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Molecular cloning and expression analysis of a zeta-class glutathione S-transferase gene in sugarcan


African Journal of Biotechnology Vol. 10(39), pp. 7567-7576, 27 July, 2011 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 ? 2011 Academic Journals

Full Length Research Paper

Molecular cloning and expression analysis of a zeta-class glutathione S-transferase gene in sugarcane
Youxiong Que1, Jinxian Liu1,2, Liping Xu1*, Jinrong Guo1, Jingsheng Xu1 and Rukai Chen1
1

Key Lab of Sugarcane Genetic Improvement, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China. 2 Department of Tea Science and Biology, Wuyi University, Wuyishan 354300, Fujian Province, China.
Accepted 10 May, 2011

Glutathione S-transferases (GSTs) play an important role in stress tolerance in plants. This is the first report of cloning and characterization of a zeta-class GST gene in sugarcane (GenBank Accession number: GQ246461). Sequence analysis showed that the cDNA sequence of Sc-GST gene was 829 bp, contained a 621 bp open reading frame (ORF), the 5’ untranslated region (UTR) of 65 bp and 3’UTR of 143 bp, plus the typical AATAA region and poly (A) tail. It encoded the 206 amino acid residues with a molecular mass of 23.1 KD and isoelectric point of 6.10. Protein domain prediction and multiple sequence alignment demonstrated that the conserved domain in Sc-GST at N-terminus was SSCXXRXRIA, while that at C-terminus was quebec platelet disorder (QPD), both of which were specific for zeta-type GST in eukaryotes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and enzyme activity assay indicated that the prokaryotic expression product was a fusion protein with a molecular weight of about 30 KD, which also possessed GST enzyme activity. It was revealed in real-time quantitative polymerase chain reaction (qPCR) analysis that the Sc-GST gene had induced expression under H2O2 and Ustilago scitaminea stresses, while it was inhibited and then induced by salicylic acid (SA) stress, suggesting that it is a type of stress-tolerant gene playing a certain role in sugarcane resistance response. Key words: Saccharum officinarum, glutathione S-transferase, homology, prokaryotic expression, real-time quantitative PCR. INTRODUCTION Glutathione S-transferases (GSTs, EC 2.5.1.18) are a group of multifunctional enzymes, which play a key role in cellular detoxification. They can catalyze the conjugation of the tripeptide glutathione (GSH; γ-Glu–Cys–Gly) into a variety of hydrophobic, electrophilic, and usually cytotoxic substrates, and thus reduce glutathione (GSH) and protect the cellular macromolecules from attack of these toxic compounds. Since the first GST gene was cloned from Zea mays, a number of GST genes or its homologous sequences had been identified and annotated from a variety of plants (Shimabukuro et al., 1970; Basantani and Srivastava, 2007; Chi et al., 2010). According to their protein homology and genomic structure, the GSTs in plants were classified into 8 types, including the phi, tau, theta, zeta, lambda, dehydroascorbate reductase (DHAR) and tetrachlorohydroquinone dehalogenase (TCHQD) which were soluble (cytoplasmic) plus the microsomal one (Dixon et al., 2002a; Moons, 2005; Basantani and Srivastava, 2007). Among them, two classes, phi and tau, were specific to plants and belonged to the most numerous and the abundance of these enzymes; three smaller classes, zeta and theta (also found in animals and fungi) and lambda were widely distributed in eukaryotes, while both types of DHAR and TCHQD were only recently

*Corresponding author. E-mail: queyouxiong@yahoo.com.cn or xlpmail@yahoo.com.cn. Tel: 0086-591-83772604. Fax: 0086-591-83851742. Abbreviations: GST, Glutathione S-transferase; IPTG, isopropyl-β- D-thiogalactopyranoside; DHAR, dehydroascorbate reductase; TCHQD, tetrachlorohydroquinone dehalogenase; HR, hypersensitive response; SAR, systemic acquired resistance; QPD, quebec platelet disorder; qPCR, quantitative polymerase chain reaction; SA, salicylic acid.

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reported in Arabidopsis, rice and soybean (Dixon et al., 2002a). Besides, the GST gene was a kind of multi-gene family, and so far it had been found that, Arabidopsis thaliana had 48 GST genes, Glycinemax had 25 GST genes, Zea mays had 42 GST genes and Oryza sativa had 59 GST genes, respectively (Dixon et al., 2002b; Moons, 2003; Soranzo et al., 2004; Chi et al., 2010). Previous studies have demonstrated that plant GSTs play an important role in various stress responses. Several researches revealed that, the expression of GSTs in plants was highly responsive to biotic and abiotic stress and a wide variety of stress-associated chemicals, including those arising from pathogen attack, oxidative stress and heavy-metal toxicity, such as microbes, plants, insects and vertebrates and other synthetic and natural auxins, salicylic acid, methyl jasmonate, abscisic acid and H2O2 from the toxic effects of electrophilic compounds (Dixon et al., 2002a; 2002b; Moons., 2005). Tobacco seedlings which over-expressed a tobacco tau class GST gene were found to be more tolerant to chilling and osmotic stress than wild-type plants (Roxas et al., 1997), while a GST gene discovered in black grass participated in oxidative stress tolerance (Cummins et al., 1999). A tomato tau class GST gene was reported to confer resistance to oxidative stress (Kampranis et al., 2000). Cho et al. (2007) revealed, the expression of a phi class GST gene from O. sativa appeared to play a role in the conjugation of herbicide and GPOX activity. Expression profiling of the GST family in sorghum and other higher plants indicated that this gene family played a role not only in stress-related biological processes but also in the sugar-signalling pathway (Chi et al., 2010). All the earlier mentioned researches represent an era of attempting to assign functions for GSTs in endogenous metabolism and development. Sugarcane is an important commercial or cash crop of tropical and sub-tropical regions. Kurama et al. (2004) identified three expressed sequence tag (EST) clusters homologous to GST consensus sequence in sugarcane EST genome through the sequencing of ESTs of a sugarcane database derived from libraries induced and not induced by pathogens. A comparison of the deduced amino acid sequence of these three sugarcane EST clusters with cloned GST genes in GenBank database revealed a high degree of similarity with the phi class GST gene. Expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR) showed that all these EST expressed in sugarcane leaves inoculated with Puccinia melanocephala. To our knowledge, this is the only report about GST gene identification and characterization in sugarcane. Until now, there is still no report about the molecular cloning and characterization of the full-length GST gene in sugarcane, which should be helpful in knowing the stress tolerance mechanism and improvement of sugarcane varieties by molecular breeding approach. The objective of this study was to clone and characterize one full-length GST gene in

sugarcane.
MATERIALS AND METHODS Sugarcane variety FN 22, sugarcane stem full-length cDNA library, Escherichia coli DH5α and BL21 (DE3), and the prokaryotic expression vector were provided by Key Laboratory of Sugarcane Genetic Improvement, Ministry of Agriculture, People’s Republic of China. Restriction enzyme EcoR I, Sal I, T4 DNA ligase, Taq enzyme, DNA and protein molecular marker, SYBR?Premix Ex TaqTM were purchased from TaKaRa (Japan); Isopropyl-βD-thiogalactopyranoside (IPTG) and Reverse Transcription kit were from Promega Corporation (USA); and the instrument used in the real-time qPCR analysis was the ABI PRISM7500 real-time PCR system. Strong and evenly growing sugarcane stalks were soaked and sterilized in water with 0.1% potassium permanganate for 24 h and then planted into autoclaved fine sand in 10 trays, with 40 shoots in each tray. These trays were then placed into the illumination incubator where each day they received daylight for 12 h at 29°C and nightlight for 12 h at 26° with a light intensity of 440 mol m-2 s-1. C When growing to the height of 15 cm, they were transferred into the greenhouse for cultivation and sprayed with Hongland nutrient solution. In the six leaves period, evenly growing stalks were selected and then the sand was removed. After rehydration for 2 days, the stalks were cultivated in Hongland nutrient solution for one week and then treated as follows: In group one, 5 m mol·L-1 SA and 10 mmol·L-1 H2O2 were sprayed onto the leaves, respectively (Que et al., 2009a). In group two, spore suspension with concentration of 5 × 106 spores ml-1 was inoculated through stabling bud tissues (Que et al., 2009a). The sampling times were 0, 12, 24, 36, 48, 60 and 72 h for SA and H2O2 treatment and 0, 12, 24, 36, 48 and 60 h for Ustilago scitaminea treatment. All the samples collected were immediately fixed in liquid nitrogen, and stored in a refrigerator at -85°C until RNA extraction.

Obtaining and sequence analysis of full-length cDNA of Sc-GST gene The large-scale sequencing and bioinformatics analysis were conducted for the full-length cDNA library of sugarcane stem (Private bulletin). The library clones which contained EST highly homologous to GST gene in National Center for Biotechnology Information (NCBI) database were selected and sequenced completely to obtain sugarcane full-length GST gene. For the full-length cDNA sequence of GST gene, open reading frame (ORF) was predicted with the online tool ORF Finder from NCBI (http://www.ncbi.nlm.nih. gov/gorf/gorf.html). The BLAST program in NCBI was used for the homology analysis. Besides, the ProtParam (http://cn.expasy.org/tools /protparam.html) was applied to analyze the basic properties of the encoding protein. Multiple sequence alignment and phylogenetic analysis were performed with DNAMAN software (version 6.0) and MEGA3.1. InterProScan (http://ebi.ac.uk/InterProScan) and SMAR (http://smart.emblheidelberg.de) were used to analyze the putative domain of the encoding protein of GST gene. The gene sub-cellular localization was carried out with SubLoc V1.0 (http://www.bioinfo.tsinghua. edu.cn/SubLoc/) and the signal peptide prediction was performed with SignalP3.0 (Error! Hyperlink reference not valid.). SOPMA was adopted for the secondary protein structure prediction (http://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.pl?page=npsa_sopma. html) and the analysis of the repetitive sequence of the amino acid was performed with the REP searcher (http://www.embl-heidelberg. de/~andrade/papers/rep/search.html).

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Construction of prokaryotic expression vector of Sc-GST gene pET29a (+) was used as the prokaryotic expression vector and the primer pairs amplifying the ORF of Sc-GST were designed. The primer sequences were as follows, of which the underlined parts were the restriction endonuclease site of EcoR I and Sal I, respectively: Sc-GST-F: 5’-TTGGATCCATGGCAGAGGCGGAG-3′; Sc-GST-R: 5′-TTGTCGACGGAAGAGGAAGGGG-3′. The primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services CO., Ltd. With plasmid DNA extracted from the library clone containing Sc-GST gene as the template, the PCR amplification was carried out in a 50 ?l PCR reaction. It was composed of 5?l 10 × PCR buffer, 4 ?l 2.5 mmol·L-1 deoxynucleotide triphosphates (dNTPs); 2 ?l 10 ?mol·L-1 of forward and reserves primers, respectively; 2 ?l plasmid DNA; 0.25 ?l Taq enzyme (5 U·?l); ddH2O was added as the supplement. PCR amplification program was pre- denatured for 5 min at 94° C; denaturation for 30 s at 94° annealing for 30 s at 55° and C, C extension for 1 min at 72° 30 cycles; followed by final extension C, for 10 min at 72°C. When the reaction finished, 1% agarose gel electrophoresis was performed and the target PCR product was recovered. After receiving the double digestion with EcoR I and Sal I, the obtained ORF sequence was ligated by T4 DNA ligase into pET29a (+) which had also executed the double digestion with EcoR I and Sal I. The recombinant was then transferred into E. coli DH5α. The plasmid DNA of several clones selected by blue/white colony screening was extracted. A positive clone validated by PCR and double digestion was just the prokaryotic expression vector termed the pET29a-ScGST (Que et al., 2009b). SDS-PAGE analysis of prokaryotic expression product and enzyme activity assay pET29a-ScGST and empty pET29a (+) were both transformed into E. coli BL21 (DE3). The single colony was inoculated into the LB medium containing the ampicillin (100 ?g·ml-1) for overnight shake culture at 150 r/min at 37°C. In the following day, 1% of the medium was inoculated into the LB medium containing 100 ?g·ml-1 ampicillin and shake cultured. When OD600 reached 0.4 to 0.6, 1 ml of LB liquid medium was collected as the control, and the remaining medium was added with IPTG with the final concentration 1 mmol·L-1. The LB medium with pET29a-ScGST (BL21) was induced for 2, 4 and 6 h, respectively at 37° and 1 ml medium was collected at each time C, point; the LB medium with empty pET29a (+) (BL21) was induced in IPTG for 6 h and 1 ml medium was collected. The medium collected was used for the SDS-PAGE with 25 ?l sample loading and also for GST enzyme activity assay according to Habig et al. (1974). When the electrophoresis finished, the gel was colored with Coomassie brilliant blue and imaged (Que et al., 2009b). Expression profile of Sc-GST gene under various exogenous stresses 25S rRNA was selected as the control gene in the real-time qPCR analysis (Que et al., 2009c). According to the sequence of Sc-GST and 25S rRNA (BQ536525), two pairs of real-time quantitative polymerase chain reaction (qPCR) primers were designed with GenScript Online PCR Primers Design Tool (http://www.genscript. com/cgi-bin/tools/primer_genscript.cgi). The forward and reverse primers of Sc-GST gene were 5′-CTCGACTTCGAGATCATCCC-3′ and 5′-AGGTAGAGCA GGTAGGACGC-3′, respectively. For the 25S rRNA, they were 5′-GCAG CCAAGCGTTCATAGC-3′ and 5′-CCTATTGGTGGGTGAACAATCC-3′, respectively. The 20 ?l reverse transcription system contained 4 ?l 25 mmol·L-1 MgCl2, 2 ?l 5 × RT reaction buffer, 2 ?l 10 mmol·L-1 dNTPs, 0.5 ?l RNase inhibitor, 0.5 ?l Random primers (0.5 ?g), 15 U AMV reverse

transcriptase and 4 ?l total RNA (1 ?g) as the template and RNase free H2O as the supplement. Reverse transcription program was as follows: it was firstly kept at room temperature for 10 min, then incubated at 42°C for 15 min and 95° for 5 min, and then kept at 0 C to 5°C for 5 min. In real-time qPCR amplification, the RT-PCR product was used as the template and the total volume of the reaction system was 25 ?l, including SYBR Primix Ex TaqTM (2×), 12.5 ?l; Rox Reference DYE II, 0.5 ?l; forward and reverse primers of 10 ?mol·L-1, 0.5 ?l each; cDNA template, 2.5 ?l; and sterile water, 8.5 ?l. Three replicas were set for each sample. The PCR reaction conditions were pre-denatured at 95° for 10 s, and then 40 cycles C with 94° for 5 s and 60° for 25 s. When the reaction was C C completed, the melting curve was analyzed. The method of 2- CT was adopted to analyze the real-time qPCR results (Livak and Schmittgen, 2001; Que et al., 2009d).

RESULTS Cloning and sequence analysis of Sc-GST gene Through the large-scale sequencing and bioinformatics analysis, one EST, which had 87% homology to a GST gene from Z. mays (EU970141), was obtained from sugarcane stem cDNA library. The library clone containing this EST was then sequenced completely and a full-length cDNA sequence of GST gene in sugarcane was obtained and termed as Sc-GST. It was submitted to the NCBI with the GenBank Accession number of GQ246461. The Sc-GST had a full length of 829 bp, contained a ORF of 621 bp, 5’ UTR (untranslated region) of 65 bp and 3’UTR of 143 bp, and the typical AATAA and poly (A) tail could also be found in the 3’UTR (Figure 1). The primary structure of the protein predicted was shown in Figure 1. The ORF of Sc-GST gene encoded 206 AA residues with the molecular weight of encoding protein of 23.1 KD and isoelectric point of 6.10. Protein domain prediction showed that it had the conserved domain both at the N- and the C-terminus (Figure 2). Sequence homology analysis of Sc-GST gene The blastn alignment analysis indicated that, the homology between the Sc-GST gene and the zeta-type GST gene in Z. mays (EU970141) was 84% (671/796). It also showed that the homology of the Sc-GST encoding protein with that of the zeta-type GSTs in Z. mays (ACG42259), O. sativa (ABI17930) and Brassica napus (AAO60040) were 87% (196/224), 71% (161/225) and 54% (122/222), respectively. Therefore, it could be initially determined that the Sc-GST gene obtained in this study was of zeta type. Furthermore, through the Conserved Domain Database (CDD) search of the protein conserved domain in NCBI, it could be found that the GST protein encoded by the obtained Sc-GST had the typical characteristics of the zeta-type GST structure both at the C- and N-terminus, which further determined that this Sc-GST was of zeta type (Figure 3). The zeta-type GSTs from different species were

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Figure 1. Nucleotide sequence of Sc-GST gene and primary structure of Sc-GST protein. Capital and small letters represent amino acid and nucleotide, respectively; *represents stop codon; the underline shows the polyadenylation signal AATAA.

Hits only found by BLAST are indicated by for hits in the schnipsel database. Disordered regions detected by DisEMBL ( ).

Figure 2. Putative domain of Sc-GST protein.

searched and downloaded from the NCBI, and DNAMAN was used for the multiple sequence alignment towards the amino acid of these zeta-type GSTs. The results of multiple sequence alignment were shown in Figure 4, in

which the shaded part was the conserved region. Multiple sequence alignment showed that the N-terminus of all proteins was highly conserved with the typical SSCXXRXRIA domain, which was specific in zeta type

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Figure 3. Protein domain analysis of Sc-GST protein.

GST in eukaryotes. These proteins also had the quebec platelet disorder (QPD) conserved region at the C-terminus, which again indicated that the Sc-GST was of zeta type. Using the MEGA3.1 software, the phylogenetic tree of the Sc-GST and the zeta-type GSTs from other species was constructed. Figure 5 showed that these zeta-type GST proteins consisted of two branches, one from animals and the other from plants. In the branch of plant, the Sc-GST and the GST of the Z. mays were classified into one group while that from A. thaliana and B. napus were classified into the other group, showing that the homological evolutionary relationship between the two within one group was close. Besides, it could be found that the relationship between the zeta-type GST of Gramineae and Cruciferae in the category of plant was not that close, maybe they evolved from the same ancestor along different pathways. Construction of the expression vector Sc-GST gene prokaryotic

expression products are shown in Figure 7. It showed that the Sc-GST protein began to express in the second hour with the weight of 30 KD, which was basically consistent with the deduced molecular weight of the fusion protein of 30.1 KD, including Sc-GST encoding protein of 23.1 KD plus the His ? Tag peptides of 7 KD, while no target protein was expressed in the empty vector. Enzyme activity assay indicated that the prokaryotic expression product possessed the GST enzyme activity. From the aforementioned, it indicated again that this prokaryotic expression vector was successfully constructed, without wrong coding or reading-frame shift of the amino acid sequence, and Sc-GST gene had the successful expression in the BL21 (DE3). Expression profile of Sc-GST gene under different kinds of exogenous stresses Real-time PCR was used to examine the expression profile of Sc-GST gene under various exogenous stresses. Figure 8 shows that the expression profile of Sc-GST gene varied with different types of stresses. Under the stress of SA, the expression of Sc-GST gene was firstly inhibited, which lasted at the time points of 12 and 24 h, and the expression of Sc-GST gene was induced but not obviously at the time points of 36, 48 and 60 h with the expression value almost equal to that at 0 h. At the time point of 72 h, the expression of this gene was inhibited again. Under the H2O2 stress, the expression of Sc-GST gene was induced during the whole period with the "increasing-declining-increasing" trend. At the time point of 12 h, the expression was 1.7 times that of the control at 0 h, and decreased to be equal to that of the control at the time point of 24 h. It was up-regulated gradually after the time point of 36 h and reached the peak at the 72 h time point which was 5.6 times of that of control at 0 h. After the U. scitaminea inoculation, the Sc-GST gene expression in sugarcane was constantly induced, and the induction was rather strong at the time point of 12 h with the expression 6.9 times that of control

After the PCR product from the ORF amplification of ScGST gene had been recovered, it received the double digestion with BamH I-Sal I. The target fragment was then recovered to ligate by T4 DNA ligase into the pET29a (+) which also received the double digestion by BamH I and Sal I, and the positive recombinant pET29a-ScGST was then transformed into the E. coil DH5a. The fragment with the same size as that of the target could be obtained from pET29a-ScGST, through the identification with PCR amplification and BamH I + Sal I digestion. The results of the agarose gel electrophoresis are shown in Figure 6, indicating the successful construction of the prokaryotic expression vector pET29a-ScGST. SDS-PAGE analysis of the prokaryotic expression product and enzyme activity assay The SDS-PAGE electrophoresis results of the prokaryotic

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M.musculus O.sativa R.norvegicus Sc-GST Z.mays A.thaliana B.napus D.rerio H.sapiens Consensus M.musculus O.sativa R.norvegicus Sc-GST Z.mays A.thaliana B.napus D.rerio H.sapiens Consensus M.musculus O.sativa R.norvegicus Sc-GST Z.mays A.thaliana B.napus D.rerio H.sapiens Consensus M.musculus O.sativa R.norvegicus Sc-GST Z.mays A.thaliana B.napus D.rerio H.sapiens Consensus M.musculus O.sativa R.norvegicus Sc-GST Z.mays A.thaliana B.napus D.rerio H.sapiens Consensus

. .. . . .. . . .. . . .. M QA G . .. . . .K P IL Y S YF R S SC S W RV R IA L A LK G I DY E I VP I NL I M AS S G SP E A RQ T H GE I AG A A AP E R .R L KL Y S FW R S SC S Y RV R IA L S LK G L DY E Y KP I NL L . .. . . .. . . .. . . .. M QA G . .. . . .K P VL Y S YF R S SC S W RV R IA L A LK G I DY E I VP I NL I . .. . . .. . . .. . . .. M AE A E AT T G .R L RL Y S YW R S SC S H RA R IA L N LK S V DY E Y KA V NL L . .. . . .. . . .. . . .. M AE A E AT V G .R L ML Y S YW R S SC S H RA R IA L N LK G V DY E Y KA V NL L . .. . . .. . . .. . . .. M AN S G EE K . .. L KL Y S YW R S SC A H RV R IA L A LK G L DY E Y IP V NL L . .. . . .. . . .. . . .. M AN L G EE K K EK L KL Y S YW R S SC A H RV R IA L S LK G L EY D Y IP V NL L . .. . . .. . . .. . . .. M SA T V AR L I .K P VL Y G YY R S SC S W RV R IA F A LK G I EY E Q KP I NL I . .. . . .. . . .. . . .. M QA G . .. . . .K P IL Y S YF R S SC S W RV R IA L A LK G I DY E T VP I NL I ly r s sc r r ia lk y nl K DG G Q QF T E EF Q T LN P MK Q V PA L K ID G .I T I VQ S L AI M E YL E ET R P IP R L LP Q D PQ K RA I A .. N E QS H P EF E K LN P MK Y V PA L V DG D DT V V VD S F AI L L YL E DT Y P QH P L LP Q D PK M KA L K DG G Q QF S E EF Q T LN P MK Q V PA L K ID G .I T I GQ S L AI L E YL E ET R P IP R L LP Q D PQ K RA I K .. G E QS D P EF V K LN P MK F V PA L V DG D .R V I GD S Y AI A L YL E DK Y P EP P L LP Q D LQ K KA L K .. G E QS D P EF V K LN P MK F V PA L V DG S .S V I GD S Y AI T L YL E DK Y P EP P L LP Q D LQ K KA L K .. G D QF D S DF K K IN P MG T V PA L V DG D .V V I ND S F AI I M YL D EK Y P EP P L LP R D LH K RA V K .. G E QS D P DF K K IN P MG T V PA L V DG D .V V I SD S L AI V M YL D EK Y P EP P L LP P D LH K RA V K DG G Q QL T D QF K A IN P MQ Q V PA V S ID G .I T L SQ S L AI I Q YI E ET R P EP R L LP A D PM Q RA H K DG G Q QF S K DF Q A LN P MK Q V PT L K ID G .I T I HQ S . .. . . .. . .. . . .. . . .. . . .. . .. . q f npm vp s V RM I S DL I A SG I Q PL Q NL S V LK Q V GQ E N. . . .Q M Q WA Q K VI T SG F N AL E K .. I L QS T AG K N IQ I A SI V G SS I Q PL Q NN S V LD F I EE K LD S Q EK V N WI Q Y HL N RG F T AL E K .. M L KG C TT T V RM I S DL I A SG I Q PL Q NL S V LK Q V GQ E N. . . .Q M P WA Q K AI T SG F N AL E K .. I L QS T AG K N HQ . . .. . . .. . . .. . .. . . .R F I DQ K VG A G ES V L WT Q Q QI E RG F T AI E N LI Q L KG C AG K N HQ I A SI V A SG I Q PL H NL T V LR F I DQ K VG A G ES V L WT Q Q QI E RG F T AI E N LI Q L KG C AG K N YQ A M SI V L SG I Q PH Q NL A V IR Y I EE K IN V E EK T A WV N N AI T KG F T AL E K .. L L VN C AG K N FQ A A SI V L SG I Q PH Q NL G V IK F I EE K IN S E EK T A WV T N AI T KG F T AL E K .. L L VS C AG K V RI I C DI I A SG I Q PL Q NL Y V LQ K I GE D .. . . .K V Q WA Q H FI N RG F Q AL E P .. V L KE T AG K . .. . . .. . . .. . . .. . NL S V LK Q V GE E M. . . .Q L T WA Q N AI T CG F N AL E Q .. I L QS T AG I w gf a e l Y CV G D EV S M AD V C LV P QV A N .A E R FK V DL S P YP T I SH I N KE L LA L E VF Q V SH P R RQ P DT P Y AT G D EI Q L GD L F LE P QI Y G GI K R FG I DM T N YP T L AR L H EA Y ME H P AF Q A AL P E RQ P DA P Y CV G D EV S M AD V C LA P QV A N .A E R FK V DL S P YP T I SH I N KA L LA L E AF Q V SH P C RQ P DT P Y AT G D EV Q L AD V F LA P QI Y A AI E R TK I DM S N YP T L SR L H SE Y MA H P AF V A AL P G RQ P DA P Y AT G D EV Q L AD V F LA P QI Y A AI E R TK I DM S N YP T L AR L H SE Y MS H P AF E A AL P G KQ P DA P H AT G D EI Y L AD L F LA P QI H G AI N R FQ I NM E P YP T L AK C Y ES Y NE L P AF Q N AL P E KQ P DA P H AT G D EV Y L AD L F LA P QI Y G AI N R FQ I NM E P YP T L AK C Y ES Y KD L P AF Q N AA P E KQ P DA P Y CV G D EI S M AD I C LV P QV Y N .A D R FK V DM T Q YP T I RR L N QT L VE I E AF K A SH P S RQ P DT P Y CV G D EV T M AD L C LV P QV A N .A E R FK V DL T P YP T I SS I N KR L LV L E AF Q V SH P C RQ P DT P gde d l pq r yp t f p qpd p A EL R S SP E A EL R S SS . S SS . S ST I A ST S D DL R T EL R

39 59 39 44 44 42 45 44 39

98 117 98 101 101 99 102 103 72

152 175 152 143 161 157 160 156 110

211 235 211 203 221 217 220 215 169

215 239 215 206 224 221 224 219 173

Figure 4. Multiple sequence alignment of zeta class GST proteins isolated from different species. The big frame shows the conservative domain of zeta GST; the small frame shows the QPD domain. M. musclus (NP_034493); O.sativa (ABA92395); Rattus norvegicus (NM_001109445); Z. mays (ACG42259); A. thaliana (AAO60039); B. napus (AAO60040); Danio rerio,(NM_001002481); Homo sapiens (NM_145871).

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99 100 86 46 100

Rattus norvegicus (NM 001109445) GSTZ Mus musculus (NP 034493) GSTZ Homo sapiens (NM 145871) GSTZ Sus scrofa (XP 001926158) GSTZ Danio rerio (NM 001002481) GSTZ Anopheles gambiae str (EAA08171) GSTZ

100

Arabidopsis thaliana (AAO60039) GSTZ Brassica napus (AAO60040) GSTZ Triticum aestivum (AAD09190) GSTZ

63 65 100

Oryza sativa (ABA92395) GSTZ Sc-GST Zea mays (ACG42259) GSTZ

0.1

Figure 5. Phylogenetic tree of zeta class GST isolated from different species.

A

B

Figure 6. Validation of pET29a-ScGST by PCR and double digestion with BamHⅠ+SalⅠ. A: M, DL2000; 1, negative control; 2, PCR production. B: M, DL2000; 1, digested with BamHⅠ+SalⅠ.

at 0 h. Although the expression was still induced during the following time points, it began to decrease gradually. Finally, at the time point of 60 h, the expression was up-regulated again, reaching 5.7 times that of the control. Based on the real-time qPCR analysis, it could be found that the Sc-GST had the induced expression under the stress of H2O2 and U. scitaminea, while it was inhibited

and then induced under the stress of SA, which was in accordance with the previous studies, indicating that the biotic and abiotic stress could induce the expression of GST (Dixon et al., 2002a; Dean et al., 2005; Marrs, 1996; Gonneau et al., 1998). From the aforementioned, it was inferred that the Sc-GST gene might play a certain role in the mechanism of sugarcane detoxification and thus

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30 KD

Figure 7. The expression of pET29a-ScGST in BL21. M, protein maker; 1, BL21 without induction; 2, pET29a (+) induction for 6 h; 4, pET29a-ScGSTwithout induction; 5, 6 and 7, pET29a-ScGSTinduction for 2, 4 and 6 h, respectively.

disease resistance. DISCUSSION To date, GSTs were found to diverge significantly in terms of overall sequence, nucleotide or amino acid, even within a certain class. Within the phi and tau classes, the sequence identity in both classes could be as low as 40% and the identity between the two classes was even less than 25% in plants. However, as demonstrated by the crystallization and X-ray diffraction of GST proteins from bacterial, plant and animal, all GSTs characterized so far displayed a striking level of structural conservation (Dixon et al., 2002b). It is noteworthy that, the structures of all known GSTs exhibit a two-domain fold, of which one was a conserved N-terminal domain for glutathione binding and thioredoxin folding and the other was even more divergent C-terminal four-helix bundle fold domain containing the determinants for second-substrate specificity (Sheehan et al., 2001). DNA sequences corresponding to zeta-class GSTs had also been described in mammals, plants and Caenorhabditis elegans (Board et al., 1997). In the present study, a full-length GST gene was obtained from the sugarcane stem cDNA library, and was termed as Sc-GST, with GenBank Accession number GQ246461. The encoding protein of this gene had 87% homology with the zeta-type GST protein in Z. mays (ACG42259). In comparison with the zeta-type GST genes cloned in Z. mays (ACG42259), O. sativa (ABI17930) and B. napus (AAO60040), the homologies of the putative amino acid sequences were relatively high, indicating that the GST gene was rather conservative

among these species. Protein domain analysis showed that, the structure of this Sc-GST encoding protein had the characteristics of that of the zeta-type GST, the typical SSCXXRXRIA domain at its N-terminus and the QPD region at its C-terminus, which was specific in zeta-type GST. From the aforementioned, it indicated that the Sc-GST gene obtained in this study belongs to the zeta class type. Despite the numerous associations of GSTs with stress responses and even much greater progress that has been made in the function survey of GSTs in a range of plants recently, the function of GSTs enzymes in sugarcane remains elusive. Whereas, plant GSTs had well described a variety of functions, which included defending the invasion of the exogenous and the endogenous toxin (Edward and Dixon, 2000; Dixon et al., 2002a; Moons, 2003), acting as the binding protein or the ligand (Zettl et al., 1994; Gonneau et al., 1998), protecting the tissue from injury (Berhane et al., 1994; Roxas et al., 1997; Cummins et al., 1999; Dixon et al., 2002a), regulating the stable redox status and the programmed aging of cell (Kampranis et al., 2000; Moons, 2005) as well as acting as the stress signaling protein (Loyall et al. 2000) or catalyzing the isomerization reaction (Dixon et al., 2000). In plants, the expression of most GST genes was constitutive, while that of some were tissue-specific and could also be regulated by biotic and abiotic stresses (Dixon et al., 2002a). Coldness, heat, heavy metals, salt, polyethylene glycol (PEG), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), naphthalene acetic acid (NAA), H2O2 and methyl viologen (MV), which is also termed paraquat, may all induce the expression of a certain type of GST. Previous researches had also

Que et al.

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Figure 8. Expression profile of Sc-GST gene under various exogenous stresses.

revealed that, the expression of the zeta-type GST genes could be induced by the cell division, indole-3-acetic acid (IAA) and cytokinin (Marrs, 1996; Gonneau et al., 1998). Several GSTs were known to be induced by plant hormones, suggesting that plant hormones could regulate GSTs or GSH-conjugating activity was regulated by plant hormones (Marrs, 1996; Gonneau et al., 1998). SA and H2O2 were proven to play important roles in signal transduction and resistance response of plant at early stages, such as hypersensitive response (HR) and

systemic acquired resistance (SAR) (Scott et al., 1999). In the present study, with the application of pET29a (+)/BL21 (DE3) prokaryotic expression system, the fusion protein expression of Sc-GST gene was successfully induced and the protein was found to have GST-specific enzyme activity. Real-time qPCR analysis demonstrated that the expression of Sc-GST gene was induced both by H2O2 and U. scitaminea stress, and firstly inhibited and then induced by SA treatment. Although further study is necessary, the expression of Sc-GST gene was supposed

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to be regulated by all these three kinds of exogenous stresses and Sc-GST gene was thus assumed to play a certain role in sugarcane disease resistance, which was similar to the results from previous studies in several other plant species (Dixon et al., 2002a). Free oxygen radicals, such as O2-, H2O2 and OH- , would be generated under the stress of H2O2 and U. scitaminea. These radicals had strong toxin and could cause great harm to plant cells. While the expression of Sc-GST gene was greatly induced under the stress of H2O2 and U. scitaminea, it was reasonable to assume that Sc-GST gene was a type of stress-tolerant gene playing a certain role in detoxification and thus disease resistance in sugarcane. This is the first report of cloning and characterization of a full-length zeta-class GST gene in sugarcane. The results should be helpful in the ongoing exploitation of resistance genes and their application in sugarcane. ACKNOWLEDGEMENTS This research was funded by the earmarked fund for Modern Agro-industry Technology Research System (CARS-20); Research Fund for the Doctoral Program of Higher Education (20103515120006); Natural Science Foundation of Fujian province (2010J01078) and type A Research Fund of Fujian Education Department (JA10115).
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