Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina

Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.


Introduction
Dunaliella salina, one member of the genus Dunaliella (Chlorophyceae, Volvocales), is an extremely halotolerant, unicellular, green, and motile algae. The genus Dunaliella is unique in its remarkable ability to survive in the media with a wide range of NaCl concentrations, from about 0.05 M to saturation (around 5.5 M), while maintaining a relatively low intracellular sodium concentration [1,2]. This remarkable osmotic adaptability is mediated primarily by the massive de novo synthesis of the compatible solute, the glycerol, following salt stress [3]. These characteristics make D. salina an obvious applicable value as a model organism in studying the mechanism of osmoregulation under salt environment conditions. In addition, under high salt stress, D. salina could accumulate large amounts of b-carotene in cells, which makes it one of the best sources of natural b-carotene [4][5][6][7].
Glycerol is an important osmotically compatible solute in Dunaliella under salt stress [8,9]. The extracellular osmotic pressure is released in Dunaliella by changing intracellular glycerol content. The glycerol is synthesized rapidly when the concentration of saline increases, and the glycerol transforms to starch when the concentration of saline drops [10][11][12]. At high salinity, D. salina accumulates massive amounts of glycerol and the level of intracellular glycerol is proportional and osmotically equivalent to the external NaCl concentration, reaching about 8 M or 55% (w/v) of the cell weight at saturated NaCl concentrations [13,14]. Moreover, the green alga Dunaliella tertiolecta could also adapt the different concentration of saline by synthesizing or eliminating the intracellular glycerol to balance the osmotic potential of intracellular and extracellular [15,16]. Nicotinamide adenine dinucleotide (NAD + )-dependent glycerol-3-phosphate dehydrogenase (G3PDH) plays a major role in the osmoregulation process in Dunaliella [12,17]. In glycerol biosynthesis pathway, G3PDH catalyze dihydroxyacetone phosphate (DHAP) to form glycerol-3-phosphate, which is converted to glycerol finally by glycerol-3-phosphate phosphatase [18,19].
It was found that there are five isozymes of G3PDH in D. salina, and these isozymes respectively take effects in different salinities and play important roles in glycerol metabolism [12]. Chen et al found that four loci produced different G3PDH isozymes functioning under different salinity conditions [12]. In this study, we isolated the cDNA of a NAD + -dependent G3PDH from D. salina, which is one isozyme with highly homology of previously isolated G3PDH in this alga [20]. Subsequently, a series of bioinformatics tools were employed for the analysis of its physicalchemical characteristic, conserved structural domain, transmembrane and signal sequence condition, secondary and spatial structure, phylogenesis, and so on. Finally, this G3PDH was subcloned in the pET-32a(+) vector and undergone prokaryotic expression to further elucidate the pathway of glycerol metabolisms in Dunaliella.

Cultivation of D. salina under Salt Stresses
Cells of D. salina strain 435 (UTEX 200) conserved in our laboratory were cultivated in the culture medium according to Chen et al [21]. Cells grown at the late log phase were harvested by centrifugation at 5,000 g for 15 min at 4uC for next experimental procedure.
Isolation of cDNA for G3PDH in D. salina The total RNA was prepared from 10 mL of D. salina cells grown at the late log phase with using E.Z.N.A. Total RNA Kit II (OMEGA) according to the manufacture's instruction. Subsequently, the total RNA was treated by DNase I (RNase Free) (TaKaRa) and was dissolved in 0.1% (v/v) diethyl pyrocarbonate solution (TaKaRa) [7,22].
The first strand of cDNA was synthesized from the total RNA using PrimeScript TM RT-PCR kit (TAKARA) according to the manufacturer's instructions [7,22]. Reverse transcription (RT) reaction was performed with the parameters set as follows: 42uC for 30 min, followed by 70uC for 15 min. Primers Dsgpdh1-F and Dsgpdh1-R were used to amplify the conserved fragment of the D. salina G3PDH cDNA by using Premix Ex Taq (TaKaRa) following the manufacturer's instructions. The PCR procedure is as the following: 1 cycle of 94uC, 5 min; 30 cycles of 94uC, 30 s, 51uC, 30 s, and 72uC, 1 min; and 1 cycle of 72uC, 10 min; used primers listed in Table 1.
Based on the obtained conserved cDNA fragment sequence, gene specific primer Dsgpdh3'F was designed and 39 RACE was conducted with oligo dT-Adaptor primers using RNA PCR Kit (AMV) Ver.3.0 (TaKaRa). The first-strand cDNA was amplified by LA Taq (TaKaRa) with the parameters set as follows: 94uC, 4 min; 30 cycles of 94uC, 30 s, 46uC, 30 s, and 72uC, 1 min with a final extension at 72uC for 10 min.
The 59 RACE operation was accomplished with SMART TM MMLV Reverse Transcriptase (Clontech) and synthesized primers SMARTAO and 59-RACE CDS. The second 59RACE was conducted using the Dsgpdh5'F2primer designed according to the fragment obtained from the first 59RACE reaction with Dsgpdh5'F1 primer. Other handlings including touchdown PCR were employed according to the SMARTer TM RACE cDNA Amplifcation Kit User Manual, with the exception of LA Taq DNA polymerase (TaKaRa) for touchdown PCR, rather than Advantage 2 Polymerase Mix.
The full-length G3PDH cDNA was obtained with specific primes (Dsgpdh-F and Dsgpdh-R, Table 1) corresponding to the 59 and 39 ends of the G3PDH gene. The PCR procedure to amplify the G3PDH cDNA fragment is as follows: 1 cycle of 94uC, 5 min; 30 cycles of 94uC, 30 s, 55uC, 30 s, and 72uC, 1 min; and 1 cycle of 72uC, 10 min. All amplified fragments were cloned into pCR2.1 vector (Invitrogen) and undergone Sanger sequencing.
All PCR products were separated by electrophoresis in 1.5% (w/v) agar gels, cloned in the pMD18-T vector (TaKaRa) and sequenced before the further experiments. Plasmid preparations, transformations, and other standard molecular biology techniques were carried out as described previously [23].

Plasmid Construction and Protein Expression
Plasmid pET-32a-G3pdh was constructed by insertion of the D. salina G3PDH open reading frame (ORF) into the BamH I and Xho I (all restriction endonucleases are products of TaKaRa, Japan) restriction sites of expression vector pET-32a(+) (Novagen, Darmstadt, Germany). The G3PDH cDNA fragment was used as templates to synthesize the G3PDH ORF by using PrimSTAR HS DNA Taq (TaKaRa, Japan) with forward primer ORF-F and reverse primer ORF-R, which will introduce the BamH I and Xho I site into the 59 and 39 end of the ORF, respectively. The PCR procedure is as the following: 1 cycle of 94uC, 30 s; 30 cycles of 94uC, 30 s, 60uC, 30 s, and 72uC, 2 min; and 1 cycle of 72uC, 10 min. PCR product was separated by electrophoresis in 1.5% (w/v) agar gels, cloned in the pMD18-T vector and transformed into E. coli JM109, which was verified by double digestion of BamH I and Xho I. Both plasmids pET-32a and pMD18-T-JM109-G3pdh were prepared from E. coli BL21 and JM109, then digested by BamH I and Xho I at 30uC for 45 min or 2 h. The digested products were separated by electrophoresis in 1% (w/v) and 1.5% (w/v) agar gels, and the ORF and pET-32a fragments were recycled using E.Z.N.A. kit (OMEGA, USA). T4 DNA ligase (0.5 mL containing 200 NEB units; New England Biolabs, USA) was then added, and samples were incubated at 16uC for 12-16 h. 5 mL of the ligation mixture was used to transform electronically competent E. coli (100 mL of BL21(DE3)). The correct strain of E. coli BL21 (DE3)/pET-32a-G3pdh was verified by double digestion of BamH I and Xho I. E. coli strains BL21 (DE3) were grown in LB medium at 37uC in darkness on a platform shaker at 230 cycles min 21 . Ampicillin (100 mg mL 21 ) was used for selection or maintenance of plasmids. To induce the expression of the D. salina G3PDH, a final concentration of 1.0 mmol L 21 isopropyl-b-D-thiogalactopyranoside (IPTG) was added to the E. coli culture when the optical density (OD) value reached 0.4-0.6, and the culture was allowed to continue growing for 3-4 h before harvesting by centrifugation.

SDS-PAGE Electrophoresis Analysis
The transformed cells with plasmid pET-32a-G3pdh or pET-32a was disrupted by ultrasonication in PBS buffer containing 1 mmol L 21 phenylmethysulfonyl fluoride (PMSF). The supernatants were collected by centrifugation at 12,000 g for 30 min at 4uC. Protein concentration was estimated as described [24]. Then supernatant in each samples were boiled for 5 min after adding 56SDS loading butter (4:1, v:v); protein molecular weight marker was also boiled for 3 min before loading samples. 10 mg of each samples were subjected to SDS-PAGE in a Bio-Rad protein electrophoresis system (Bio-Rad, Hercules, CA, USA), which was carried out at 80 V, and increased to 120 V after 1 h. The concentration of separating gel and stacking gel were 12% (w/v) and 5% (w/v). Protein electrophoretic profiles in gels were visualized through Coomassie Blue R-250 staining procedure.

Isolation of cDNA for G3PDH from D. salina
A pair of specific primers were designed to obtain G3PDH cDNA conserved fragment from D. salina on the basis of the G3PDH gene sequences of D. salina, Chlamydomonas reinhardii, Arabidopsis thaliana, Pichia stipitis, zygosaccha and four predicted G3PDH gene sequences. When using total RNA from D. salina cells as RT substrate, an expected 583 bp fragment amplified with primers Dsgdph1-F/R was cloned and sequenced (Fig. 1a).
On the basis of this cDNA conserved fragment, the 39 end fragment was amplified by 39 RACE reaction, which was 884 bp in length (Fig. 1b); and then two 59 RACE reactions were fulfilled resulting two fragments with 984 bp in the first step (Fig. 1c) and 1253 bp in the second step (Fig. 1d), respectively. Then, based on the sequence assembly, full-length G3PDH cDNA was amplified using specific primers Dsgpdh-F/R, which was 2100 bp (Fig. 2).

Bioinformatics Analysis of G3PDH cDNA and Amino Acid Sequence
Nucleotide sequence analysis showed that the D. salina G3PDH cDNA contained 2100 bp nucleotides with an ORF of 2100 bp, which contained 19.29% A, 31.48% G, 29.05% C and 20.18% T. The ORF encoded a 699-amino-acid-long peptide including 78 basic amino acids (lysine, arginine), 83 acidic amino acids (aspartic acid, glumatic acid), 266 hydrophobic amino acids (isoleucine, leucine, phenylalanine, tryptophan, valine) and 144 polar amino acids (Asparagine, cysteine, serine, threonine, tyrosine). Analysis by ProtParam tool revealed that the molecular weight of this peptide was 76.6 kDa, the isoelectric point was 6.49.
A complete homologous search by BLAST demonstrated that the nucleotide and putative protein sequence had, respectively, sequence identities of 91% and 95% with the published D. salina G3PDH with NAD + as coenzyme (AY845323.1), 83% and 78% with D. viridis G3PDH1 (EU624406.1), 76% and 72% with D. viridis G3PDH2 (EU624407.1), which indicated that the protein encoded by the obtained cDNA in this study might belong to the G3PDH family with NAD + as coenzyme.
The Conserved Domain Database (CDD) provided by NCBI was employed to predict the structural and functional region (Fig. 2), which manifested that the putative polypeptide from D. salina contained three conserved regions including phosphoserine phosphatase (SerB) domain, N-and C-terminals binding to NAD + (Fig. 2). Using Pfam database to search and predict the structural and functional domain of this putative polypeptide gained a similar result as shown by Fig. 3. The difference from CDD conservative structure speculated by NCBI was that SerB domain was affiliated to similar hydrolase domain family. The point also elucidated the first 330 amino acids should possess hydrolase function, and the latter amino acids played the main role of glycerol-3-phosphate dehydrogenase. Consequently, the results predicted that this deduced protein was a NAD + -dependent G3PDH (EC 1.1.1.8). Eukaryotic neural network (NN) search by SignalP 3.0 showed that this polypeptide had no signal peptide. It was also predicted to be non-secretory protein by Markov models (HMM) of SignalP 3.0. TMHMM Server v. 2.0 predicted that the G3PDH had no transmembrane domain. Presumption of subcel-

Prediction of Protein Structure
The secondary structure of the protein was deduced by NPS@ service PHD, GOR1, SOPMA and PREDATOR methods. On account of the distinct emphases of various methods, the predicted results were also different. So these methods were made a comparison and PredictProtein was adopted to analyze online ( Table 2). As shown by Table 2, the G3PDH protein had abundant a-helixes, some extended strands, many random coils and a few b-turns, but had no 3 10 -helix, p-helix and other rare secondary structure.
Three-dimensional structure of the D. salina G3PDH was predicted by 3D-JIGSAW comparative modeling program based on homologues of known structures automatically, and was visualized using RasMol software (Fig. 4). A big gap could be observed in the center of the protein by Fig. 4 using both the cartoon and ribbon models, which was regarded as the possible
The phylogenetic tree for the complete homologous G3PDHs was constructed using neighbor-joining method by MEGA 4.0.2 software. As Figure 6 shown, the G3PDH obtained in this study (indicated by red arrow in Fig. 6) share highest evolutionary position with other homologues from the Dunaliella genus, they all clustered into the green algae group with Chlamydomonas reinhardtii and Chlorella variabilis.

Prokaryotic Expression, Protein Purification and Enzymatic Assay
The G3PDH ORF sequence of 2100 bp was amplified by PCR with D. salina cDNA as templates. The cDNA was subsequently subcloned in the pET-32a(+) expression vector in the BamH I/Xho I sites. The constructed prokaryotic expression vector pET-32a-G3pdh was transformed into E. coli strain BL21 (DE3) and IPTG was used for induction. The G3PDH in transgenic strains were analyzed by SDS-PAGE. As shown by Fig. 7, a clear protein expression band could be observed at the position of 100 kDa (the addition of target gene 76.6 kDa and histidine marker 23 kDa), which was consistent with the theoretical value. Whereas, an obvious protein expression band appeared at the location of 20 kDa in positive control and no band was found in the samples due to the inhibition of the protein expression by the introduced target gene (Fig. 7).

Discussion
As is known, the osmotic adjustment response of Dunaliella (especially D. salina) functioned by varying the intracellular concentration of a compatible solute glycerol to balance the osmotic pressure inside and outside of cells. When subjected to hyperosmotic shock D. salina cells rapidly shrink followed by synthesis of glycerol to increases the internal osmolarity for resuming original volume of cells. Under hypoosmotic shock, it was found rapid swells followed by a decrease in internal glycerol and volume resumption [21]. The biosynthesis of glycerol in D.    salina involved the key enzymes G3PDH, which convert DHAP to glycerol-3-phosphate. Some studies have been performed to investigate the G3PDH for elucidating the mechanism of osmotic stress tolerance by glycerol. In a study, a novel G3PDH (NAD + ) (EC1.1.1.8) gene (PfGPD) was cloned from halotolerant yeast Pichia farinosa, and the PfGPD gene was induced by salt stress [25]. In another study, He et al cloned the cDNA encoding a NAD +dependent G3PDH from D. salina, and the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis [20]. In addition, He et al have cloned two novel chloroplastic G3PDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively [26]. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression.
In the present study, the cloned 2100 bp G3PDH cDNA from D. salina acts with NAD + as coenzyme, and the comparative study of conservative regions discovered NAD + and 3-phosphoglycerate binding sites in the G3PDH protein, theoretically testifying the cloned cDNA encodes G3PDH of osmosis-adjusting type. The G3PDH protein deduced from G3PDH cDNA contains 699 amino acids, of which the molecular weight is 76.6 kDa and the isoelectric point is 6.49, with 23.33 charge in pH 7.0. This G3PDH gene and protein had 91% and 95% identity with the putative G3PDH gene sequence (AY845323.1) and protein (AAX56341.1) [20]. The putative G3PDH protein contained 701 amino acids, with the molecular weight of 76.9 kDa, isoelectric point of 6.1 and 26.08 charge in pH 7.0 environment. So, it was showed some certain structural differences between them, and also reflected the diversity and complicity of G3PDH isoenzyme with NAD + as coenzyme. It was reported that three isoforms of G3PDH have been separated from D. tertiolecta [27,28]. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The first chloroplast form was the major form when the cells were grown on high NaCl, and it has been a form for glycerol production for osmoregulation. The second form increased in specific activity when inorganic phosphate was increased and played roles in stimulating cell growth and glyceride synthesis. The presumption of subcellular localization by WoLF PSORT showed that G3PDH in the present study may situate in the chloroplast as the osmoregulation form in glycerol production. Similarly, it was thought that the G3PDH in the study by He et al was also an osmoregulation form in chloroplast [20]. However, another study cloned and sequenced a cDNA encoding the D. salina FAD-G3PDH, which situated in the mitochondrial. The expression of FAD-G3PDH was enhanced by salt treatment. Its catalytic site facing toward the cytosol, combined action of this enzyme with the cytosolic NAD +dependent G3PDH forms the glycerol-3-phosphate shuttle [29]. In this shuttle, cytosolic NAD + -dependent G3PDH oxidizes cytosolic NADH to NAD + , and catalyzes the reduction of DHAP to glycerol-3-phosphate. Subsequently glycerol-3-phosphate passes the outer mitochondrial membrane and is oxidized to DHAP by FAD-G3PDH, simultaneously delivers its electrons to the respiratory chain [30]. Due to the essential role in glycolytic pathway, G3PDH is one of the typically constitutive housekeeping genes in living organisms [31,32]. Consistently, results of sequence alignment showed that G3PDH genes were conservative between plants and algae. Phylogenetic analysis indicated that G3PDHs of green algae clustered into one group. Difference of G3PDHs functions between plant and green algae will be interesting in coming study considering the unicellular green algae maybe more sensitive to the salinity of environmental conditions.
According to the analysis of conservative region of the G3PDH in the present study, it could be speculated that the G3PDH functional domain originate at the 331 amino acid of the amino sequence and the first 330 amino acids are potentially correlated with other properties of the protein. Namely, G3PDH protein has two independent functional domains, SerB and G3PDH domains. SerB (EC 3.1.3.3) and glycerol-3-phosphate phosphatase (EC 3.1.3.21) are attributed to the same type of hydrolase, and two enzymes have similar functions due to their active centers of similar size. Therefore, it was speculated the G3PDH might also have glycerol-3-phosphate phosphatase activity and can catalyze DHAP to glycerol directly without glycerol-3-phosphate phosphatase. Similarly, in the studies by He et al and He et al, protein domain analysis revealed that DsGPDH2 in D. salina and DvGPDH1 and DvGPDH2 in D. viridis all encoded unique bidomain proteins with C-terminal G3PDH domains and additional N-terminal SerB domains [20,26]. It has been reported that such bi-domain G3PDHs only exist in green alga, but not in higher plants or other species, such as yeasts and animals [26]. For example, only one catalytic domain has been found exist in polypeptide chain of G3PDHs in yeasts Debaryomyces hansenii, Candida glycerinogenes and Candidamagnoliae [33][34][35]. The existence of unique bi-domain G3PDHs in these green algae might be the evolutionary consequence, which maintained a unique osmoregulation mechanism in green algae for survival in severe environments [26].
Some key enzyme genes related to glycerol metabolism, such as the cDNA of fructose-1, 6-diphosphate aldolase (DsALDP) and NAD + -G3PDH are cloned from D. salina. These genes have been transferred into bacteria or plants to increase the salt-tolerance of these species. Zhang et al transferred the DsALDP gene into E. coli cultured in media with different NaCl concentration to analyze its expression [36]. As a result, the bacteria expressing DsALDP exhibited a higher salt tolerance with increasing NaCl concentration than bacteria with no DsALDP expression. Moreover, Zhang et al transferred the DsALDP gene into tobacco by Agrobacterium tumefaciens, and DsALDP gene was expressed effectively in transgenic tobacco, which exhibited a higher salt tolerance [37]. In another report, a G3PDH gene from D. salina has been transferred into led discs cells of tobacco. RT-PCR analysis showed that G3PDH gene integrated into tobacco genome has produced mRNA [38]. In the present study, the prokaryotic expression vector pET-32a-G3pdh was constructed and transferred into E. coli strain BL21 (DE3). The analysis by SDS-PAGE showed that the G3PDH protein was expressed successfully in transgenic strains, and the further work would emphasize on transforming this G3PDH gene into other higher plants to improve their salt tolerance.
In conclusion, in the present research the cDNA of a NAD + -G3PDH was successfully isolated from D. salina. The cDNA was 2100 bp long, which encoded a deduced protein sequence of 699 amino acids with an estimated molecular weight of 76.6 kDa. Protein domain analysis revealed that G3PDH protein has two independent functional domains, SerB and G3PDH domains. The D. salina G3PDH was a nonsecretory protein that may be located in the chloroplast. The D. salina G3PDH had a closer relationship with Dunaliella G3PDHs than with those of other species in the phylogenetic analysis. The secondary and three-dimensional structure of the D. salina G3PDH is predicted. In addition, the prokaryotic expression vector pET-32a-G3pdh was constructed and transferred into E. coli, in which G3PDH protein was expressed successfully. To fully understand glycerol metabolism and osmotic adjustment based on glycerol in Dunaliella, future investigation should focus on the gene clone of some enzymes related to glycerol metabolism and the application of transgenic technology to increase salt-tolerance of other plants by transferring these cloned genes.