Agrobacterium tumefaciens mediated fused egfp-hph gene expression under the control of gpd promoter
Microbiological Research166(2011)
314—322
Available online at https://www.sodocs.net/doc/f88998480.html,
www.elsevier.de/micres Agrobacterium tumefaciens mediated fused
egfp-hph gene expression under the control of gpd promoter in Pleurotus ostreatus
Yi Ding a,Shen Liang a,Jinghang Lei a,Liguo Chen b,
Erika Kothe c,Aimin Ma a,?
a College of Food Science and T echnology,Huazhong Agricultural University,1Lion Hill Road,Wuhan430070,China
b College of Plant Science and T echnology,Huazhong Agricultural University,1Lion Hill Road,Wuhan430070,China
c Microbial Phytopathology,Friedrich-Schiller-University,Jena D-07743,Germany
Received28May2010;received in revised form24June2010;accepted3July2010
KEYWORDS Pleurotus ostreatus; Agrobacterium tumefaciens; Transformation; eGFP;
gpd promoter Summary
A transformation system for the basidiomycete Pleurotus ostreatus was established using agrobacterium-mediated infection.Following P.ostreatus glyceraldehyde-3-phosphate dehydrogenase gene analysis,its promoter region including two introns was used as cis-regulatory element to drive expression of enhanced green?uores-cent protein(eGFP).As a selection marker,the hygromycin phosphotransferase(hph) gene cassette was used in the binary vector pPEH.Mycelia without pretreatment were found to be the most ef?cient recipients in transformation experiments while fruiting body tissue or basidiospores showed lower transformation rates.A transfor-mation ef?ciency of75%was achieved.After subculturing,putative transformants were screened by PCR and Southern blot analysis showing the expected ectopic integration of the transforming DNA.At the same time,the promotor region was shown to drive expression of selection marker as well as eGFP that could be visual-ized,which will be helpful for future investigation using Agrobacterium tumefaciens mediated transformation for functional characterization of genes in the mushroom forming basidioymcete P.ostreatus.
?2010Elsevier GmbH.All rights reserved.
?Corresponding author.T el.:+862762229767.
E-Mails:maaimin@https://www.sodocs.net/doc/f88998480.html,, maaiminfood@https://www.sodocs.net/doc/f88998480.html,(A.Ma).1.Introduction
The white-rot basidiomycete fungus Pleurotus ostreatus,or oyster mushroom,has signi?cant value as an edible fungus which is commercially used world-wide.Moreover,many factors render
0944-5013/$–see front matter?2010Elsevier GmbH.All rights reserved. doi:10.1016/j.micres.2010.07.001
Agrobacterium tumefaciens mediated fused egfp-hph gene expression in Pleurotus ostreatus315
P.ostreatus a good model for understanding bio-chemical and physiological processes,including the production of active compounds as well as many enzymes in a short production cycle.These include the hydrophobins involved in hydrophobicity(Ma et al.2008),the medical effects(Sunagawa and Magae2001)and the of broad substrate adapt-abilities especially the high lignin degradation(Ha et al.2001),agro-industrial waste bioconversion (Cohen et al.2002),and toxic heavy metal biosorp-tion activities(Pan et al.2005)which are widely applied in environmental protection.In recent years,the genetic analysis of P.ostreatus has gained considerable interest(Moussa2009;Pérez et al.2009),however,the need for an easy and ef?cient transformation system became obvious to allow functional genomics in P.ostreatus.
In previous attempts,a variety of transforma-tion methods,including polyethylene glycol/CaCl2 (PEG/CaCl2)(Honda et al.2000),restriction enzyme mediated integration(REMI)(Irie et al. 2001)and particle bombardment(Sunagawa and Magae2001)were used to introduce DNA into P. ostreatus.The main drawbacks of these methods were their low transformation ef?ciency,hetero-geneous integration into genomic loci and the need for protoplasts,although improved procedures with enhanced ef?ciencies were published by Li et al. (2006).
During the previous decades,Agrobacterium tumefaciens has been used to establish transfor-mation protocols for numerous prokaryotic and eukaryotic organisms(Soltani et al.2008).In higher fungi,more and more higher fungal intact cells were successfully transformed by A.tumefaciens mediated transformation(AMT),yielding high ef?-ciencies with DNA integrated at random sites in single-copy(Chen et al.2000;Cho et al.2006; Grimaldi et al.2005;Murata et al.2006;Wang et al.2008).Key factors in?uencing transformation of higher fungi via A.tumefaciens are the choice of vectors with appropriate promoters,as well as the selection of recipient cells in a life stage,where transformation is possible at higher ef?ciencies.
Glyceraldehyde-3-phosphate dehydrogenase (GPD)is a crucial enzyme in glycolysis,which is re?ected by the fact that it represents more than5%of the total cellular protein in Saccha-romyces cerevisiae.The gene is expressed at high levels throughout the exponential growth phase in yeasts.This expression is driven by a strong and constitutive promoter(Holland and Holland 1979).Homologous as well as heterologous gpd promoters have been used widely to express genes of interest,including several basidiomycetous gpd promoters used in different higher fungi for,e.g.,expression of laccase(Alves et al.2004;
Kilaru et al.2006),peroxidase(Li et al.2001),
multi-functional cellulase gene(Cheng et al.2009),?-glucuronidase gene(Kuo and Huang2008)or the bacterial hygromycin resistance gene(hph)
(Chen et al.2000;Irie et al.2001;Kuo et al.2004),
and even in post-transcriptional gene silencing
(Eastwood et al.2008;Kemppainen et al.2009).
Furthermore,auto?uorescent protein genes were
also available,driven by these gpd promoters,
as versatile reporters to monitor transcriptional
localization of gene products in situ in real time
(Lugones et al.1999;Burns et al.2005;Murata et
al.2006).
In this study,we cloned the promoter of the P.
ostreatus gpd gene to drive expression of a gene
fusion of a double reporter gene consisting of egfp
and hph.We optimized AMT to transfer heterolo-
gous genes into P.ostreatus in order to enhance
the possibility of biotechnological applications in P.
ostreatus as a bene?cial resource.
2.Materials and methods
2.1.Strains and culture media
The P.ostreatus dikaryotic strain Pd739was pro-
vided by the Mushroom Spawn Center,Huazhong
Agricultural University.The vegetative mycelium
was maintained at25?C on potato dextrose agar
(PDA;Difco,USA)covered with autoclaved cello-
phane membrane in a Petri dish,for producing fruit-
ing bodies and germinated spores.For the selection
and maintenance of transformants,PDA medium
was supplemented with hygromycin(Roche,Ger-
many)at50?g/ml.A.tumefaciens strains AGL-1
and GV3101(IMCAS,China),grown in YEB medium
(5g/l tryptone,1g/l yeast extract,5g/l nutrient
broth,5g/l sucrose,0.49g/l MgSO4·7H2O)contain-ing100?g/ml kanamycin and50?g/ml rifampicin (Sigma—Aldrich,USA)were used to transform P. ostreatus.Escherichia coli DH5?(T akara,Japan) was manipulated as described by Sambrook and Russell(2001).
2.2.Cloning of the gpd gene in P.ostreatus
P.ostreatus genomic DNA was isolated with the DNeasy Plant Mini Kit(Qiangen China,Shang-hai).For cloning of the partial P.ostreatus gpd gene,a pair of degenerated primers LgF and LgR were designed based on the conserved GPD cod-ing regions of published basidiomycetous fungi (Kuo et al.2004)using total DNA as template. Fungal total RNA was isolated from2-week-old
316Y.Ding et al. Table1.Oligonucleotide primers used in this study.
Oligonucleotide name Sequence a
LgF5 -GGYCGTATYGTMYTCCGNAATGC-3
LgR5 -CCCCAYTCRTTRTCRTAC-3
5gpdSp15 -TAGACCTAAGCATGGACCGATCG-3
5gpdSp25 -AGTCAGCACTTCTAAGATCGGCG-3
5gpdSp35 -GTCATCCAAGCGACATGNNNNNNNNNCTATTC-3 3gpdSp15 -GGTTTGTCCTTCCCGTGTCCCC-3
3gpdSp25 -CGTCGCCGTTATCAAGGAAGCC-3
3gpdSp35 -CTCCGTAGTCTCCACCNNNNNNNNNNGAAGAC-3 EgfpF5 -ATGGTGAGCAAGGGCGAG-3
EgfpR5 -CTTGTACAGCTCGTGGA-3
pgEF5 -CGGAATTCCGTGTCGTTCGTGACTCGCAATA-3 pgBR5 -CGGGATCCCGTGGACAGGCTTTTGGGAATA-3 hygSF5 -ACATGCATGCATATGAAAAAGCCTGAACTCAC-3 hygXR5 -CCGCTCGAGCGGCTATTCCTTTGCCCTCGGAC-3 a DNA letter codes:M=(A,C);N=(A,T,G,C);R=(A,G);Y=(C,T).
mycelium homogenized with liquid nitrogen using TRI Reagent?(MRC,USA).RT-PCR using LgF and LgR (T able1)as primers was used for partial gpd gene ampli?cation.The PCR product was puri?ed and cloned into pMD18-T(T aKaRa,Japan)for sequenc-ing(Invitrogen China,Shanghai).
T wo gene speci?c primers and one partially degenerate primer were designed for self-forming adaptor(SEFA)PCR to amplify upstream or down-stream gpd sequences with minor modi?cations (Wang et al.2007).Brie?y,EasyT aq DNA polymerase (TransGen Biotech,China)was used in a50?l reac-tion mixture including5?l10×Easy T aq buffer,2?l 10mM dNTPs,proper amounts of template,primers of5gpd(sp1,sp2,sp3)(T able1)and3gpd(sp1,sp2, sp3)(T able1)while the extension time was reduced to3min.The product that was obtained from one round SEFA PCR and two rounds of thermally asymmetric PCR was separated for sequencing. The promoter sequence was further analyzed by Neural Network Promoter Prediction websoftware at www.fruit?https://www.sodocs.net/doc/f88998480.html,/seq tools/promoter.html and transcription factor binding sites searching tool at http://molsun1.cbrc.aist.go.jp/research/db/ TFSEARCH.html.
2.3.Plasmids and vector construction
The vector pHg/pBks used in this study was kindly provided by Professor Alejandro G.Pardo(National University of Quilmes,Bernal,Argentina).It con-tains the selectable hygromycin B marker gene under the control of the Agaricus bisporus gpd promoter and a bla gene allowing for plasmid rescue.For detection and analysis of heterolo-gous gene expression in P.ostreatus,a new A.tumefaciens binary vector pPEH harboring egfp and the hph fusion gene based on pCAMBIA1300 (Cambia,Australia),was constructed as follows. The egfp coding region without stop codon was ampli?ed using the primers of EgfpF and EgfpR (T able1)and was cloned into pMD18-T to generate pMD-E.Then a1133bp P.ostreatus gpd amplicon obtained by PCR ampli?cation with gene speci?c primers pgEF and pgBR(T able1)?anking a Eco RI and Bam HI restriction sites was introduced in pMD-E to form the plasmid pMPE.Subsequently, the ampli?ed hph gene,in which Sph I and Xho I restriction sites were added using primers of hygSF and hygXR(T able1),was digested and ligated into the Eco RI-Sph I excised vector pMPE to generate the egfp-hph fusion expressing cassette driven by the homologous P.ostreatus gpd promoter.This fusion construct was then inserted into the pCAMBIA1300 vector backbone after removing the CaMV35S promoter-hph by digestion with Eco RI and Xho I, generating the binary vector pPEH.All vectors were introduced into A.tumefaciens strains AGL-1 and GV3101through electroporation(BioRad,USA).
2.4.Agrobacterium-mediated transformation of P.ostreatus
Medium composition and bacteria preparation were carried out mainly as described by Chen et al. (2000).The A.tumefaciens strains AGL-1and GV3101,each harboring pCAMBIA1300,pHg/pBks or pPEH,were cultivated at28?C in YEB in the pres-ence of the proper selective antibiotics to an OD600 of0.6—0.8.Enlarged bacterial cells were collected by centrifugation,suspended in induction medium (IM,200?M acetosyringone plus,without antibi-
Agrobacterium tumefaciens mediated fused egfp-hph gene expression in Pleurotus ostreatus317
otic)to an OD600of0.2and incubated for additional 6h at28?C with shaking at150rpm to pre-induce virulence of A.tumefaciens.
As transformation explants,fungal mycelia were grown on the middle of sterile microporous membranes(0.45?m,25mm)on PDA for3 d. Alternatively,nearly mature,small fruitbodies, especially the mushroom caps(diameter of ca.
0.4cm)that just developed from the primordium were cut.For the transformation of P.ostrea-tus spores,5ml liquid IM was added to wash off the spores in autoclaved?asks.Serial dilu-tions to concentrations of105—107spores/ml were mixed with virulence pre-induced A.tumefaciens and plated onto cellophane disks on solid IM for co-cultivation.After3d incubation at25?C,the cellophane membranes with fungal and bacte-rial colonies were transferred onto selection agar medium(SM)containing50?g/ml hygromycin and 200?g/ml cefotaxime.As for mycelium transfor-mation,the membranes inoculated with fungal colonies were dipped into the culture of pre-induced bacteria for20min and then placed on an IM agar plate at25?C for5d,after which the co-cultivated mycelia were transferred to SM plates to select putative transformants.For AMT of the fruitbodies,P.ostreatus tissues newly excised were dipped into ethanol and then quickly immersed into liquid IM of pre-induced A.tumefaciens.The bacteria-fungus mixture was co-cultivated at25?C with shaking of100rpm for48h.The tissue pieces were then separated from the liquid mixtures, washed with autoclaved,double-distilled water and transferred to cellophane on a solid IM sur-face for another day before being transferred to SM for selecting potential transformants.Controls using untransformed bacteria and wild type fun-gus for selection were used in all experiments.The percentage of regenerating fungal colonies on SM from mycelia colonies grown on sterile microporous membranes,from tissue pieces and from spores was seen as the transformation ef?ciency.
2.5.Stability test and molecular analysis of transformants
Genomic DNA was extracted from?ve-round sub-cultural mycelia of transformants and wild type P.ostreatus and assessed by PCR analysis for the existence of hph using primers hygSF and hygXR (T able1)and the performance of egfp-hph with primers EgfpF and hygXR(T able1).The ampli?-cation procedures were carried out as follows:an initial denaturation at94?C for5min;30cycles of 94?C denaturation for30s,63?C or58?C anneal-ing for30s,72?C elongation for2min;and a?nal extension at72?C for10min.For Southern blot analysis,approximately3—5?g DNA of transformed strains and negative control were digested with Hin dIII.Standard protocols for size fractionated gel electrophoresis,denaturation and neutraliza-tion are described by Sambrook and Russell(2001), after which the gel was blotted by capillary transfer with20×SSC onto Hybond-N+membrane(Amer-sham Biosciences,UK).The hph fragment obtained previously was digoxigenin(DIG)labeled as a spe-ci?c probe for signal detecting.Probe labeling, hybridization,and immunological detection were all done according to the manufacturer’s protocol (Roche,Germany).
2.6.Fluorescence microscopy
Analysis of the eGFP expression was carried out by preparing the hyphae of random P.ostreatus transformants,to glass slides after5d growth on agar plates without or with hygromycin B.Samples were then analyzed by a Nikon Eclipse80i?uores-cence microscope(Nikon,Japan)with a B-2A?lter. Images were taken under40×objective for ran-dom transformed mycelial colonies and processed with NIS-Elements BR3.0imaging software(Nikon, Japan).
3.Results
3.1.Assay of the GPD gene and its5
?anking region
Following the degenerated PCR ampli?cation with primers LgF and LgR,the partial gpd coding frag-ment of a size of1192bp from P.ostreatus genomic DNA and of920bp incomplete cDNA sequence generated from total RNA revealed remarkable homology to known gpd genes.Subsequently,the entire gpd clone and complete cDNA sequence were obtained by5 and3 SEFA PCR with an upstream region of3680bp and a534bp downstream frag-ment adjacent to the obtained gpd DNA clone, respectively.The coding sequence of1011bp trans-lating to336amino acids was disrupted by six introns(GenBank accession number GU062704). The protein was highly conserved with respect to known GPD proteins with an amino acid identity of87.3%to GPD of other basidiomycetes(GenBank accession nos.AAA32634,AF515622,BAA83550, AAA33732,AAD52091).
As for the gpd promoter,a potential transcrip-tion start site was located at47bp upstream of the
318Y.Ding et
al.
Figure1.The upstream nucleotide sequence for P.ostreatus gpd to identify promoter elements.Possible pro-moter elements are framed,and the predicted transcriptional start site is underlined(Gene bank accession number GU062704).
start codon in a CT-rich region,in front of which a typical TATA-box was found(Fig.1).T wo possible CAAT-boxes(Fig.1)can be found in the upstream region,though they are located farther upstream than usual(Kilaru and Kües2005).A similar case, however,was reported for Flammulina velutipes (Kuo et al.2004).
3.2.Agrobacterium-mediated transformation
A.tumefaciens strains AGL-1and GV3101were found to result in similar transformation ef?cien-cies with P.ostreatus using the binary plasmids. The two constructed vectors were pPEH,based on pCAMBIA1300(Fig.2A),and pHg/pBks,derived from pBGgHg with an A.bisporus gpdII promoter (Fig.2B).Of all P.ostreatus recipient explants, the lowest ef?ciency was observed for germinated spores,averaging2Hyg r colonies,performing only at bacterium-to-spore ratio of100:1in favor of pPEH and pHg/https://www.sodocs.net/doc/f88998480.html,ing mycelia colonies,pPEH and pHg/pBks transformed P.ostreatus at rates of75%and35%,respectively,indicative of better functioning of the construction.In a third attempt, pieces of mushrooms were used for transformation. Approximately7%of the fruiting body pieces
were https://www.sodocs.net/doc/f88998480.html,ed transformation vectors based on the A.tumefaciens right border(RB)and left border(LB)and different T-DNA,including restriction sites.(A)Schematic representation of the cloning strategy for the egfp-hph fusion cassette cloned into the T-DNA region of the binary vector pPEH.The expression cassette is2954bp in size and consists of genes encoding enhanced green?uorescent protein(eGFP)and translational fusion to hygromycin phosphotransferase(hph).The promoter consists of913bp P.ostreatus glyceraldehyde-3-phosphate dehydrogenase (gpd)promoter(Ppgpd)followed by the initial200bp untranslated region of gpd which contains two introns.The cauli?ower mosaic virus terminator(T-35S)was fused to the Hph C-terminus.(B)Organization of the T-DNA region in the binary plasmid pHg/pBks(Kemppainen et al.,2008).The hph gene is located between approximately280bp of the A.bisporus gpd promoter(Pagpd)and T-35S.Plasmid rescue is enabled by including a replication origin(ori)and amphicillin resistance gene(amp r)from pBluescript KS+.
Agrobacterium tumefaciens mediated fused egfp-hph gene expression in Pleurotus ostreatus
319
Figure3.PCR analysis of total DNA prepared from independent P.ostreatus transformants and non-transformed wild https://www.sodocs.net/doc/f88998480.html,nes3—5:hph ampli?cation pattern of transformants generated using AGL-1and pHg/pBks,lanes6—8:hph ampli?cation pattern of transformants generated using GV3101and pHg/pBks,lanes10—12:egfp-hph ampli?cation pattern of transformants generated using AGL-1and pPEH,lanes13—15:egfp-hph ampli?cation pattern of transfor-mants generated using GV3101and pPEH,lanes2and9:negative control using wild type DNA ampli?ed by hph and egfp-hph primers,respectively,lane1:molecular size marker(kb).
found to be transformed to hygromycin resistance, with superior performance from pPEH compared to pHg/pBks.Thus,mycelia were used for all subse-quent transformation experiments.As a control, pCAMBIA1300was also used for transformation, where it did not transform either of the recipients as expected.
3.3.Analysis of P.ostreatus transformants The hygromycin resistant transformant colonies appeared after4d.Five rounds of selection were made by transferring margins of the colonies to fresh selection plates,resulting in mycelia with 15—20d needed for full growth on the plate of selection medium.This is in contrast to wildtype which grew to the same diameter within10d on PDA out of resistance pressure.
The transformants were then subcultured on PDA as samples for PCR and Southern blot analy-sis.Both pPEH and pHg/pBks derived transformants yielded the expected～1kb and～1.7kb target bands(Fig.3).The6randomly selected pPEH derived,and5pHg/pBks derived transformants studied by Southern blot analysis yielded single bands of different sizes,indicating random and single-copy insertion of the T-DNA into the fungal genome(Fig.4).
3.4.Detection and visualization of eGFP
T o verify the expression of the introduced egfp reporter gene,mycelium of separate transfor-mants grown either with or without antibiotic were inspected by?uorescence microscopy.The sim-ilar pattern(Fig.5)revealed stable and highly ef?cient eGFP expressed in the individual trans-formants.Thus,the protein fusion was suf?cient to allow both hygromycin resistance and eGFP ?uorescence.
4.Discussion
In this study,we have identi?ed the complete gpd gene from P.ostreatus and shown
that Figure4.Southern blot analysis of P.ostreatus transformants.Genomic DNA(3—5?g)digested with Hin dIII was probed using～1kb DIG-labeled https://www.sodocs.net/doc/f88998480.html,ne1:P.ostreatus wild type(wt),lanes2—6:transformants using pHg/pBks, lanes7—12:transformants using pPEH.The positions of molecular DNA size markers(kb)are shown on the left.
320Y.Ding et
al.
Figure5.Expression of eGFP in separate P.ostreatus transformants without(A)or with(B)hygromycin.Detection for?uorescence under UV light(left column)and corresponding phase-contrast micrographs(right column)are shown with the same microscope equipment of a excitation?lters at450—490nm,a dichroic?lter at505nm and an emission ?lter at520nm.Images were taken with40×?elds of view,bar=60?m.
it can be used for transformation experiments. In many basidiomycetous fungi,gpd genes are found in single-copy in the genome(Kilaru and Kües2005).However,more than one copy was reported for A.bisporus,where gpdII is exclu-sively transcribed(Harmsen et al.1992),and Coprinopsis cinerea(https://www.sodocs.net/doc/f88998480.html,/ annotation/fungi/coprinus cinereus/)which fea-tures a tandem duplication.In higher basid-iomycetes,5—10introns have been reported for gpd genes(Kilaru and Kües2005),and this is now also found to be the case for the P.ostreatus gpd gene.
The A.tumefaciens based transformation sys-tem was?rst demonstrated as a good mediator for genetic transformation of Pleurotus species,P. ostreatus.Transformation results showed that the choices of recipient materials as well as vectors directly led to the variation in?nal ef?ciencies.In this study,fungal mycelium without pretreatment was found to give the best results in transfor-mation experiments as compared to spores or fruiting bodies.In the edible mushroom P.ostrea-tus,small mushroom caps that just developed from primordium were chosen,while the transformation ef?ciency of single gill tissues from A.bisporus or F.velutipes were reported to be approximately40% and16%,respectively(Chen et al.2000;Cho et al.2006).Indeed,when we used gill tissues,no transformants could be obtained.The emergence of resistant colonies from the basidiospore-bacterium mixture would determine the transformants with-out the false positives caused by tissue tolerance to antibiotics(Kuo et al.2004),but the low ef?-ciency seemed to limit the transformation of spores via A.tumefaciens in P.ostreatus that we did not pursue.The PCR analysis of transformants for hph and egfp-hph sequences supported the notion that the transgenes were mitotically sta-ble.
This is the?rst report of stable egfp expres-sion in fusion with the hph gene in the white-rot basidiomycetous mushroom P.ostreatus,under the control of the P.ostreatus gpd promoter.Li et al.(2006)reported that a transient green?uo-rescence was visible in transformed protoplasts (by PEG method)which,however,disappeared 30h after transformation.Our transformants,in contrast,shared strong and transparent green?u-
Agrobacterium tumefaciens mediated fused egfp-hph gene expression in Pleurotus ostreatus321
orescence during several rounds of selection.We chose the homologous gpd promotor and genomic DNA,since homologous cis-regulators(Chen et al. 2000)and introns needed for mRNA accumula-tion(Lugones et al.1999;Scholtmeijer et al. 2001)have been shown to increase transformed expression in other fungi while heterologous pro-moters were less successful to drive expression (Gold and Alic1993).Murata et al.(2006)success-fully used two different promoters and terminators to drive hph and an auto?uorescent protein gene expression in Suillus bovinus to ensure that DNA rearrangement should not be promoted during A. tumefaciens infection.Additionally,careful design of the promotor construct was performed to express the auto?uorescent protein.The latter seemed necessary as successfully expressed GFP in basid-iomycetes coherently share introns in the expressed mRNA(Lugones et al.1999;Burns et al.2005; Sun et al.2009).Considering these aspects,our binary vector pPEH was developed to contain an egfp::hph fusion cassette under the control of the homologous gpd promoter,followed by a200bp sequence containing the?rst two introns of the gpd non-translated genomic region.This special design seems to be a more appropriate combination,cov-ering the critical aspects(Hellens et al.2000)for eGFP expression in the P.ostreatus transformation system.
Different recipient materials from spore,mush-room tissue and mycelium were used to test transformation ef?ciency in this work.With heterokaryons carrying both transformed and untransformed nuclei,the screening for transgene-associated phenotypes could be tedious,implying the use of spores.However,spores showed lower transformation ef?ciencies,which may be asso-ciated with the spore wall or the expression of hygromycin resistance in germinating transformed spores.Indeed,unequal penetration of the trans-gene in Tuber borchii(Grimaldi et al.2005)has been reported,which led us to investigate pene-tration and mitotic stability in our transformation system.While the single signals of Southern blot hybridization indicated that the T-DNAs inserted at single,random sites,the fraction of trans-formed nuclei was suf?cient for mycelial survival and visualization of eGFP expression driven by the hph-egfp cassette.The established AMT trans-formation system for P.ostreatus will enhance research including expression of active compounds as well as enzymes for biotechnological applica-tions.At the same time,the fungus can be used to introduce genes of interest for basic research, or for environmental remediation technologies or industrial use.Acknowledgements
This work was partially supported by the grant of National Natural Science Foundation of China (NSFC)(Grant No.30771502)to Aimin Ma.We thank Professor Alejandro G.Pardo for providing the plasmid pHg/pBks and Professor T anya Dahms for critical review.
References
Alves AM,Record E,Lomascolo A,Scholtmeijer K,Asther M,Wessels JG,et al.Highly ef?cient production of lac-case by the basidiomycete Pycnoporus cinnabarinus.
Appl Environ Microbiol2004;70:6379—84.
Burns C,Gregory KE,Kirby M,Cheung MK,Riquelme M, Elliott TJ,et al.Ef?cient GFP expression in the mush-room Agaricus bisporus and Coprinus cinereus requires introns.Fungal Genet Biol2005;42:191—9.
Chen X,Stone M,Schlagnhaufer C,Romaine CP.A fruiting body tissue method for ef?cient Agrobacterium-mediated transformation of Agaricus bisporus.Appl Environ Microb2000;66:4510—3.
Cheng S,Yang P,Guo L,Lin J,Lou N.Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters.
Bioresour T echnol2009;100:4475—80.
Cho JH,Lee SE,Chang WB,Cha JS.Agrobacterium-mediated transformation of the winter mushroom Flammulina velutipes.Mycobiology2006;34:104—7. Cohen R,Persky L,Hadar Y.Biotechnological applica-tions and potential of wood-degrading mushrooms of the genus Pleurotus.Appl Microbiol Biotechnol 2002;58:582—94.
Eastwood DC,Challen MP,Zhang C,Jenkins H,Henderson J,Burton KS.Hairpin-mediated down-regulation of the urea cycle enzyme argininosuccinate lyase in Agaricus bisporus.Mycol Res2008;112:708—16.
Gold MH,Alic M.Molecular biology of the lignin-degrading basidiomycete Phanerochaete chrysosporium.Micro-biol Rev1993;57:605—22.
Grimaldi B,de Raaf MA,Filetici P,Ottonello S,Ballario P.
Agrobacterium-mediated gene transfer and enhanced green?uorescent protein visualization in the mycor-rhizal ascomycete Tuber borchii:a?rst step towards truf?e genetics.Curr Genet2005;48:69—74.
Ha HC,Honda Y,Watanabe T,Kuwahara M.Production of manganese peroxidase by pellet culture of the lignin-degrading basidiomycete,Pleurotus ostreatus.Appl Microbiol Biotechnol2001;55:704—11.
Harmsen MC,Schuren FH,Moukha SM,van Zuilen CM,Punt PJ,Wessels JG.Sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase genes from thebasidiomycetes Schizophyllum commune, Phanerochaete chrysosporium and Agaricus bisporus.
Curr Genet1992;22:447—54.
Hellens R,Mullineaux P,Klee H.A guide to Agrobacterium binary Ti vectors.Trends Plant Sci2000;5:446—51.
322Y.Ding et al.
Holland JP,Holland MJ.Primary structure of a glyceraldehyde-3-phosphate dehydrogenase gene from Saccharomyces cerevisiae.J Biol Chem 1979;254:9839—45.
Honda Y,Matsuyama T,Irie T,Watanabe T,Kuwahara M.
Carboxin resistance transformation of the homoba-sidiomycete fungus Pleurotus ostreatus.Curr Genet 2000;37:209—12.
Irie T,Honda Y,Hirano T,Sato T,Enei H,Watanabe T,et al.Stable transformation of Pleurotus ostrea-tus to hygromycin B resistance using Lentinus edodes GPD expression signals.Appl Microbiol Biotechnol 2001;56:707—9.
Kemppainen M,Duplessis S,Martin F,Pardo AG.T-DNA insertion,plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.Micro-bial Biotechnol2008;1:258—69.
Kemppainen M,Duplessis S,Martin F,Pardo AG.RNA silencing in the model mycorrhizal fungus Laccaria bicolor gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus.Environ Micro-biol2009;11:1878—96.
Kilaru S,Hoegger PJ,Majcherczyk A,Burns C,Shishido K,Bailey A,et al.Expression of laccase gene lcc1in Coprinopsis cinerea under control of various basidiomycetous promoters.Appl Microbiol Biotechnol 2006;71:200—10.
Kilaru S,Kües https://www.sodocs.net/doc/f88998480.html,parison of gpd genes and their pro-tein products in basidiomycetes.Fungal Genet Newsl 2005;52:18—23.
Kuo CY,Chou SY,Huang CT.Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes.
Appl Microbiol Biotechnol2004;65:593—9.
Kuo CY,Huang CT.A reliable transformation method and heterologous expression of?-glucuronidase in Lentin-ula edodes.J Microbiol Methods2008;72:111—5.
Li DM,Youngs HL,Gold MH.Heterologous expression of
a thermostable manganese peroxidase from Dichomi-
tus squalens in Phanerochaete chrysosporium.Arch Biochem Biophys2001;385:348—56.
Li G,Li RX,Liu QY,Wang Q,Chen M,Li BJ.A highly ef?cient polyethylene glycol-mediated transforma-tion method for mushrooms.FEMS Microbiol Lett 2006;256:203—8.
Lugones LG,Scholtmeijer K,Klootwijk R,Wessels JGH.
Introns are necessary for mRNA accumulation in Schizophyllum commune.Mol Microb1999;32:681—9.Ma AM,Shan LJ,Wang HJ,Du ZP,Xie BJ.Partial charac-terization of a hydrophobin protein Po.HYD1puri?ed from the oyster mushroom Pleurotus ostreatus.World J Microbiol Biotechnol2008;24:501—7.
Moussa TA.Molecular characterization of the phenol oxidase(pox2)gene from the ligninolytic fungus Pleurotus ostreatus.FEMS Microbiol Lett2009;298: 131—42.
Murata H,Sunagawa M,Yamazaki T,Shishido K, Igasaki T.Expression of the auto?uorescent protein, DsRed2,in the recombinants of the ectomycor-rhizal basidiomycete,Suillus grevillei,generated by Agrobacterium-mediated transformation.Mycorrhiza 2006;16:407—12.
Pan XL,Wang JL,Zhang DY.Biosorption of Pb(II)by Pleu-rotus ostreatus immobilized in calcium alginate gel.
Process Biochem2005;40:2799—803.
Pérez G,Pangilinan J,Pisabarro AG,Ramírez L.
T elomere organization in the ligninolytic basid-iomycete Pleurotus ostreatus.Appl Environ Microbiol 2009;75:1427—36.
Sambrook J,Russell DW.Molecular Cloning.3rd ed.New York:Cold Spring Harbor Laboratory,Cold Spring Har-bor;2001.
Scholtmeijer K,W?sten HAB,Springer J,Wessels JG.
Effect of introns and AT-rich sequences on expression of the bacterial hygromycin B resistance gene in the basidiomycete Schizophyllum commune.Appl Environ Microbiol2001;67:481—3.
Soltani J,van Heusden GP,Hooykaas PJ.Agrobacterium-mediated transformation of non-plant organisms.In: Tz?ra T,Citovsky V,editors.Agrobacterium:From Biology to Biotechnology.New York:Springer;2008.
p.649—75.
Sun SJ,Chen DX,Xie BG,Hu FP,Zheng JG.Iso-lation of GPD promoter from Tremella fuciformis and driving expression of EGFP gene.DNA Cell Biol 2009;28:65—70.
Sunagawa M,Magae Y.Transformation of the edible mush-room Pleurotus ostreatus byparticle bombardment.
FEMS Microbiol Lett2001;211:143—6.
Wang S,He J,Cui Z,Li S.Self-formed adaptor PCR:a simple and ef?cient method for chromosome walking.
Appl Environ Microbiol2007;73:5048—51.
Wang J,Guo L,Zhang K,Wu Q,Lin J.Highly ef?cient Agrobacterium-mediated transforma-tion of Volvariella volvacea.Bioresour T echnol 2008;99:8524—7.