基因克隆和表达

Cloning and expression of peroxisomal Ascorbate Peroxidase genefrom wheatYaping Chen,Huazhong Wang,Xiue Wang,Aizhong Cao&Peidu Chen*State Key Laboratory of Crop Genetics and Germplasm Enhancement,Nanjing Agricultural University, Nanjing210095,People’s Republic of China;*Author for correspondence(Phone:+86-25-84396026;E-mail: pdchen@)Accepted24October2005Key words:peroxisomal ascorbate peroxidase,powdery mildew,SSH,wheatAbstractA full-length cDNA encoding wheat peroxisomal ascorbate peroxidase(pAPX)was cloned by Suppression Subtractive Hybridization(SSH)and in silico approach.The cDNA was1027bp in length and contained a complete ORF of876bp,which encodes a protein of292amino acid residues.Its deduced amino acids sequence had84%identity with that of pAPX from barley.The gene was designated as Ta-pAPX.The Ta-pAPX homologous genes were mapped on wheat chromosome7A and7D using Chinese Spring nulli-tetrasomic lines analysis.Northern analysis indicated that,after inoculation by Erysiphe graminis Dc.f.sp. tritici,the expression of Ta-pAPX gene in Yangmai5was enhanced,but its expression in wheat-Haynaldia villosa6VS/6AL translocation lines changed a little.The results implied that Ta-pAPX may be related to susceptibility of wheat to powdery mildew.The complete coding sequence of Ta-pAPX was cloned into an expression vector pET32(a+)and a protein with the same deduced molecular weight(MW)was expressed in E.coli BL21(DE3),which showed ascorbate peroxidase activity.Abbreviations:APX–ascorbate peroxidase;ESTs–expressed sequence tags;IPTG–isopropyl-beta-D-thiogalactopyranoside;MW–molecular weight;ORF–open reading frame;pAPX–peroxisomal ascorbate peroxidase;SSH–Suppression Subtractive Hybridization.IntroductionAscorbate peroxidase(APX),found in higher plants,cyanobacteria,and algae[1],is the key enzyme in degradation hydrogen peroxide.So far, at leastfive APX isoforms have been identified in plants:cytosolic isoforms,mitochondria isoforms, peroxisomal/glyoxysomal isoform and two chlo-roplastie isoforms,one in stroma and the other associated with the thylakoid membranes,all of which catalyze the reaction:2ascorbate peroxidaseþH2O2!2monodehydroascorbateþ2H2OAPXs activity increased in response to a num-ber of stress conditions,such as drought[2],salt [3],high temperature[4]and pathogen infection [5].Relationship between different stress condi-tions and changes of APX activity were observed.Powdery mildew caused by E.graminis DC.f.sp.tritici is one of the most serious diseases of common wheat in China and many other countries.The Triticum aestivum(‘‘Yangmai5’’)–Haynaldia villosa6VS/6AL translocation line carrying powdery mildew resistance gene Pm:21 confers effective resistance to all current powdery mildew races.To investigate the mechanism ofMolecular Biology Reports(2006)33:207–213DOI10.1007/s11033-005-4536-1ÓSpringer2006resistance to powdery mildew in6VS/6AL trans-location line,suppression subtractive hybridiza-tion(SSH)was performed between translocation line carrying Pm21and its recurrent parent Yangmai5to isolate resistance relative genes. Among the ESTs obtained by SSH analysis,a cDNA fragment that encodes a polypeptide homologous to barley peroxisomal ascorbate peroxidase gene did express differentially between the resistant and susceptible lines.In the present paper,we report the cloning and characterization of the full-length wheat peroxisomal APX gene by in silico approach,which is thefirst reported in wheat.The expressions of Ta-pAPX in Esc-herichia coli and pathogen infected leaves were also analyzed.Materials and methodsMaterialsPlant materials:Powdery mildew susceptible common wheat variety‘‘Yangmai5’’and resistant wheat–Haynaldia villosa6VS/6AL translocation line were provided by Cytogenetics Institute, Nanjing Agricultural University and grown in a isolation environment under room temperature for7days,leaves were then harvested at0,6, 12,24,36h after inoculation by E.graminis DC.f.sp.tritici for RNA isolation.Chinese Spring nulli-tetrasomic lines were grown under room temperature for DNA isolation.Bacterial strain and vector:pET32(a+)and BL21(Escherichia coil)were provided by Cytoge-netics Institute,Nanjing Agricultural University.Suppression subtractive hybridization(SSH)Suppression subtractive hybridization was per-formed according to Diachenko et al.[6].Total RNA was extracted by Trizol(GIBCO BRL).Poly (A)+RNA was prepared by Promega oligo(dT) kit.First-strand cDNAs were synthesized using oligo(dT)16as a primer.cDNA of Yangmai5un-inoculated by E.graminis DC.f.sp.tritici was used as Driver,cDNA of6VS/6AL translocation line inoculated for different time was used as Tester. PCR fragments obtained by SSH were cloned into a pGEM-T Easy vector(Promega).The putative clone was sequenced by Bioasia Company.A partial fragment encoding part of Ta-pAPX was obtained according to the BLAST homology searching.Cloning and sequencing of cDNAA full-length cDNA sequence encoding peroxi-somal ascorbate peroxidase(pAPX)was obtained by searching for database and splicing,Primers were then designed according to the sequences. APXF:5¢-TAGGGTCGTCCGCGA TGG-3¢AP XR:5¢-CCCCTTACTTGCTCCTC TT-3¢;RT-PCR screenings were performed using thefirst-strand cDNA of translocation line as templates. PCR were conducted by DNA Thermal Cycler (Perkin-Elmer)in25l l reaction mixture;The amplification reaction was for1cycle of3min at 94°C;33cycles of30s at94°C,40s at58°C; 2min at72°C;finally extended at72°C for7min. The PCR products were reclaimed and cloned into pGEM-T Easy vectors.The DNA sequences were determined based on BLAST program in GenBank. Expression and enzymatic activity of Ta-pAPXin E.coliThe ORF of Ta-pAPX were amplified by PCR and cloned into the pET-32(a+)expression vector. Primers were designed with an EcoRI cloning site at5¢end(5¢-TCGGAATTCATGGCGGCTC C-3¢) and with a NotI cloning site at3¢end(5¢-A GGCGGCCGCACTAGTGATT-3¢).The ampli-fied products were EcoRI/NotI restricted,ligated to EcoRI/NotI restricted pET-32(a+)and trans-formed into the strain BL21(DE3).The correct orientations of the inserts were verified by restric-tion endonuclease analyses.Individual colonies were inoculated into10ml of liquid medium with 100l g/ml of ampicillin,and allowed to continue to grow for about12h before IPTG was added to the liquid medium(1mMfinal concentration).Bac-terial cells were harvested about0.5,1,1.5,2h after IPTG added.Protein was extracted accord-ing to the protocol provided by Invitrogen.SDS-PAGE analysis followed Sambrook et al.[7].For in vitro detection of Ta-pAPX activity,the packed cells were resuspended at0.1of the original culture volume in25mM Tris buffer(pH7.5),containing 5%(w/v)glycerol,1mM EDTA,2mM ascorbic acid and250mM PMSF.After sonication,the soluble proteins were obtained by centrifugation at20812,000Âg for20min.The supernatant,which contained about50l g of protein,was directly loaded onto a native polyaciylamide gel for elec-trophoresis[8].The detection of Ta-pAPX activity in gels was the same as described by Ron Mittler et al.(1993)[9].Chromosome localization of Ta-pAPXPrimers were designed based on the sequence of Ta-pAPX gene:APX1:5¢-GTGATTCGTCAGTT TGTCC-3¢,APX2:5¢-CCCTTACTTGCTCCTC TT-3¢.PCR was performed using the DNA of Chinese Spring nulli-tetrasomic lines as templates. The amplification reaction was for1cycle of3min at94°C,33cycles of30s at94°C,30s at55°C; 2min at72°C,finally extended at72°C for 7min.PCR products were analyzed by30% PAGE gels,which stained by silvery.Assay for APX activity and Northern blotting analysisSoluble leaf protein was extracted by grinding0.5g of fresh leaf tissue and pestle in10ml of100mM sodium phosphate buffer(pH7.0)containing 5mM ascorbate and1mM EDTA,membrane and other cell debris were removed by centrifuge at 12000g for20min(4°C).The supernatant was used for APX activity analysis.Measurement of APX activity was performed as described previ-ously[10].The decrease in A290was measured when ascorbate was oxidized.Total RNAs were extracted from leaves.The equal quantity of total RNA(15l g/lane)was Figure1.Sequence of the Ta-pAPX gene and its deducted amino acid sequence(The sequence underlined is the suggested trans-membrane domain).209fractionated in 1%formaldehyde agarose gel,and blotted to nylon membranes as described by Sam-brook et al.[7].32P-dCTP-labeled 18S rRNA se-quences were used as probe for control,Northern blotting was according to Ausubel et a1.1995[11].ResultsCloning and sequencing of Ta-pAPXBy SSH,a cDNA fragment that express differen-tially between translocation line and Yangmai5was obtained.After sequencing,this fragment was found to have high homology to APX genes according to BLAST homology searching.The full-length cDNA clone was subsequently isolated from translocation line by RT-PCR using in silico approach.The cDNA was 1027bp in length and contained an open reading frame encoding a pre-dicted polypeptide of 292amino acids (Figure 1).Its deduced amino acids sequence had 84%homology to that from the Hordeum vulgare pAPX gene and 79%homology to that from Oryza sativa peroxisome type ascorbate peroxi-dase (Figure 2).Both the characteristicC-terminalFigure 2.Alignment of the deduced amino acid sequence of Ta-pAPX from wheat and the sequence of APX of barley(AB063117.1),rice(AK104490.1)and upland cotton (gb|AAB52954.1|).Black boxes indicate sequence identity;Dark gray boxes indicate sequencesimilarity.Figure 3.Analysis Chinese Spring nulli-tetrasomic lines chromosome localization of Ta-pAPX using.210part (from Phe-244to Lys-291)and the single transmembrance domain (from Thr-260to Tyr-282)as predicted by SOSUI [12]were found and conserved.Therefore,the isolated clone encodes the pAPX ,a basic protein of 31.73kD with a predicted pI of 7.74,and was hence designated as Ta-pAPX .PCR using DNA of Chinese Spring nulli-tetrasomic lines as templates showed that the Ta-pAPX homologous genes were located on wheat chromosome 7A and 7D,respectively.(Figure 3).Expression of Ta-pAPX in E.coli and assay for Ta-pAPX activity in vitroAfter induction with IPTG of different time (0.5,1,1.5,2h),31kD fusion protein,similar to the MW of barley HvAPX ,was successfully expressed in BL21(DE3)(Figure 4),the amount of expressed protein was enhanced when the time induced by IPTG was prolonged.This suggested that the Ta-pAPX was correctly expressed in E.coli .However,fusion protein was not expressed in the PET vector (without Ta-pAPX )alone.When pET 32(a+)–Ta-pAPX was trans-formed into E.coli,Ta-pAPX activity was detected in bacterial extracts by native-PAGE,whereas extracts from culture that contained the pET vec-tor alone (without Ta-pAPX )showed no APX activity (Figure 5).Expression of Ta-pAPXThe activities of APX were measured in order to investigate the change of expression of APX in-duced by E.graminis .As shown in Figure 6,slightly reduced activity of APX in the induced resistant line was observed at about 12h post-infection.And yet,under the same conditions APX activity significantly increased in susceptible lines (significance level 0.01)(Table 1).In addition,there were different APX activities between the analyzed resistant and susceptible line,the resis-tant line held obviously higher APX activity than susceptible line in a long time.NorthernblottingFigure 4.12%SDS-PAGE analysis of the expression of Ta-pAPX protein after IPTG induced.(Lane 1,0.5h;2,1h;3,1.5h;4,2h;M,Mark;CK,pET.).Figure 5.Enzymatic activity of Ta-pAPX expressed in BL21.Line 1pET-32(a+)-Ta-pAPX ;CKpET-32(a+).Figure 6.Changes of the activities of APX in the detached wheat leaves inoculated by E.graminis for 0,6,12,24,48h respectively.211analysis confirmed the above results(Figure7). Ta-pAPX expression of the inoculated susceptible line increased at about12h post-infection but decreased at about24h.Ta-pAPX expression of resistant line decreased slightly at about12h post-infection and maintained a relatively stable level at the subsequent time.DiscussionSSH technique is applicable to many molecular genetic and positional cloning studies for the identification of differentially expressed genes.In this research,the resistant6VS/6AL translocation line,used as tester in SSH,was inoculated by E. graminis for different time and the susceptible ‘‘Yangmai5’’was not inoculated.Some differen-tially expressed ESTs was obtained after SSH, Among them,a cDNA,which contains an ORF encoding a polypeptide homologous to pAPX from other species,was isolated from wheat.It contains a putative C-terminal transmembrane domain that may be anchored to a specific mem-brane(e.g.the plasma membranes or organelle membranes).Since it was found by Foyer and Halliwell in 1976,ascorbate peroxidase has been studied by many researchers on various aspects.Up to now, at leastfive APX isoforms have been identified in plants.Many studies focus on cytosolic isoforms, and a few on peroxisomal isoforms.Recently, pAPX(or gAPX)was cloned from cotton[13],A. thaliana[14],spinach[15],barley[6]and wheat in our study.Pathogen infection can result in oxidative stress in which more H2O2is generated in the microbody matrix and may readily diffuse into the cytosol. APX,which binds to the outside of the mem-brance,was considered to play an important role in decomposing H2O2to protect cell from oxida-tive damage.It was shown previously that APX accumulated clearly in wheat susceptible line[16]. In our results,expression of Ta-pAPX and activity of APX also increased in susceptible variety Yangmai5.Constitutive expression of pAPX and activity of APX in resistance line was higher than that of susceptible line,which indicated that resistance line maybe preformed sufficiently to eliminate ROI (reactive Oxygen intermediate).It has been re-ported recently that during the interaction of plants and pathogens,the expression of ROI detoxifying enzymes such as APX is suppressed and this sup-pression plays a key role in elevation cellular ROI levels,thereby inducing the initiation of PCD and other defenses during pathogen attack[17].These may explain why expression of Ta-pAPX and activity of APX have no change or even decrease in translocation line after inoculation.It should be investigated further that how much ROI should be hold or eliminated to keep cellular balance.Ta-pAPX was mapped onto the homologous group7,7A and7D,by wheat nulli-tetrasomic analysis and correctly expressed in bacterial cells. Further detailed analyses on the over-expression of the Ta-pAPX in common wheat are in pro-gressing in our laboratory.Table1.APX activity analysis of resistant and susceptible lines after infection of E.graminisHours after infection Resistant line Significance of difference Susceptible line Significance of difference00.9898a0.8600a60.9795a0.9637b120.9758a0.9535b240.9688a0.9488b480.9670a0.9442bFigure7.Northern blot analysis Ta-pAPX and18SrRNA.Line1,2,3Yangmai5was inoculated by E.graminis for0,12,24h;4,5,6,7Translocation lines was inoculated by E.graminis for0,12,24,48h.Hybridization of18SrRNA used as a control isshown in the bottom panel.212AcknowledgementsThis research was supported by grants from the Chinese High Tech Program of China(2001AA 222152),the Chinese High Tech Program of China (2004AA222140),the National Natural Science Foundation of China(30270828),and Program for Changjiang Scholars and Innovative Research Team in University.References1.Asada K(1992)Physiol.Plant.85:235–241.2.Mittler R&Zilinskas B(1994)Plant J.5:397–405.3.Lopez F,Vansuyt G,Case-Delbart F&Fourcroy P(1996)Physiol.Plant.97:13–20.4.Shi WM,Muramoto Y,Ueda A&Takabe T(2001)Gene273:23–27.5.Mittler R,Feng XQ&Cohen M(1998)Plant Cell10:461–473.6.Diachenko L,Lau YC&Campbell AP(1996)Proc.Natl.A93:6025–6030.7.Sambrook J,Fritsch EF&Maniatis T2nd ed.,New York:Cold Spring Harbor Laboratory press.8.Charles R,Caldwell J,Tyrano F&McMahon BM(1998)Planta.204:120–126.9.Mittler R&Zilinskas AB(1993)Anal.Biochem.212:540–546.10.XU LL&YE MB(1989)J.Nanjing Agric.Univ.12:82–83.11.Ausubel FM,Brent R,Kingston RE,Moore DD,SeidinanJG,Smith JA&Struchl K(1995)Short Protocol in Molecular Biology(3rd ed.).John Wiley&Sons,Media, Pennsylvania,USA.12.http://sosui.proteome.bio.tuat.ac.jp/sosuiframe.html.13.Bunkelmann JR&Trelease RN(1996)Plant Physiol.110:589–598.14.Zhong H,Wang J,Nickel U,Randy DA&MG HowardPlant Mol.Biol.34:967–971.15.Ishikawa T,Yoshmura K,Sakai K,Tamoi M,Takeda T&Shigeoka S(1998)Plant Cell Physiol.39:23–34.16.Chen LF,Ye MB,Chen YX&Xu LL(1997)Acta Phy-topathol.Sinica27(2):113–118.17.Ron M,Elza HH,Bjorn LO,Wim VC,Hilde W,Dirk I&Brian EE(1999)A24:14165–14170.213。