Identification, sequence and phylogenetic analysis of CeDGAT1 in C. ellipsoidea

Based on the expressed sequence tag (EST) data of C. ellipsoidea, a full-length cDNA fragment of C. ellipsoidea DGAT1, designated as CeDGAT1, was cloned and identified. The nucleotide sequence has a full CDS of 2,142 bp, encoding a polypeptide of 713 amino acid residues with a calculated molecular mass of 81.76 kDa. It was registered in GenBank (ID No. KT779429). CeDGAT1 shared no more than 40% identity with DGAT1s of higher plants, such as G. max (40%), Z. mays (39%), R. communis (38%), Arabidopsis (38%), B. napus (36%), V. fordii (35%) and J. curcas (34%).

To examine the relationships among different sources of DGAT, a phylogenetic tree was generated from an alignment of the deduced amino acid sequences of CeDGAT1 with 44 DGAT homologues from other species (including membrane-bound DGAT1 and DGAT2 and cytosolic DGAT3). The dendrogram grouped CeDGAT1 with members of the DGAT1 family. DGAT2 and DGAT3 formed a separate cluster different from DGAT1 branches (Fig. 1). In detail, CeDGAT1 clustered together with APDGAT1 (Auxenochlorella protothecoides) and CvDGAT1 (Chlorella vulgaris) and formed a distinct lineage. Furthermore, CeDGAT1 is more closely related to DGAT1s from animals and fungi than to DGAT1s from plants.

Protein analysis with the TMpred program [41] predicted nine strongly hydrophobic transmembrane regions, which is consistent with the nine transmembrane domains predicted for B. napus DGAT1, T. majus DGAT1 and Arabidopsis DGAT1 [6, 9, 20]. Using the PROSITE database [42], a number of putative functional motifs, including N-glycosylation, cAMP- and cGMP-dependent protein kinase phosphorylation, protein kinase C phosphorylation, casein kinase II phosphorylation, tyrosine kinase phosphorylation, N-myristoylation and amidation sites were identified in CeDGAT1 (Additional file 1: Table S1). Compared with AtDGAT1, N-glycosylation and amidation sites were found only in CeDGAT1, while a leucine zipper pattern was detected only in AtDGAT1. It remains to be determined whether these sites are important for the functional regulation of the enzyme in vivo.

As previously reported [43, 44], a consensus amino acid sequence for an acyl-CoA binding motif (Fig. 2, I) and a conserved sequence for the DAG-binding motif (Fig. 2, III) of DGAT were also found in CeDGAT1. There was also a fatty acid-binding protein signature spanning residues Ala571 to Asn587 (Fig. 2, II) containing a putative tyrosine phosphorylation site: Tyr582 [6]. The CeDGAT1 protein contained an invariant proline (Pro381), which is thought to participate in presenting the fatty acyl group to the active site for esterification to (diacyl) glycerol and is critical for DGAT1 activity [45]. A highly conserved serine residue (Ser410) was essential for the activity of acyl-CoA: cholesterol acyltransferase, an enzyme closely related to DGAT1 [24, 46]. CeDGAT1 showed a leucine zipper motif with only one conserved leucine (Leu386) in the sequence. A visual examination of CeDGAT1 also revealed the sequence of a putative C-terminal ER retrieval motif (YYHDW, Fig. 2, IV), which is similar to other DGAT1 proteins in plants [11].

Lipid analysis and expression pattern of CeDGAT1 in C. ellipsoidea

To understand the relationship between CeDGAT1 expression and lipid synthesis, we investigated the expression pattern of CeDGAT1, the variation in biomass, and the total fatty acid (TFA) and TAG contents in heterotrophic cultures of C. ellipsoidea on nitrogen-replete and nitrogen-depleted (1/4 N) media (Fig. 3). As shown in Fig. 3a, the algal cell biomass increased more slowly in nitrogen-depleted cultures than in nitrogen-replete cultures, but both the TFA content and TAG content increased rapidly from 19% and 9% to 44% and 23%, respectively, from 36 to 108 h (Fig. 3b and c). On the contrary, TFA and TAG contents were not significantly changed in the growth process of nitrogen sufficient condition. So there was a 108% and 212% increase in TFA content and TAG content, respectively at the 108^th hour under nitrogen-depleted culture condition than under nitrogen sufficient culture condition.

Quantitative real-time PCR was performed to examine the expression profiles of CeDGAT1 in C. ellipsoidea cells under nitrogen-replete and nitrogen-depleted (1/4 N) conditions (Fig. 3d). 18S rRNA was used as an internal reference control. We noted that CeDGAT1, which catalyses the last committed step in TAG biosynthesis, was downregulated under nitrogen-replete conditions. Nevertheless, CeDGAT1 showed transient upregulation, with its transcript level peaking at 84 h following the onset of nitrogen depletion and declining thereafter. The upregulation of CeDGAT1 was concomitant with the increase in the TFA and TAG contents under nitrogen deprivation, suggesting that CeDGAT1 was highly induced by nitrogen deprivation and that its increased expression coupled with lipid content change may play an important role in TAG accumulation.

CeDGAT1 can recover the TAG synthesis of the quadruple mutant yeast strain H1246

To verify the diacylglycerol acyltransferase activity of CeDGAT1, the CeDGAT1 gene was heterologously expressed in the TAG-deficient S. cerevisiae quadruple mutant strain H1246 [47], which lacks the four genes DGA1, LRO1, ARE1 and ARE2 encoding DGAT, PDAT (phosphatidylcholine: diacylglycerol acyltransferase), ASAT1 (acyl-CoA: sterol acyltransferase 1) and ASAT2 (acyl-CoA: sterol acyltransferase 2), respectively. These four genes are essential for the formation of neutral lipids. Lipid bodies can be formed by the expression of at least one of four genes. INVSc1 and H1246 cells harbouring an empty pYES2.0 vector were used as positive and negative controls, respectively.

Nile Red was used to stain lipid bodies in yeast cells. Lipid bodies were present in the wild-type yeast strain INVSc1 or the mutant strain transformed with the CeDGAT1 gene but were undetectable in the quadruple mutant strain carrying the empty expression vector (pYES2.0) (Fig. 4a). The total lipids were extracted from yeast cells and then subjected to TLC (Thin-Layer Chromatography) analysis. Upon expression of CeDGAT1, a prominent band corresponding to TAG appeared on the TLC plate as expected, whereas no TAGs were identified in mutant yeast cells lacking the endogenous yeast DGAT and PDAT activities (Fig. 4b), which is consistent with the results from Nile Red staining (Fig. 4a). These results suggest that CeDGAT1 can successfully restore the ability of the quadruple mutant strain H1246 to form neutral lipids and confirm that CeDGAT1 encodes a functional protein capable of catalysing the last step of TAG biosynthesis.

Heterologous expression of CeDGAT1 can more significantly increase the total fatty acid content in the wild-type yeast than DGAT1s from some higher plants

We separately transferred CeDGAT1 and another three DGAT1 genes from higher plants, including oil crop G. max, A. thaliana and B. oleracea, into the wild-type yeast (INVSc1). RT-PCR results showed that the DGAT1 genes had nearly identical expression patterns in transgenic yeast (Additional file 2: Figure S1). The fatty acid contents of yeast carrying DGAT1 genes were measured by GC. As shown in Fig. 5, the total fatty acid contents of yeast significantly increased due to the expression of different DGAT1s when compared to the yeast transformed with pYES2.0. In detail, the total fatty acid content in yeast carrying AtDGAT1, GmDGAT1, BoDGAT1 and CeDGAT1 was 234.7 μg/mg, 243.3 μg/mg, 258.5 μg/mg and 290.0 μg/mg, respectively. Among the yeast expressing different DGAT1 genes, the total fatty acid content in the yeast transformed with CeDGAT1 increased most remarkably, by 142%. In contrast (compared with the yeast expressing CeDGAT1), the total fatty acid contents of the yeasts transformed with the three DGAT1 genes from higher plants significantly decreased by 19% (AtDGAT1), 16% (GmDGAT1) and 11% (BoDGAT1), respectively. These results suggest that CeDGAT1 may function in improving the oil content of plants, especially oil crops.

Over-expression of CeDGAT1 enhances the seed oil content and seed weight in higher plants

To explore CeDGAT1 as a tool to manipulate acyl-CoA pools and to engineer TAGs in plants, CeDGAT1 was over-expressed in Arabidopsis and B. napus var. Westar under the control of the constitutive NOS promoter. In Arabidopsis, three independent homozygous lines were selected for advancement to the T4 generation and used for detailed analysis. RT-PCR results showed that the CeDGAT1 transcript was expressed in transgenic lines over-expressing CeDGAT1 (Additional file 3: Figure S2). The transgenic lines did not show any visible morphological difference from untransformed control plants (data not shown). GC analysis revealed that the transformation of wild-type Arabidopsis with CeDGAT1 leads to a higher seed oil content (Fig. 6a). The average total fatty acid content in wild-type Arabidopsis seeds was 31.5 mg/100 mg of seed, but increased to 33.9–43.1 mg/100 mg in the transgenic lines. Thus, the transgenic seeds displayed an approximately 8–37% higher oil content than that of wild-type plants. In addition, the 1,000-seed weight of transgenic plants was 9–15% greater than that of the control (Fig. 6b).

In B. napus, twelve transgenic plants were identified by PCR (Additional file 4: Figure S3), and four of the twelve independent T3 transgenic B. napus lines were chosen for further analysis. The RT-PCR results showed that the CeDGAT1 transcript was expressed in transgenic lines (Additional file 3: Figure S2). Again, transgenic B. napus lines did not show any visible morphological difference from wild-type plants (data not shown). The fatty acid content in the seeds of wild-type B. napus and transgenic lines was measured using GC. As shown in Fig. 7a, the average total fatty acid content in wild-type B. napus seeds was 39.6 mg/100 mg of seed. In lines expressing CeDGAT1 under the NOS promoter, the total fatty acid content increased to 44.5–46.8 mg/100 mg of seed, representing an increase of 12–18% over the control. Moreover, the average 1,000-seed weight in the CeDGAT1 transgenic lines increased by 6–29% compared to that of wild-type plants (Fig. 7b). These results indicated that the CeDGAT1 gene can stimulate fatty acid biosynthesis and enhance seed weight.

Subcellular localization of CeDGAT1

EGFP-tagged CeDGAT1 was expressed in tobacco BY-2 suspension cells and then examined under confocal laser-scanning microscopy. As shown in Fig. 8, the distribution pattern of EGFP-CeDGAT1 was similar to that of endogenous ER stained with ER-tracker™ Red, and the signal was visualized around the nucleus and thread-like ER networks, indicating typical ER localization.

Strains and growth conditions

Chlorella ellipsoidea was initially cultured mixotrophically in 1 L flasks containing 500 mL of sterilized Endo medium [57] and incubated at 25 °C under illumination (100 μmol photons/m²/s) for one week with shaking at 160 r/min. These pre-cultured cells were transferred to Erlenmeyer flasks (3 L), each containing 1 L of fresh medium to a final volume of 1.5 L, and incubated for another 4 days. These pre-cultured cells, after centrifugation and washing with sterilized water, were sampled as the starting point (0 h). Then, the collected cells were resuspended at a density of approximately 9.5 g/L in a 20 L BioFlo 415 fermentor (New Brunswick Scientific, USA) containing 14 L of modified Endo medium, in which urea was used to replace KNO₃ in the original Endo medium. The media containing urea at 0.4 g/L and 1.6 g/L were named N-depleted (1/4 N) and N-repletion media, respectively. The culture conditions were maintained at 25 °C, and a thermocirculator was used to maintain a constant temperature in the fermentor by circulating water through the jacket. The fermentor was aerated with filtered ambient air at a flow rate of 0.5 vvm, and the pH was maintained at 6.8 using 1 M KOH. The cultures were sampled 24, 36, 60, 84 and 108 h after they were initiated using N-depleted (1/4 N) or N-repletion medium. The cells were harvested by centrifugation (5,000 g at 4 °C for 5 min). Aliquots for RNA analysis and gene cloning were frozen in liquid nitrogen and stored at −80 °C if not immediately used, while those for lipid analysis were washed with water and freeze-dried.

The wild-type yeast strain INVSc1 (Invitrogen, UK), the H1246 mutant strain (Matαyor245c::KanMX4 lro1::TRP1 are1::HIS3 are2::LEU2 ADE2 ura3) [47], Arabidopsis thaliana (ecotype Columbia) and Brassica napus (Westar) were used to determine the function of CeDGAT1 by heterologous expression.

Cloning of a cDNA encoding DGAT1 from C. ellipsoidea

Total RNA was isolated from algae cells of the exponential growth phase of C. ellipsoidea using the EasySpin RNA Extraction Kit (Aidlab Biotech, Beijing, China), and cDNA was prepared from 5 μg of total RNA-template with the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). The coding sequence of CeDGAT1 was amplified using the gene-specific primers P1 and P2 (Additional file 6: Table S2) based on the expressed sequence tag (EST) data of C. ellipsoidea. The 25 μL final reaction volume used for PCR contained 2.5 μL of 10× PCR buffer with MgCl₂, 1 μL of each primer (10 μM), 2.0 μL of 2.5 mM dNTPs, 1 μL of cDNA sample, 0.5 μL of EasyPfu DNA polymerase (TransGen Biotech, Beijing, China), and 17 μL of double-distilled water. The reaction conditions for PCR were as follows: denatured at 95 °C for 10 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; and a final extension step of 72 °C for 10 min. The amplified cDNA was cloned into the pEASY-Blunt vector (TransGen Biotech, Beijing, China), and the corresponding clones were verified by PCR and DNA sequencing.

Yeast expression vector construction and transformation

The full-length CeDGAT1 ORF was amplified using primers P3 and P4 (Additional file 6: Table S2) and subcloned between the Hind III and EcoR I sites of the pYES2.0 yeast expression vector. The Saccharomyces cerevisiae strains (the wild-type strain INVSc1 and the mutant strain H1246) were transformed using the LiAc method [58]. We also separately transferred another three DGAT1 genes from higher plants, including the oil crop Glycine max (accession no. AY496439.1), Arabidopsis thaliana (accession no. NM_127503.2) and Brassica oleracea (unpublished data from our laboratory) into the wild-type yeast (INVSc1). The primers that were used for DGAT1 gene cloning are shown in Additional file 6: Table S2. Transformants were selected on synthetic complete medium lacking uracil (SC-ura). For heterologous expression studies, the yeast strains were transferred into liquid SC-ura medium containing 2% (w/v) glucose at 30 °C overnight and then induced by adding 2% (w/v) galactose and 1% (w/v) tergitol NP-40 (Sigma, Taufkirchen, Germany) for an additional 72 h at 20 °C. The expression of DGAT1s in transgenic yeast was verified at the transcript level by RT-PCR (for the RT-PCR primers see Additional file 6: Table S2).

Plant vector construction and transformation

The complete CeDGAT1 was cloned into the plant expression vector pCAMBIA2301 under the control of the nopaline synthase (NOS) promoter and nos terminator, yielding pCAMBIA2301-CeDGAT1 (Additional file 7: Figure S5). The final binary vector was verified and then transferred into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method [59]. Arabidopsis plants were transformed by vacuum infiltration [60]. Brassica napus var. Westar was transformed using hypocotyl explants and the modified method of DeBlock et al. [61]. T1 generation seeds were selected on kanamycin (50 mg/L), and then the selected transformed plants were transferred to soil. T3 transgenic B. napus lines and homozygous T4 transgenic Arabidopsis lines were used for seed and oil analyses. Genomic DNA was isolated from B. napus var. Westar leaf material. The stable integration of the NOS: CeDGAT1: nos cassette into the genome of transgenic B. napus was checked by PCR amplification using the specific primers P21 and P22 (Additional file 6: Table S2). GUS histochemical staining of the leaves from the transgenic lines was also conducted as described by Jefferson et al. [62]. In the meantime, the expression of CeDGAT1 in Arabidopsis and B. napus was detected by RT-PCR using the CeDGAT1-specific primers P11 and P12 (Additional file 6: Table S2). The Arabidopsis housekeeping gene actin (primers P23 and P24) and the B. napus housekeeping gene GAPDH (primers P25 and P26) were used as internal controls.

Nile Red staining and microscopy

The Nile Red staining was used to visualize the intracellular lipid bodies as an indicators of TAG formation [63]. For yeast cell staining, a 500 μL suspension of yeast cells in the culture medium was stained with 5 μL of Nile Red (1 mg/mL in acetone stock), incubated in the dark for 5 min, and immediately used for microscopic analysis.

Lipid analysis by TLC and GC-MS

For the analysis of lipids from yeast and C. ellipsoidea, the cells were harvested by centrifugation, and the resulting cell pellets were ground to a fine powder under liquid nitrogen and subsequently treated with isopropanol at 80 °C for 10–15 min to stop the lipolytic activity. Isopropanol was evaporated under nitrogen gas before lipid extraction. The total lipids were extracted according to a modified version of the Bligh and Dyer method [64], and TAG was separated from the total lipids by thin-layer chromatography (TLC) on Silica Gel 60 plates (Merck, Darmstadt, Germany). The solvents that were used were hexane/diethyl ether/glacial acetic acid (70:30:1, v/v). The lipids were visualized by spraying Primuline (Sigma, 10 mg/100 mL acetone: water (60:40 v/v)) and exposing the plate to UV. Triolein (Sigma) was used as the standard. TAGs were recovered from the TLC plates and then trans-esterified with 5% H₂SO₄ in methanol at 85 °C for 1 h. The fatty acid methyl esters (FAMEs) were extracted with hexane and analysed by GC-MS following the methods described in the following section.

Fatty acid analysis

Cellular fatty acids were extracted by incubating 10 mg of dried seeds of control and transformed plants or 50 mg of yeast powder and freeze-dried algae powder in 3 mL of 7.5% (w/v) KOH in methanol for saponification at 70 °C for 4 h. After the pH was adjusted to 2.0 with HCl, the fatty acid were subjected to methylesterification with 2 mL of 14% (w/v) boron trifluoride in methanol at 70 °C for 1.5 h. A phase separation was produced by adding 1 mL of 0.9% (w/v) NaCl and 4 mL of hexane. The upper phase was dried under a nitrogen gas flow and resuspended in 0.3 mL of acetic ether prior to GC analysis. An analysis of fatty acid methyl esters (FAME) was performed by GC-MS (gas chromatography–mass spectrometry, TurboMass, PerkinElmer, USA) equipped with a capillary column (BPX-70, 30 m × 0.25 mm × 0.25 μm). Hydrogen was used as the carrier gas at a flow rate of 1.0 mL/min. The injector and detector temperatures were held at 250 °C. The column oven was temperature-programmed from 100 to 190 °C at 15 °C/min, where the temperature was held for 1 min increased to 220 °C at 10 °C/min, and then held for 4 min. The total FA content was quantified using heptadecanoic acid (C17:0) (Sigma) as an internal standard added to samples prior to extraction.

Dry weight determination

For dry weight determination, the algal cells were collected by filtering the culture through pre-weighed Whatman GF/C filter paper (1.2 μm pore size). Then, the filter paper was dried at 80 °C in an oven until the weight was constant.

Quantitative real-time PCR detection of CeDGAT1 expression in C. ellipsoidea

The total RNA was isolated from the cells of C. ellipsoidea at six growing points (0 h, 24 h, 36 h, 60 h, 84 h, and 108 h) during the time course of nitrogen depletion or repletion. All of the real-time reactions were performed on a LightCycler® 480 Real-Time PCR System (Roche Applied Science, Mannheim, Germany) using the LightCycler® 480 SYBR Green I Master Mix Kit (Roche Applied Science) according to the manufacturer’s instructions: 95 °C for 30 s and then 40 cycles of 95 °C for 10 s, followed by 55 °C for 10 s, and 72 °C for 20 s. All of the qRT-PCR experiments were performed in triplicate. The primers that were used for the qRT-PCR of CeDGAT1 are P27 and P28 (Additional file 6: Table S2). To normalize the transcript levels in each sample, 18S rRNA was used as the internal standard (primers P29 and P30). The relative expression was computed following the formula (Cta-Ctb), where Cta and Ctb are the average Ct values of the reference and target genes, respectively.

Alignment and molecular phylogenetic analysis

Multiple alignments were performed using MAFFT v6.847b [65] with the L-INS-i algorithm. A phylogenetic tree was reconstructed with FastTree using the approximate maximum-likelihood method [66]. For testing the robustness of the tree, 1000 bootstrap replicates were carried out. The transmembrane regions in the CeDGAT1 protein were predicted with the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html). Three-dimensional structures of the DGAT1 proteins (CeDGAT1, AtDGAT1, GmDGAT1, and BoDGAT1) were predicted by I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/).

Subcellular localization of CeDGAT1 in tobacco BY-2 Cells

To determine its subcellular location, CeDGAT1 without the stop codon was amplified from cDNA, cloned into the GATEWAY donor vector pGWC according to the method described by Chen et al. [67] and sequenced. The CeDGAT1 was then introduced in the destination vector pGWB5 with infusion with EGFP under the CaMV 35S promoter by LR reaction following the manufacturer’s instructions (Invitrogen). The final construct CeDGAT1::EGFP was transferred into Agrobacterium tumefaciens strain GV3101. The Agrobacterium-mediated transformation of tobacco BY2 cells was performed according to An [68] and Genschik et al. [69]. Briefly, BY2 cells were incubated for 3 days at 27 °C in the dark (without shaking) with Agrobacterium GV3101 containing CeDGAT1::EGFP. Subsequently, cells were plated on medium containing two antibiotics: timentin (500 mg/L) to kill off Agrobacterium and kanamycin (100 mg/L) to select for transformed cells. Transformed cells appeared after one month as a callus on plates and were then transferred to fresh plates once a month. A suspension culture was obtained by the addition of small transformed callus clumps to liquid culture medium containing kanamycin. The transformed BY-2 suspension cells were stained with ER-tracker™ Red following the manufacturer’s instructions (Invitrogen) and then observed and photographed using a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Germany).

Statistical analysis

All of the experimental data were statistically compared using a one-way analysis of variance (ANOVA) with the software Statistical Product and Service Solutions (SPSS) v19.0, followed by a post-hoc test to determine the significant difference among the treatment means.