Molecular Cloning, Expression Analysis, and PreliminarilyFunctional Characterization of the Gene Encoding ProteinDisulfide Isomerase from Jatropha curcas

Haibo Wang1,2 & Zhurong Zou1& Ming Gong1

Received: 23 December 2014 /Accepted: 12 March 2015 /Published online: 1 April 2015# Springer Science+Business Media New York 2015

Abstract Reactive oxygen species (ROS) in plants, arising from various environmentalstresses, impair the thiol-contained proteins that are susceptible to irregular oxidative formationof disulfide bonds, which might be alleviated by a relatively specific modifier called proteindisulfide isomerase (PDI). From our previous data of the transcriptome and digital geneexpression of cold-hardened Jatropha curcas, a PDI gene was proposed to be cold-relevant.In this study, its full-length cDNA (JcPDI) was cloned, with the size of 1649 bp containing theentire open reading frame (ORF) of 1515 bp. This ORF encodes a polypeptide of 504 aminoacids with theoretical molecular weight of 56.6 kDa and pI value of 4.85. One N-terminalsignal peptide (−MASKGSIWSCMFLFSLI VAISAGEG-) and the C-terminal anchoringsequence motif (−KDEL-) specific to the endoplasmic reticulum, as well as two thioredoxindomains (−CGHC-), are also found by predictions. Through semi-quantitative RT-PCR, theexpression of JcPDI was characterized to be tissue-differential strongly in leaves and roots, butweakly in stems, and of cold-induced alternations. Furthermore, JcPDI overexpression in yeastcould notably enhance the cold resistance of host cells. Conclusively, these results explicitlysuggested a considerable association of JcPDI to cold response and a putative applicationpotential for its correlated genetic engineering.

Keywords Jatropha curcas . Protein disulfide isomerase .Molecular cloning . Expressionanalysis . Yeast overexpression

Appl Biochem Biotechnol (2015) 176:428–439DOI 10.1007/s12010-015-1585-3

* Ming Gonggongming6307@163.com

1 Engineering Research Center of Sustainable Development and Utilization of Biomass Energy,Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology ofYunnan Province, School of Life Sciences, Yunnan Normal University, Kunming 650500, China

2 College of Biological Resources and Environmental Science, Qujing Normal University,Qujing 655011 Yunnan, People’s Republic of China

Introduction

Jatropha curcas, a shrub or small tree belonging to the family of Euphorbiaceae [1, 2], iswidely distributed in tropical and subtropical areas, especially in central and south America,Africa, Southeast Asia, India, and China [3]. J. curcas can be used for producingbiodiesels and preventing further degradation of grid lands, due to its high content(40–60 %) of seed oil and intrinsic drought-tolerance. In addition, it can be grown asan economic species, with medicinal value of anti-cancer effects. So, it has multipleapplication potentials for biofuel production, medicine development, environmentalimprovement, and etc. [4].

Protein disulfide isomerase (PDI), belonging to the family of oxidoreductases, is mainlyreserved in the lumen of the endoplasmic reticulum (ER), especially of the tissues specifiedwith abundant synthesis and secretion of disulfide bond-enriched proteins (accounted for0.4 %). PDI is a multifunctional enzyme which catalyzes the formation of disulfide bondsbetween cysteine residues and isomerization of misformed disulfides during the early stages ofprotein folding into its three-dimensional structure and secretion in the endoplasmic reticulum[5]. Furthermore, PDI was evidenced with other biological roles, e.g., participating in collagensynthesis and lipid transport, due to its functions as proline-4-hydroxylase and microsomaltriglyceride transfer protein, respectively [6]. This enzyme was also involved in plantresponse and resistance to a number of stresses such as drought, cold, salt, and radiation,through the metabolic participation of dehydroascorbic acid reduction [7]. From our previousdata of the digital gene expression (DGE) profiling of cold-hardened J. curcas, theassociation of PDI with cold tolerance was again implicated by its upregulated expressionunder cold exposure [8]. Herein, we firstly reported the cloning of a full-length cDNA encodingPDI from J. curcas (JcPDI), according to our existent chilling transcriptome and DGE results[8, 9]. Moreover, the expression patterns of JcPDI in different tissues of J. curcas underdifferent periods of chilling treatments were evaluated by semi-quantitative RT-PCR, and itsfunction was preliminarily characterized by recombinant expression in yeast Saccharomycescerevisiae.

Materials and Methods

Plant Materials and Treatments

Seeds of J. curcas were surface-sterilized in 1.5 % CuSO4 for 20 min and rinsed thoroughlywith sterile distilled water according to our previous methods [10], and then soaked in distilledwater for 24 h. The imbibed seeds were sown on six layers of wetted filter papers in trays andgerminated in climate chamber at 26 °C in the dark for 5 days. Then, the geminated seeds weretransferred to pots containing sterilized soil with perlite, peat, and sand (1:2:1) in climatechamber with the parameters of 26/20 °C (day/night), 75 % relative humidity (RH), and 16 hphotoperiod, and sequentially grown for 14 days.

For cold treatment, 2-week-old J. curcas seedlings were subjected to chilling at 12 °C for12, 24, and 48 h, respectively, as described in our early study [11, 12]. The roots, stems, andleaves from each treatment as well as the control seedlings (continually under normal growthconditions) were harvested and frozen in liquid nitrogen and stored at −80 °C until RNAextraction.

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Cloning of the Full Length of JcPDI cDNA

Total RNAwas extracted from the seedlings of J. curcas using a plant RNA isolation reagent(Tiangen Biotech, Beijing) following the manufacturer’s instructions. After DNase I treatmentto remove traces of the contaminant genomic DNA, 5 μg of total RNAwas used to synthesizethe first-strand cDNA by oligo(dT)18. Based on the matched Unigene sequence in ourGenbank-deposited transcriptome data (accession number: GAHK01013601) [9], twospecific primers (forward: 5′-ACTGCAAAAACAGAGAGAGC-3′, and reverse: 5′-TAAAGGGGCTAAGTAAAC-3′) were designed for amplifying the full length ofJcPDI cDNA. Using leaf reversely transcribed cDNA as template, the PCR was performedas follows: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 50.3 °C for 30 s and 72 °Cfor 90 s; and a final extension step of 72 °C for 10 min. After purification, the PCRproducts were cloned into the pEASY-T1 vector (TransGene, Beijing) by TA cloningto generate the recombinant plasmid pEASY-T1-JcPDI that was further verified bysequencing.

Bioinformatic Analyses of JcPDI

For the deduced protein (JcPDI) of the cloned cDNA, the theoretical isoelectric point (pI) andmolecular weight were determined using the ProtParam tool (http://web.expasy.org/protparam/), the signal peptide was predicted with SignalP 3.0 (http://www.dtu.dk/services/SignalP/), and the secondary structure, subcellular location, and hydrophilic/hydrophobiccharacteristics were predicted using SOPM, WolfSport, and TMHMM programs, respectively.In addition, the three-dimensional (3-D) structure of JcPDI was predicted by homology basedSwiss modeling and Phyre program (Version 2.0), and displayed by the VMD program(version 1.8.7). Multiple sequence alignment of JcPDI and other plant PDIs was performedby ClustalWand presented via GenDOC program (version 2.6.002), of which the phylogenetictree was further constructed using the MEGA program (version 4.0) with the neighbor-joiningmethods. The conserved domain analysis was performed by the CDD tool of the NCBI server(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The prediction of cis-acting elementswithin the promoter region of JcPDI was also performed by online programs, PlantCARE andPLACE.

Expression Analysis of JcPDI in J. curcas by Semi-quantitative RT-PCR

Semi-quantitative RT-PCR was utilized to evaluate JcPDI mRNA expression in differenttissues of J. curcas during cold-hardening, using 18S ribosomal RNA (18S rRNA) (GeneBankaccession no AY823528) as an internal standard, and gene-specific primers for JcPDI(JcPDI_F1: 5′- ATACCCATTGTGACCCTC −3′ and JcPDI_R1: 5′- TTCCCACTTGAAGACCTG −3′) and 18S rRNA (Jc18S_F: 5′- AGAAACGGCTACCACATC −3′ and Jc18S_R: 5′-CCAAGGTCCAACTACGAG −3′). PCR reactions with triplicates were conducted under thefollowing conditions: 94 °C for 5 min; 26 cycles of 94 °C for 30 s, 55 °C (for JcPDI) or 51 °C(for 18S rRNA) for 30 s, and 72 °C for 60 s; and 72 °C for additional 10 min. Three microlitersof PCR products were detected on 1.0 % agarose gels, and the visualized target bands wereestimated for their intensities by using Quantity One (Version 4.6.2, Bio-Rad). Then, theamount of JcPDI was normalized against that of 18S rRNA to obtain the relative values thatwere further averaged (with±SD) for comparison.

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Expression Analysis of JcPDI in S. cerevisiae (Yeast)

By BamH I and Xho I digestion, the JcPDI cDNA fragment was subcloned from plasmidpEASY-T1-JcPDI into the vector pYES2 to obtain the recombinant yeast expression vectorpYES2-JcPDI that was then transformed into the yeast strain of INVSc1.

A single clone of the recombinant yeast (INVSc1-pYES2-JcPDI) or control yeast(INVSc1-pYES2) was grown in 1 % yeast extract, 2 % peptone, 2 % glucose (YPD) liquidmedium overnight at 30 °C. The cells were harvested by centrifuging at 4000 rpm for 1 min,resuspended in 20 ml 1 % yeast extract, 2 % peptone, 2 % galactose (YPG) liquid mediumuntil the optical density (OD600) of 0.4, and then induced for 36 h at 30 °C. The different yeastcell cultures were adjusted with an identical OD600 value of 2.0. Then, the yeast suspensionswere serially diluted into 1, 1/10, 1/20, 1/40, 1/80, and 1/160, inoculated with 2 μl each ontothe YPG solid medium, and cultured at 30 °C and 18 °C for 2–3 days. In parallel, the yeastcells were diluted by adding 60 ml YPG liquid medium until the OD600 of 0.2, and cultured at30 °C and 18 °C. The OD600 values of yeast suspension were detected every 3 h. Threeindependent measurements were carried out simultaneously. The growth profiles of the yeastcells were examined by comparing their growth curves with horizontal ordinate of time (h) andlongitudinal coordinates of optical density (OD600).

Results

Cloning of the Full-Length cDNA of JcPDI

The full-length cDNA of JcPDI were amplified from young leaves of J. curcas under chillinghardening at 12 °C for 24 h, and inserted into a TA cloning vector to generate the recombinantplasmid pEASY-T1-JcPDI that was identified by clony PCR (Fig. 1).

Bioinformatic Analyses of JcPDI

The sequence of JcPDI cDNA was determined and submitted into the GenBank of NCBI(accession no. KJ670153.1), consisting of 1649 bp with a complete open reading frame (ORF)

Fig. 1 Cloning of JcPDI cDNA. a Full-length cDNA amplification. b Colony PCR of recombinant plasmid(pEASY-T1-JcPDI). c Recombinant plasmid of pEASY-T1-JcPDI. d Restriction enzyme digestion by BamH Iand Xho I; M. Trans 2 K Plus II DNA Marker (TransGene, Beijing, China)

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of 1515 bp (Fig. 2). Its matched genomic DNA sequence (Jc4S06094, 31,037 bp) from thedatabase (http://www.kazusa.or.jp/jatropha/) contains 10 exons and 9 introns. Additionally, thepromoter prediction of JcPDI identified the CpG island, core regulatory elements, transcriptionstart site (TSS), and the CRT/DRE (C-repeat/dehydration-responsive element) element (CCGAC), implying its cold-relevance.

The deduced JcPDI protein is a hydrophobic polypeptide of 504 amino acids (aa), withtheoretic molecular mass of 56.6 kDa and pI value of 4.85. From predictions, this protein is

Fig. 2 Nucleotide and deduced amino acid sequence of JcPDI cDNA. The start codon (ATG) and stop codon(TAA) are underlined

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composed of 148 aa for α-helix (36.90 %), 121 aa for β-sheet (24.01 %), 49 aa for β-turn(9.72 %), and 186 aa for random coil (26.74 %). It has a N-terminal signal peptide of 25 aa(−MASKGSIWSCMFLFSLIVAISAGEG-) and C-terminal ER-anchoring signal (−KDEL-)that are responsible for targeting PDI into ER to execute the catalytic activities of disulfideformation, reduction, and isomerization. Furthermore, JcPDI has three potential transmem-brane regions located in 161–167, 244–245, and 300–304, two of which are however not fullymembrane-penetrated. This protein also contains two sites of O-glycogylation (Ser139, Ser205).

Multiple protein sequence alignment of JcPDI with other plant PDIs demonstrated a certaindegree of sequence variations in both N- and C- terminals, but with a considerably conservedcentral region (Fig. 3a). Conserved domain analysis indicates JcPDI is a multidomain protein,consisting of four consecutive thioredoxin domains (−a-b-b′-a′-) (Fig. 3b). Both the N-terminala ( − 5 0 V V E F YA P W C G H C K N L A P Q Y 6 8 - ) a n d C - t e r m i n a l a ′(−395LLEFYAPWCGHCKKLAPIL413-) domains have catalytic disulfide/dithiol active cen-ters, while the two internal domains (b, b′) are structural, providing additional interactions forprotein isomerisation, and thus called thioredoxin-like domains. In addition, the x junctionpeptide (−480RDEDKV485-) and c functional domain (−486EEEDKETLEESEET500-) can befound at its C-terminal (Fig. 3).

Further constructed phylogenetic tree indicates that JcPDI has high identity to its orthologsof evolutionally close species such as Ricinus communis (80.47 %) and Populus trichocarpa(77.22 %), but contrastively low homology to those of monocot plants including rice(59.73 %), corn (57.92 %), and wheat (56.15 %) (Fig. 4).

Molecular homologous modeling of JcPDI at the Swiss-Model server yielded a 3-Dstructure that is similar to that of yeast PDI (Fig. 5a). Therein, both of domains a, a′ arecomposed of several secondary structures in the order of β1-α1-β2-α2-β3-α3-β4-β5-α4, andthe core sequence motif ‘-CGHC-’ is located between β2 and α2. Furthermore, the domains b,b′ are composed of structural elements in the order of β1-α1-β2-α2-β3-β4-β5-α3 andα1-β1-α2-β2-α3-β3-β4-α4, respectively. The functional domain c was also found in JcPDIto form an α-helix rich in acidic amino acids (Asp and Glu, accounted for 60 %), serving as themain binding region of Ca2+ and the regulation areas (Fig. 5a). In addition, the PROCHECKprogram was used to examine the modeling and calculate for Ramachandran plotting. Theresults showed that 98.4 % of amino acid residues were located in the energy stable regions(Fig. 5b), implying that the simulated 3-D structure of JcPDI is reliable.

Differential Expression of JcPDI in Different Tissues of J. curcas Under ChillingStress

The expression of JcPDI in different tissues of J. curcas during chilling hardening wasanalyzed by semi-quantitative RT-PCR. The results showed that JcPDI expression could be

�Fig. 3 Sequence alignment of PDIs from J. curcas with other plant species and the corresponding CDDprediction. a Sequence alignment. The catalytic thioredoxin domains (a, a′) are underlined, with the core ‘-CGHC-’ residues marked by double underlines. The endoplasmic reticulum anchoring sequence ‘-KDEL-’ ismarked by asterisks. b Domain structure of JcPDI by NCBI CDD prediction. Alignment was performed by theGenDOC program with the organisms such as Arabidopsis thaliana, NP_177875.1; Brassica carinata,ABB17025.1; Daucus carota, BAI67717.1; Gossypium hirsutum, ABO41851.1; Ipomoea batatas,AAT39459.1; Jatropha curcas, JcPDI; Oryza sativa, AAX85991.1; Populus trichocarpa, EEE80253.1; Ricinuscommunis, AAB05641.1; Triticum aestivum, AAA19660.1; Vitis vinifera, XP_002285864.1; and Zea mays,ABP88739.1

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transcriptionally detected in all examined tissues, but with differential levels: strongly in leavesand roots, weakly in stems. Moreover, the expression level of JcPDI was increased in roots by82 % as compared with the control (CK) after 12 h, 24 h of 12 °C cold exposure, but droppeddramatically to an amount less than CK after 48 h. In leaves, its expression was decreased after12 h of cold exposure, but rapidly restored after 24 and 48 h (Fig. 6).

Fig. 5 The 3-D structure of JcPDI constructed by homology-based modeling. a Cartoon model of the 3-Dstructure predicted by Swiss-Modeling. b Ramachandran map of the JcPDI modeling structure generated byPROCHECK, Phi, and Psi represent the dihedral angle of amino acid residues

Fig. 4 Phylogenetic tree analysis of PDIs from J. curcas and other plant species by ClustalW alignment andMEGA mapping via neighbor-joining method. Plant PDIs include those indicated in Fig. 3 and additional ones(Datisca glomerata, AAD28260.1; Glycine max, BAG16714.1; Medicago sativa, CAA77575.1)

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Expression and Functional Evaluation of JcPDI in Yeast

The recombinant yeast expression construct pEASY-T1-JcPDI and empty vector pYES2 weretransformed into the yeast strain of INVSc1, the positive clones of which were verified byyeast colony PCR.

The function of JcPDI was preliminarily evaluated by comparing the growth of therecombinant yeast strain INVSc1-pYES2-JcPDI and the control yeast strain INVSc1-pYES2under normal (30 °C) and cold stress (18 °C) conditions. At 30 °C, both of the yeast strains

Fig. 6 Expression of JcPDI from J. curcas in different tissues and different time under low temperature. a ThePCR product for the JcPDI gene and 18S rRNA from roots, stems, and leaves of J. curcas. b Relative expressionof JcPDI gene mRNAs detected per lane were normalized using 18S rRNA intensities

Fig. 7 Growth comparison by serial dilution point-plating and growth curves of the recombinant yeast strainINVSc1-pYES2-JcPDI and the control yeast strain INVSc1-pYES2 under normal condition of 30 °C (a, c) andcold stress of 18 °C (b, d)

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were shown with similar growth appearance on plates and no obvious dilution effects (Fig. 7a),and the trends of the growth curves were nearly consistent in YPG medium (Fig. 7c). Incontrast, the growth of the recombinant yeast cells remarkably outperformed the control cellsat 18 °C, although both of them appeared to be somewhat inhibited by cold stress. Theirgrowth difference was distinct on plates of serial dilutions (Fig. 7b), and also reflected bycomparing their growth curves (Fig. 7d). These results indicated that the yeast overexpressionof JcPDI could enhance the cold resistance of the recombinant yeast cells, to some extentreinforcing a functional relevance of JcPDI to the cold tolerance in J. curcas.

Discussion

The PDI, a main disulfide bond modifier for its antioxidant function, plays a curial role inmaintaining the stability of protein structure under numerous stresses such as reactive oxygenspecies (ROS) [13]. This enzyme is widely distributed in various organisms. In rice, at least 12members were found with 127–563 aa of 16.28–62.25 kDa, containing a signal peptidecommonly located at the N-terminal [14]. Furthermore, 12 in maize, 9 in wheat, 5 inChlamydomonas reinhardtii, and 3 in muscus were discovered. Because of the differences inthe constitution and arrangement of the functional domains among various PDIs, the availableclassifications of PDI proteins seem confused [15, 16]. According to a newmethod proposed bySelles (2011) [17], based on the counts of Trx domain (−CXXC-) and additional domains (TM,D, COPII, J, ARMET, and etc.), PDIs can be classified into nine types: PDI-A (−a-), PDI-B (−a-b-b′-), PDI-C (−TM-a-COPII-), PDI-D (−J-a-ARMET-), PDI-E (−a-TM-), PDI-F (−a-C-ter-),PDI-L (−a-b-b′-a′-), PDI-M (−a°-a-b-), and PDI-S (−a°-a-D-). Regarding this, the encoded PDIof JcPDI cDNA cloned in this study belongs to the type of PDI-L, as shown in Fig. 3b.

The complexity of the PDI family was consistent with the specificity and diversity of geneexpression and regulation. In 7-days Arabidopsis seedling, AtPDI1, AtPDI5, AtPDI6, AtPDI9,and AtPDI12 were found with abundant expression, while AtPDI2, AtPDI3, AtPDI4, andAtPDI7 expressed scarcely [16]. AtPDI5 mRNA was especially abundant in the flowers andimmature seeds [18]. Moreover, qPCR analysis showed that SpPDI1 in Ipomoea batatas had ahigh expression level in tuberous root, but low expression in root [19]. In addition, a number ofevidences demonstrated the effects of abiotic stresses or hormones on PDI expression. Forexamples, a PDI gene from maize capillament was characterized with induced expression bydrought, low temperature, abscisic acid (ABA), and salt [20]. In rice, ABA treatment couldinduce the expression of OsPDI1-1 in all tissues, and gibberellin (GA) could elicit theexpression of OsPDI1-2, OsPDI1-4, OsPDI5-3, and OsPDI5-4 in leaves and stems, but metalHg treatment could sharply induce the expression of PDIs in roots [21]. Based on our previousdata of the transcriptome and digital gene expression (DGE) of cold-hardened J. curcas [8, 9],as well as the RT-PCR results presented herein (Fig. 6), JcPDI was found with tissue-differential expression and cold-induced expression fluctuations. Cold stress at 12 °C for 12and 24 h revealed that JcPDI gene in root was upregulated 1.48- and 1.82-fold compared withcontrol without cold-hardening, respectively, but the truth of downregulated to an amount lessthan control after 48 h suggested that the transcript level of JcPDI potentially kept lowexpression over 48 h in root. The JcPDI expression tendency in leaves and the results inmaize [20] hinted that its expression level in leaves possibly restored to control over 48 h, andthen retained high expression level to resist the cold stress. Furthermore, JcPDI overexpressionin yeast could significantly enhance the cold resistance of host cells (Fig. 7), probably by

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alleviating the protein damages of cold-elicited oxidative stress. Collectively, these dataprovided an explicit insight that JcPDI is plausibly involved in the cold response and tolerancein J. curcas and naturally eligible for applications in its correlated genetic engineering.

Acknowledgments This work was supported by several grants from the National Foundations of NaturalSciences, China (No. 31260064, 31460059, 31460179) and the Education Bureau of Yunnan Province (No.ZD2010004).

Ethical statement This research did not include vertebrate studies or animal experiments and field studies,without any ethical risks. Our academic lab does not locate in the protected area, and no specific permissions arerequired.

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