformation effi￾ciency decreased to 71 % (Fig. 4). The results showed that the
maximum transformation efficiency occurred at 28 °C, which
is the optimal cultivation temperature of both A. tumefaciens
GV3101 and G. weberianum TZC-1. The optimal co￾cultivation temperature (28 °C) found in this experiment was
higher than the 20 °C of Ganoderma sp. RCKK-02 (Sharma
and Kuhad 2010) and 25 °C of G. lucidum (Shi et al. 2012).
Moreover, unlike in Ganoderma sp. RCKK-02 that showed
no transformations at temperature above 29 °C, the transfor￾mation efficiency of G. weberianum TZC-1 reached to 71 %
at 30 °C (Sharma and Kuhad 2010).
The co-cultivated mycelial disks were then screened on the
PDA medium containing 125 mg/L hygromycin B. After a 4-
day inoculation period at 28 °C, the daughter hyphae grew
from the margins of the transformed mycelia disks. After three
successive subcultures, single spore isolation experiments
were conducted and many discrete colonies were obtained
on the screening PDA medium containing 125 mg/L
hygromycin B.
In this experiment, the actively growing mycelia cultivated
on the PDA medium were used as the starting material and a
high transformation efficiency was achieved. Both
Fig. 7 Expression analysis of reporter genes in randomly chosen
Ganoderma weberianum transformants across ten successive
generations. a–f Fluorescence microscopy analysis of transformants
harboring the enhanced green fluorescent protein (egfp) gene. a, d
Images under bright field and fluorescence field of untransformed
mycelia, respectively. b, e Images under bright field and fluorescence of
mycelia from a transformant, respectively. c, f Images under bright field
and fluorescence field of spores from a transformant, respectively. g–i
Histochemical GUS staining analysis of transformants harboring the β-
glucuronidase (uidA) gene. g Untransformed mycelia; h, i Mycelia from
two independent transformants. Size bar, 10 μm
Fig. 6 PCR identification of the hygromycin phosphotransferase (hpt) gene in progeny mycelia of Ganoderma weberianum across ten successive
generations. Lane M, DNA marker; lanes1–9, transformants mycelia; lane 10, untransformed mycelia
Folia Microbiol
homogenized mycelia and mycelia pellets have been used as
the starting material in ATMT for macrofungi, such as
Antrodia cinnamomea (Chen et al. 2009), Pleurotus ostreatus
(Ding et al. 2010), Flammulina velutipes (Okamoto et al.
2010), Physisporinus vitreus (Schubert et al. 2013), and
Volvariella volvacea (Wang et al. 2008). The results demon￾strated that the growing mycelia were the simple and appro￾priate starting material for ATMT of G. weberianum.
Although acetosyringone can be useful for improving the
transformation efficiency of the ATMT method (Jiang et al.
2013; Michielse et al. 2005), the efficiency of this transforma￾tion method for G. weberianum without the addition of
acetosyringone has reached to 100 % (Fig. 4).
Molecular identification of transformants
Transformants of the egfp and uidA genes were randomly
chosen from several transformation experiments. Thus, they
were considered as independent transgenic lines. PCR reac￾tions were performed to confirm the transgenic lines. The
transformants of pBI-35S:egfp were amplified to display the
0.6-kb band of the hpt gene (Fig. 5a) and the 1.5-kb band
representing the fusion of the 35S promoter fragment and
the egfp gene (Fig. 5b). The transformants of pBI-35S:uidA
were amplified to display the 0.6-kb band of the hpt gene
(Fig. 5c) and the 1.5-kb band representing the fusion of the
35S promoter fragment and the uidA gene (Fig. 5d). The re￾sults indicated that both the hpt gene and egfp/uidA gene were
simultaneously transformed into the fungal mycelia.
Genetic stability of transformants
To determine whether the integrated DNA was stably main￾tained or not, the transformants were subcultured for ten suc￾cessive generations in the absence of hygromycin B and were
subsequently screened for hygromycin B resistance and PCR
identification. All transformants were resistant to hygromycin
B and amplified the specific fragment of the hpt gene by PCR
(Fig. 6). The strong expression of GUS and GFP was also
detected in spores and mycelia in transformants (Fig. 7).
These results confirmed that foreign genes could be stably
maintained across ten successive generations.
Expression analysis of reporter genes in transformants
Fluorescence imaging was employed to detect the expression
level of the egfp gene, and histochemical GUS staining assay
was used to analyze the expression of the uidA gene in
transformants. A very strong fluorescence was detected in
the mycelia and spores of all tested transformants containing
both hpt and egfp genes (Fig. 7b, c, e, f), but no fluorescence
was detected in untransformed mycelia (Fig. 7a, d). In the
GUS staining assay, the mycelia of the transformants
containing both hpt and uidA genes were stained dark blue
(Fig. 7h, i) and the spores were also stained light blue; how￾ever, the untransformed mycelia were not stained blue
(Fig. 7g).
In the present experiment, the CaMV 35S promoter, which
is widely used in plant experiments was chosen to drive the
hpt, uidA, and egfp genes in the two binary vectors pBI-
35S:uidA and pBI-35S:egfp. There were earlier reports of
the CaMV 35S promoter in driving the expression of reporter
genes in G. lucidum, Pleurotus citrinopileatus (Sun et al.
2002), and P. chrysosporium (Sharma et al. 2006). These re￾sults proved again that the CaMV 35S promoter can be used to
efficiently drive the gene expression in basidiomycetes. Thus,
the CaMV 35S promoter provides an additional choice in the
molecular biological studies of basidiomycetes and allows the
construction of a shuttle vector system for alternative expres￾sion of genes in fungi.
In conclusion, a simple and efficient genetic transformation
method of G. weberianum via ATMT has been successfully
established, which included the growing mycelia of
G. weberianum, A. tumefaciens strain GV3101, vector pBI￾H1 harboring the CaMV 35S promoter and the selective hpt
marker. The key step is the co-cultivation of the bacterial lawn
and fungal mycelia at 28 °C on YEA medium. The stable
genetic transformants could be obtained by single spore iso￾lation of transformants.
Acknowledgments This work was financially supported in part by the
Science and Technology Planning project of Guangzhou Municipality,
China (Grant No. 1212011542) and the Natural Science foundation pro￾ject of Guangdong Province, China (Grant No. 8151009101000013) to
Zhou YP and the science research project of Education Bureau of Guang￾zhou Municipality (10A045) to Chen QH.
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