Development of a simple and efficient transformation system for the ...

World J Microbiol Biotechnol (2012) 28:283–291 DOI 10.1007/s11274-011-0818-z

ORIGINAL PAPER

Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum Liang Shi • Xing Fang • Mengjiao Li • Dashuai Mu • Ang Ren • Qi Tan • Mingwen Zhao

Received: 30 September 2010 / Accepted: 10 June 2011 / Published online: 22 June 2011 Ó Springer Science+Business Media B.V. 2011

Abstract In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0.2 mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research.

L. Shi  X. Fang  M. Li  D. Mu  A. Ren  M. Zhao (&) Key Laboratory for Microbiological Engineering of the Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, Jiangsu, People’s Republic of China e-mail: [email protected] Q. Tan National Engineering Research Center of Edible Fungi, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Science, Shanghai 201106, China

Keywords Ganoderma lucidum  Agrobacterium tumefaciens-mediated transformation  Promoter  Fluorescence assay  Histochemical staining assay Abbreviations GPD Glyceraldehyde-3-phosphate dehydrogenase PEG Polyethylene glycol REMI Restriction enzyme-mediated integration ATMT Agrobacterium tumefaciens-mediated transformation EGFP Enhanced green fluorescent protein GUS b-glucuronidase AS Acetosyringone MES 2-(N-morpholino)ethanesulfonic acid HPH Hygromycin B phosphotransferase

Introduction Ganoderma lucidum (Curtis: Fr.) P. Karst is a lamella-less basidiomycete belonging to the family Polyporaceae. It has been widely used in East Asia as a remedy for minor health disorders and to promote vitality and longevity (Lin 1979). Recent research has shown that G. lucidum has many biological and pharmacological activities that can be attributed to its complex chemical composition (Wang and Ng 2006; Mizushina et al. 1998; Eo et al. 1999). In addition to the studies on the pharmacological activities of G. lucidum, molecular genetics studies have also been conducted, resulting in the cloning and characterization of several of its genes (Ding et al. 2008; Xu et al. 2006; Shang et al. 2008, 2010; Zhao et al. 2007). Establishment of an efficient and convenient transformation method forms the basis for further functional studies of these genes.

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An efficient transformation method has been considered essential in the functional study of the G. lucidum genes at the molecular level, as it facilitates creating both targeted and random gene disruptions, thereby shedding light on the functional role of these genes. Recently, several different methodologies, including the use of fungal protoplast electroporation and restriction enzyme-mediated integration (REMI), have been developed for transforming G. lucidum (Sun et al. 2001; Kim et al. 2004). Agrobacterium tumefaciens-mediated transformation (ATMT) has been employed for genetically transforming a wide variety of plants (Vain 2007). It has been reported that A. tumefaciens is also able to transfer its DNA to various filamentous fungi, including members of the Ascomycetes, Basidiomycetes, and Zygomycetes phyla, as efficiently as to plants (Michielse et al. 2005; Sun et al. 2009; Chen et al. 2009). The ATMT approach generates a high number of transformants and does not require special equipment (Duarte et al. 2007). These advantages of ATMT make this method a valuable tool for molecular genetic manipulation of fungi. To our knowledge, there have been no previous successful attempts of ATMT in G. lucidum. In this study, we demonstrate a high efficiency method for the genetic transformation of G. lucidum using the ATMT method. Using this method, we have expressed enhanced green fluorescent protein (EGFP) and b-glucuronidase (GUS) genes in G. lucidum. This method will greatly facilitate future molecular genetic studies of this fungus and allow us to gain a better understanding of the genetics of this organism.

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Construction of T-DNA binary vectors The transforming binary vector pGL-GPD was derived from the A. tumefaciens binary vector pCAMBIA 1300 (CAMBIA, Canberra) by replacement of the 35S promoter from CaMV, which drives the expression of the hygromycin B phosphotransferase (HPH) gene, with the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene promoter from G. lucidum previously isolated (GenBank: DQ404345.1; Xu et al. 2006; Fig. 1A). A similar strategy was used to create the binary vectors pLE-RAS, pLE-GPD, and pAN-GPD by replacement of the 35S promoter with the ras promoter from Lentinula edodes (GenBank: AY453854.1), the gpd promoter from L. edodes (GenBank: GQ457137.1), and the gpd promoter from Aspergillus nidulans (GenBank: Z32524.1), respectively (Fig. 1A). To generate the pGL-GPE plasmid, we excised the egfp reporter gene and CaMV 3’UTR sequences from pBGgHg (Chen et al. 2000) and cloned them into the KpnI, BamHI and PstI sites of the multiple cloning site of plasmid pGLGPD, thereby placing egfp under the control of the Gl-GPD promoter (Fig. 1B). To construct the pGL-GPG plasmid, the egfp gene in pGL-GPE was replaced with the GUS gene from pBI121 (Clonetech, USA) using BamH I and Xba I double restriction enzyme digestion (Fig. 1B). The primers used for PCR amplification and subcloning are listed in Table 1. The plasmids pGL-GPE and pGL-GPG also contain the kanamycin-resistance gene as a selection marker for the Agrobacterium strain used. These constructs were introduced into LBA4404 using 20 mM calcium chloride.

Materials and methods

A. tumefaciens-mediated transformation

Strains and culture conditions

Protoplasts of G. lucidum were prepared by a method previously described (Sun et al. 2001). The number of protoplasts used for ATMT was 105. LBA4404 harboring a T-DNA binary vector was grown at 28°C on a rotatory shaker (200 rpm) in 5 mL of LB broth supplemented with 50 ug/mL rifampin and 50 ug/mL kanamycin to an optical density at 600 nm (OD600) of 0.5–0.6. Bacterial cells were harvested by centrifugation at 4,000 9 g, washed once with fresh IM, and diluted in 5 ml of fresh IM to an OD600 of 0.1. The bacterial cells were further grown at 28°C on a rotatory shaker (200 rpm) to an OD600 of 0.5–0.6. Co-culture of A. tumefaciens and G. lucidum protoplast was conducted at different ratios of bacteria to protoplast (1:1, 10:1, 100:1, and 1,000:1). The co-cultures were spread evenly on 50 mm nitrocellulose membranes laid on top of 10 mL of solidified AS-containing IM-agar plates [IM plus 1.8% agar containing various concentrations of AS (0, 0.05, 0.2, and 1 mM)] and incubated at different temperatures (22, 25, and 28°C) for varying lengths of

Ganoderma lucidum strain HG was grown at 28°C on CYM (1% maltose, 2% glucose, 0.2% yeast extract, 0.2% tryptone, 2 mM MgSO47H2O, 33 mM KH2PO4) and used as the recipient host strain for transformation. The DH5a strain of Escherichia coli was used for plasmid amplification and grown on Luria–Bertani (LB) medium containing 100 lg/mL ampicillin or 50 lg/mL kanamycin as required. LBA4404 strain of Agrobacterium tumefaciens was used as the T-DNA donor for fungal transformation of G. lucidum. Minimal medium (MM) [10 mM K2HPO4, 10 mM KH2PO4, 2.5 mM NaCl, 2 mM MgSO47H2O, 0.7 mM CaCl2, 9 lM FeSO47H2O, 4 mM (NH4)2SO4, 10 mM glucose, pH 7.0] was used for the cultivation of LBA4404. Induction medium (IM) [MM containing 0.5% (w/v) glycerol, 200 lM acetosyringone (AS), 40 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.3] was used to co-cultivate LBA4404 and G. lucidum.

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Fig. 1 Plasmid constructs for transformation. A Construction plasmids by replacement of the promoter driving expression of the HPH gene. In pGl-GPD, the hygromycin resistance marker (hph) is expressed from the G. lucidum gpd promoter, whereas in plasmid pLe-GPD, pLe-RAS, and pAn-GPD, it is expressed from the L. edodes gpd, L. edodes ras, and A. nidulans gpd promoters, respectively. LB and RB, left-border and right-border regions of T-DNA from A. tumefaciens Ti plasmid. MCS, multiple cloning site. B Construction of plasmids by insertion of egfp and gus as reporter genes driven by G. lucidum gpd promoter. In both pGl-GPE and pGl-GPG, the reporter gene is expressed from the G. lucidum gpd promoter

Table 1 Oligonucleotide primers used

The sequences with lowercase letters, ccaacatggtgg, ggtacc, ggatcc, ctgcag, and tctaga indicate BstX I, Kpn I, BamH I, Pst I and Xba I restriction sites respectively

Name

Sequence

Gl-GPD-F1 Gl-GPD-R1

50 -GATCccaacatggtggTCCAAAGCCGCTCTCATGG-30 50 -GATCccaacatggtggAGGGGGATGAAGAGTGAG-30

Le-RAS-F

50 -GATCccaacatggtggCTTCGCATAGCGGGATCA-30

Le-RAS-R

50 -GATCccaacatggtggAGAAACAGTCGGCTCCTC-30

Le-GPD-F

50 -GATCccaacatggtggCGAAGTTTGAGGTGGTTG-30

Le-GPD-R

50 -GATCccaacatggtggATTCAAGCAGTCAATGG-30

An-GPD-F

50 -GATCccaacatggtggGAATTCCCTTGTATCTCT-30

An-GPD-R

50 -GATCccaacatggtggGGGAAAAGAAAGAGAAAA-30

Gl-GPD-F2

50 -GATCggtaccTCCAAAGCCGCTCTCATGG-30

Gl-GPD-R2

50 -GATCggatccAGGGGGATGAAGAGTGAG-30

EGFP-F

50 -GATCggatccATGGTGAGCAAGGGCGAG-30

EGFP35S-R

50 -GATCctgcagTAATTCGGGGGATCTGG-30

GUS-F

50 -GATCggatccATGTTACGTCCTGTAG-30

GUS-R

50 -GATCtctagaTCATTGTTTGCCTCCCTG-30

GUS-mid

50 -GATACGTACACTTTTCCCGGC-30

HPH-DET-F

50 -TCGTTATGTTTATCGGCACTTT-30

HPH-DET-R

50 - GATGTTGGCGACCTCGTATT-30

co-culture duration (12, 36, 60, and 84 h). Following coculture, the membranes were transferred to CYM plates supplemented with 300 lg/mL cefotaxime and 100 lg/mL hygromycin B to inhibit the growth of A. tumefaciens and select for the positive G. lucidum transformants. Each experiment was repeated three times.

Extraction of genomic DNA Following growth on CYM media for 10 days at 28°C, mycelia of each strain were harvested by vacuum-filtration and washed with sterile water. Mycelia were ground to a fine powder in liquid nitrogen. Extraction of total DNA

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from the fungus was conducted by a method adapted from the CTAB method described previously (Saghai-Maroof et al. 1984). PCR and Southern blot analysis Detection of the hph gene was conducted using primers HPH-DET-F and HPH-DET-R (Table 1). Amplification included an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s, and elongation at 72°C for 1 min, and a final elongation step at 72°C for 5 min. Detection of a fusion fragment containing the Gl-GPD promoter and the EGFP or GUS gene was conducted using primers Gl-GPD-F2 and EGFP35S-R or GUS-mid (Table 1). Amplification included an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 40 s, annealing at 56°C for 40 s, and elongation at 72°C for 1 min, with a final elongation step at 72°C for 5 min. For Southern blot analysis, 30 lg of genomic DNA from untransformed G. lucidum and three randomly selected transformants was digested overnight at 37°C with BamH I (Fermentas, Canada). The pGL-GPG plasmid linearized with BamH I was used as the positive control. The digested products were separated by electrophoresis on a 0.8% TBE-agarose gel, the digested DNA transferred onto a Hybond-N? nylon membrane and probed with a 517 bplong hph fragment generated by PCR amplification using primers HPH-DET-F and HPH-DET-R. The hph-specific DNA probe labeling, hybridization and signal detection were conducted using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, Germany) according to the manufacturer’s instructions. Expression analysis of EGFP and GUS reporter genes The green fluorescence emission from EGFP was detected using a Nikon Eclipse Ti–S microscope. Images were recorded and processed using the NIS-Elements F package. For detection of GUS activity, mycelia were incubated at 37°C for 10 h in detection buffer containing 0.5% Triton X-100, 0.05 mM K3(Fe[CN]6), 0.05 mM K4(Fe[CN]6), 0.1 M sodium phosphate (pH 7) and 0.5 mg X-gluc mL-1 (Sigma, USA). Microscopy was performed using a Nikon Eclipse Ti–S microscope. Images were recorded and processed using NIS-Elements F package. Assay for mitotic stability of transformants To determine mitotic stability, thirty randomly selected transformants were cultured on CYM plates without hygromycin B for 7 days. Mycelia from the edge of the

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cultures were picked with a toothpick and grown on fresh CYM plates for another 7 days. After repeating this procedure 5 times, germinating mycelia from each transformant were transferred to CYM plates containing hygromycin B (100 lg/mL).

Results Optimization of conditions for ATMT of G. lucidum In an attempt to develop a simple, highly efficient transformation system for G. lucidum, we have tested the applicability of the ATMT method. A preliminary transformation assay was performed by spreading a mixed suspension of protoplasts and bacteria on sterile nitrocellulose membranes on IM agar plates with or without AS, followed by incubation at 28°C as described previously. After 36 h, the membranes were transferred to CYM agar plates containing 300 lg/mL cefotaxime and 100 lg/mL hygromycin B and incubated at 28°C for 7 days. At the end of the incubation period, a few fungal colonies were obtained on culture plates with AS (data not shown), demonstrating successful transformation of G. lucidum using the ATMT method. Subsequently, the effects of different co-cultivation conditions on the efficiency of ATMT of G. lucidum were examined. Growth temperature, duration of co-cultivation, ratio of bacteria to protoplast, and concentration of AS were optimized. Based on previous studies on ATMT and growth conditions of G. lucidum, 22, 25 and 28°C were chosen for our assay to optimize the co-culture temperature conditions. The number of regenerating colonies was found to be the highest at 25°C, and not at 28°C, which is the optimal cultivation temperature of both LBA4404 and G. lucidum (Fig. 2A). There was no obvious effect of the length of duration of co-cultivation on the efficiency of ATMT (Fig. 2B). However, the membranes were completely covered by the colonies after prolonged co-cultivation (over 96 h), making it difficult to count the colonies. To ensure the shortest possible duration for the successful completion of the ATMT protocol, 12 h was chosen as the duration of cocultivation for subsequent assays. The number of colonies increased with increasing ratio of bacteria to protoplasts used for co-cultivation and reached the peak at a ratio of 1,000:1 (Fig. 2C). The number of colonies obtained at this ratio was twofold higher than the number of colonies obtained when bacteria and protoplasts were both 105. The phenolic compound AS, an inducer of the expression of Agrobacterium virulence genes, is vital for efficient T-DNA transfer. The optimal concentration of AS for

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Fig. 2 Factors affecting the efficiency of ATMT of G. lucidum. A Effect of co-cultivation temperature on transformation efficiency. The growing colonies were counted as transformants. B Effect of length of duration of co-cultivation on transformation efficiency. C Effect of bacteria to protoplast ratio on transformation efficiency.

D Effect of AS concentration on transformation efficiency. E Effect of different promoters on transformation efficiency. Values reported are the mean of three independent experiments and indicate the number of transformants. Error bars indicate confidence intervals of percentages (P \ 0.05)

co-cultivation and ATMT of G. lucidum was found to be 0.2 mM (Fig. 2D). A few transformants were obtained when co-culture was conducted with 0.05 mM AS, but no positive colonies were obtained in co-cultures with either 1 mM AS or without AS.

were obtained using LBA4404 carrying the plasmid pLERAS or pLE-GPD, in which hph was driven by promoters derived from L. edodes. A few transformants were obtained by transformation with LBA4404 carrying the plasmid pAN-GPD, in which hph was driven by a promoter from A. nidulans.

Impact of promoters from different sources on transformation efficiency A comparative study of ATMT was performed by co-cultivation of G. lucidum with LBA4404 carrying one of the following plasmids: pGL-GPD, pLE-RAS, pLE-GPD, or pAN-GPD (Fig. 2E). These plasmids contain a cassette in which hph is under the control of different fungal promoters. The results showed that the best transformation efficiency was obtained using LBA4404 carrying the plasmid pGL-GPD, in which hph was expressed from the G. lucidum derived GPD promoter. A few transformants

Characterization of reporter gene integration and expression To investigate the pattern of integration of the foreign DNA fragment, three putative transformants were randomly selected for Southern blot analysis using the 517 bp PCR-amplified hph sequence as the probe. The results showed that all 3 transformants had the T-DNA insertion, and the various sized positive DNA bands obtained in the Southern blot analysis indicated that integration had occurred randomly at different sites in the genome

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(Fig. 3A). The Southern blot analysis therefore confirmed that the foreign DNA fragment from the transforming plasmid was successfully integrated into the chromosomal DNA of G. lucidum. Ganoderma lucidum transformants obtained by ATMT were also analyzed to determine EGFP and GUS expression in mycelia. The mycelia of wild type and transformants were examined by PCR analysis and expression analysis for the presence and expression of the reporter genes. PCR analysis was performed to confirm the presence of the fusion fragment containing the gpd promoteregfp/gus gene in genomic DNA isolated from the putative transformants (Fig. 3B and C). Fluorescence imaging and histochemical staining assay were used to detect expression of egfp and gus in the transformants, respectively (Fig. 4A and B). While no GFP fluorescence or blue staining could be detected in wild-type mycelia, the transformants Fig. 3 Identification of the foreign DNA fragments in randomly chosen G. lucidum transformants. A Southern Blot analysis of three randomly selected transformants. Lane N wild-type as negative control, Lane 1–3 randomly chosen transformants, Lane P pGl-GPG as positive control. The unit of number of markers and length indicator is base pair. B Amplification pattern obtained with primers for the gpd promoter-egfp fusion fragment in genomic DNA isolated from the G. lucidum transformants. Lane 1–3 randomly chosen transformants containing egfp; Lane P pGlGPE as positive control, Lane N untransformed G. lucidum as negative control, Lane M, DL 2000 DNA Marker. C Amplification pattern obtained with primers for the gpd promoter-gus fusion fragment in genomic DNA isolated from the G. lucidum transformants. Lane 1–3 randomly chosen transformants containing gus, Lane P pGlGPG as positive control, Lane N, untransformed G. lucidum as negative control, Lane M DL 2000 DNA Marker

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exhibited positive GFP signal and GUS activity to various degrees. This suggested that the transformants had not only integrated egfp and gus but also expressed these genes. However, in comparison to the blue staining of the GUSpositive transformants, the green fluorescence signal from EGFP-positive transformants was weaker. Mitotic stability G. lucidum transformants did not show any phenotypic differences compared to the wild-type strain under identical physiological conditions. To determine whether the transforming DNA was stably maintained or not in the genome of G. lucidum, 30 transformants were grown on CYM plates without hygromycin B and replated for five generations. We obtained 23 mitotically stable transformants out of 30 colonies ([70%), thus confirming the genetic stability of the

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Fig. 4 Expression analysis of the reporter gene in randomly chosen transformants of G. lucidum. A Fluorescence microscopy analysis of three randomly selected transformants harboring egfp. a and b Fluorescence and bright field images of wild-type G. lucidum mycelia respectively; c, e and g Green fluorescence of mycelia of randomly chosen transformants harboring egfp. The GFP fluorescence were shown

by arrows. d, f and h Bright field image of mycelia shown in c, e, and g; Bar 20 lm (in each image). B Histochemical staining analysis of three randomly selected transformants harboring gus. a Mycelia from wildtype G. lucidum; b, c and d Mycelia from randomly chosen gus containing transformants showing GUS expression. The blue staining were shown by arrows. Bar 0.5 mm (in each image). (Color figure online)

integrated DNA in this fungus and the suitability of the method as a tool to introduce foreign DNA into G. lucidum.

of an efficient gene transfer system. In the last decade, ATMT has been successfully applied to a few different fungal species, mainly because of its efficiency and technical simplicity (Michielse et al. 2005). The main objective of this work was to establish optimum conditions for ATMT of G. lucidum. Using the ATMT system, we obtained at least 200 stable G. lucidum transformants per 105 protoplasts. In contrast, the transformation yield was 15

Discussion Molecular genetic studies of edible fungi in general, and G. lucidum in particular, have been limited due to the lack

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transformants per 107 protoplasts using electroporation and 4–17 transformants per 107 protoplast lg DNA, using REMI (Kim et al. 2004; Sun et al. 2001). The ATMT system clearly resulted in an increased transformation frequency compared to the other methods used thus far for the transformation of G. lucidum. Furthermore, compared with other transformation systems used in G. lucidum, the ATMT system did not require complex operations and expensive equipment, such as a gene pulser apparatus. Consistent with the results of studies in Mortierella alpine (Ando et al. 2009), we observed that increasing the amount of A. tumefaciens in the co-culture mixture led to an increase in the transformation frequency. However, there is a limitation in the concentration of A. tumefaciens that can be used effectively. The highest number of bacteria used in our experiments was 108. Higher numbers of A. tumefaciens can lead to bacterial over-growth during coculture, thereby inhibiting fungal growth significantly, probably due to nutritional or space limitations (Chen et al. 2009). On the other hand, excessive addition of protoplasts can cause fungal over-growth during co-culture, which makes the subsequent isolation of single transformants difficult (Covert et al. 2001). It has been shown that an optimal co-culture temperature plays an important role in obtaining the maximum number of transformants (Combier et al. 2003). We have shown that the most favorable co-cultivation temperature is 25°C, which is different from the normal growth temperature (28°C) for G. lucidum. All the co-culture temperatures we tested resulted in positive fungal transformants suggesting that the Agrobacterium vir genes are expressed over a wide range of temperatures allowing effective T-DNA transfer and integration. Moreover, transformants were obtained only when the co-culture was induced with AS. This is consistent with earlier reports that demonstrate the essential role of AS for vir gene expression (de Groot et al. 1998; dos Reis et al. 2004; Michielse et al. 2008). Taken together, these data suggest that the Agrobacterium vir genes play a key role in ATMT (Combier et al. 2003). In this study, we tested ATMT mediated fungal transformation with several plasmids and were successful with some of the constructs. The primary difference between the constructs was the source of the promoter used to drive expression of the antibiotic resistance marker. Of all the plasmids tested, the construct carrying the promoter of Glgpd resulted in the highest transformation efficiency. The promoters Le-gpd and Le-ras, which are both from the basidiomycete L. edodes, were able to drive the antibiotic resistance gene but the numbers of colonies obtained were far fewer than that obtained with the Gl-gpd promoter. Moreover, the number of transformants obtained using the construct carrying the An-gpd promoter was only 2 ± 1.7 per 105 protoplasts, which was much lower than the

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number obtained from other constructs. This data indicated that the gpd promoter from A. nidulans, which is one of the most common regulatory sequences used for gene expression in fungi, was functional, albeit significantly less active in G. lucidum. This might be attributed to the fairly substantial genetic differences between the two fungal divisions (Berbee and Taylor 1993). Our results demonstrate that promoter sequences belonging to homologous species are more active and highlight the importance of using homologous promoters to drive gene expression (Chen et al. 2000; Godio et al. 2004). The EGFP fluorescence was detected in the transformants, although the fluorescence intensity was not particularly strong. These results indicate that either the expression or activity of EGFP is sub-optimal in G. lucidum. We compared the DNA sequence of egfp with the codon bias of G. lucidum (Xu et al. 2006). Our analysis showed that 22.5% of the codons in egfp are less frequently used in G. lucidum, which could account for lower translational efficiency and weak fluorescence signal. These data implied that egfp codon optimization based on codon bias of G. lucidum might result in a better reporter system. Although the transformants obtained from independent transformation events showed variable GUS expression, overall we found GUS to be a more robust reporter gene than EGFP. This is consistent with recent report demonstrating the use of GUS as a reporter gene to study the OSC gene promoter activity in the ganoderic acids (GA) biosynthesis pathway in G. lucidum (Shang et al. 2010). Mitotic stability analysis showed that over 70% of the transformants tested remained mitotically stable even after being subculture in the absence of hygromycin B, and they maintained antibiotic resistance conferred by the hph gene. The result of Southern blot analysis demonstrated that T-DNA integration into the chromosome, a mechanism observed in several mitotically stable filamentous fungi obtained using ATMT (dos Reis et al. 2004; Meyer et al. 2003). These data indicate that the ATMT system can be effectively used in molecular genetic studies of this fungus. In addition, using the ATMT of G. lucidum, we expressed several genes involved in the triterpenes biosynthetic pathway, including the acetyl-CoA acetyltransferase and pyrophosphomevalonate decarboxylase genes under the control of the Gl-gpd promoter. Semi-quantitative RT–PCR assay showed that the expression of these genes was significantly enhanced in the transformants. This correlated with the transformants having levels of triterpenes that were at least 30% higher compared to that of the control sample (data would be published in another report). These results suggest that this transgenic method can be used for the expression of functional genes. Our results demonstrate that ATMT improves the transformation efficiency of G. lucidum and the mitotic

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stability of transformants, thereby showing that ATMT is a powerful tool for functional genomics research in G. lucidum. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Project No. 30970042, 30871767), the Fundamental Research Funds for the Central Universities (Project No. KYZ201121), and Shanghai Committee of Science and Technology, China (Project No. 08JC1418100).

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