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Identification and characterization of a delta-12 fatty acid desaturase gene from marine microalgae Isochrysis galbana
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Xiaotian Han1, 2, 3, Shuai Wang4, Li Zheng3, 4, *, Wanshun Liu1
Acta Oceanologica Sinica | 2019, 38(2) : 107 - 113
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Acta Oceanologica Sinica | 2019, 38(2): 107-113
• Marine Biology •
Identification and characterization of a delta-12 fatty acid desaturase gene from marine microalgae Isochrysis galbana
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Xiaotian Han1, 2, 3, Shuai Wang4, Li Zheng3, 4, *, Wanshun Liu1
Affiliations
  • 1 College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
  • 2 Changjiang Estuary Marine Ecosystem Research Station, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
  • 3 Marine Ecology and Environmental Science Laboratory, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
  • 4 Key Laboratory for Marine Bioactive Substances and Modern Analytical Technology, First Institute of Oceanography, Ministry of Natural Resources, Qingdao 266061, China
Published: 2019-02-25 doi: 10.1007/s13131-019-1354-1
Outline
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The cDNA of the delta-12 fatty acid desaturase gene, IgFAD2, was cloned from the marine microalgae Isochrysis galbana, a species capable of producing docosahexaenoic acid. Sequence analysis indicated that the open reading frame measured a length of 1 158 bp and encoded 386 amino acids with a predicted molecular weight of 42.8 kDa and an isoelectric point of 9.2. Computational analysis of the protein sequence of IgFAD2 showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that were universally present among plant desaturases. Quantitative real-time PCR results showed that the abundance of IgFAD2 transcript was significantly upregulated under different environmental stresses including low temperature (15°C), high salinity (salinity of 62 and 93), and nitrogen starvation (220 μmol/L). Heterologous expression indicated that yeast cells transformed with a plasmid construct containing IgFAD2 could convert endogenous oleic acid (18:1∆9, OA) into linoleic acid (18:2∆9, 12, LA). These findings confirm that I. galbana IgFAD2 plays important roles in the biosynthetic pathways of unsaturated fatty acids.

delta-12 fatty acid desaturase  /  expression analysis  /  Isochrysis galbana
Xiaotian Han, Shuai Wang, Li Zheng, Wanshun Liu. Identification and characterization of a delta-12 fatty acid desaturase gene from marine microalgae Isochrysis galbana[J]. Acta Oceanologica Sinica, 2019 , 38 (2) : 107 -113 . DOI: 10.1007/s13131-019-1354-1
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1 Introduction
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Fatty acids in plants, as in all other organisms, are the major structural components of membrane phospholipids and triacylglycerol storage oils (Sørensen et al., 2005; Li et al., 2007). Fatty acid desaturases are enzymes responsible for the production of unsaturated and polyunsaturated fatty acids (Alonso et al., 2003). Among these enzymes, delta-12 fatty acid desaturase is the first and the rate-limiting step enzyme that introduces a double bond between the twelfth and thirteenth carbon atom of fatty acid chain and thereby converts oleic acid (18:1∆9, OA) into linoleic acid (18:2∆9, 12, LA) in fatty acid biosynthesis pathway (Wei et al., 2004; Zhang et al., 2009; Khadake et al., 2009).
Changing levels of unsaturated fatty acids (UFA) have a crucial role in maintaining the fluidity of membrane lipids when plants are subjected to abiotic stress (Allakhverdiev et al., 2001; Alonso et al., 2003). The ability of cells to regulate the desaturation of membrane lipids is mainly determined by the action of fatty acid desaturases (Mendes et al., 2012). The transcriptional level of fatty acid desaturases has association with diverse environmental factors, such as temperature, salinity, and nitrogen availability (Allakhverdiev et al., 2001; Kargiotidou et al., 2008; Lu et al., 2009; Zhang et al., 2011). Several fatty acid desaturase genes have been cloned from various organisms such as fungi (Sakai and Kajiwara, 2005; Gostinčar et al., 2009), algae (Domergue et al., 2003; Lu et al., 2009; Iskandarov et al., 2010; Wang et al., 2016), moss (Kaewsuwan et al., 2006; Chodok et al., 2013) and higher plants (Mietkiewska et al., 2006; Cao et al., 2013; Lozinsky et al., 2014). However, as an important player in fatty acid biosynthesis pathway, the effects of abiotic stress on the gene expression of delta-12 fatty acid desaturases and their heterologous expression have yet to be investigated in microalgae without cell wall.
As one of the most promising biofuel producers, Isochrysis galbana is receiving increasing attention owing to its high lipid content, photosynthesis efficiency, land avoidance and cultivation success (Demirbas and Demirbas, 2011; Sánchez et al., 2013). In addition, I. galbana, a unicellular photoautotrophic microalga without cell wall, is rich in ω-3 polyunsaturated fatty acids (PUFAs). In the present work, we reported the isolation and characterization of a delta-12 fatty acid desaturase gene (IgFAD2) from I. galbana via rapid amplification of cDNA ends (RACE) and analyzed the gene expression levels under stress conditions through quantitative real-time PCR (qRT-PCR). Moreover, the heterologous expression of this gene in yeast Saccharomyces cerevisiae was investigated.
2 Materials and methods
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2.1 Microalgae culture and stress treatments
The microalga I. galbana was obtained from the Institute of Oceanology, Chinese Academy of Sciences. The microalgae (initially 2.6×105 cell/mL) were grown in liquid f/2 medium (880 μmol/L nitrogen) (Guillard and Ryther, 1962) at 20°C and at a salinity of 31 under a constant irradiation of 100 μmol/(m2·s).
In order to determine the abundance of IgFAD2 transcript at different conditions, the microalgae were cultured at 15°C and 25°C under the stable illumination duration for 6, 12, 24, and 48 h; at the salinity of 62 and 93 for 6, 12, 24, 48, and 72 h; or in medium containing 220 μmol/L and 1 760 μmol/L nitrogen for 6, 12, 24, and 48 h.
2.2 Total RNA extraction
The microalgae were harvested for extracting cellular total RNA through centrifugation at 12 000× g and 4°C for 10 min. Algal cells were ground into powder in liquid nitrogen. Total RNA was extracted by the method of Wang et al. (2016). The concentration of total RNA was determined by measuring the UV absorbance at 260 nm using a Thermo Scientific NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and the RNA purity was checked by determining the A260/A280 ratio and 1.2% denaturing agarose gel electrophoresis.
2.3 Rapid amplification of IgFAD2 cDNA ends
The mRNA was purified from total RNA extracted with CTAB method by using an Oligotex mRNA Mini Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. The reverse transcription reaction was performed with 3′-RACE CDS Primer and 5′-RACE CDS Primer and PrimeScriptTM Reverse Transcriptase (TaKaRa, Tokyo, Japan) following the manufacturer’s protocol. The RACE was performed using IgFAD2 RC1 and RC2 (Table 1) and SMART RACE cDNA Amplification Kit (Clontech, CA, USA) following the manufacturer’s instructions. The PCR products were purified with a gel purification kit (Tiangen, Beijing, China), subcloned into pMD18-T vector (TaKaRa, Tokyo, Japan) and sequenced (Sunny Biotechnology Company, Shanghai). The full-length cDNAs were assembled on the basis of the sequences of 3′-RACE and 5′-RACE PCR fragments.
2.4 Bioinformatics analysis
The full-length cDNA sequence of IgFAD2 was assembled with SeqMan software of DNASTAR 7.1. The theoretical MW and pI of IgFAD2 were calculated with ExPASy Compute pI/Mw tool (http://au.expasy.org/tools/pi_tool.html). Multiple alignments were created using ClustalX and analyzed using BoxShade program (www.ch.embnet.org). Transmembrane regions were predicted through DAS Transmembrane Prediction server (http://www.sbc.su.se/~miklos/DAS/maindas.html). Phylogenetic tree was constructed with Mega 5.0 by using a neighbor-joining algorithm with 1 000 permutations.
2.5 Quantitative real-time PCR
The IgFAD2 expression was analyzed through qRT-PCR with Stratagene Mx3000P® qPCR system and SYBR® PrimeScriptTM RT-PCR kit (TaKaRa, Tokyo, Japan). According to the manufacturer’s instructions, a pair of primers (RT1, RT2) (Table 1) was designed and used to quantify the abundance of IgFAD2 transcript. The abundance of rbcL (RT3, RT4) was used as an endogenous control. The qRT-PCR was performed for 40 cycles of denaturation at 95°C for 10 s and annealing at 51°C for 10 s and at 72°C for 10 s. All of the reactions were conducted in at least three duplicates. The qRT-PCR data were examined with comparative Ct (2–∆∆Ct).
2.6 Expression in yeast
The open reading frames were amplified with specific primers, IgFAD2 F1 and IgFAD2 R1 (Table 1), and subcloned into the yeast expression vector pYES2.0 (Invitrogen) under the control of GAL1 promoter. The 5′ end of the F1 and R1 contained an EcoRI or a BamHI restriction site (italicized) to facilitate subsequent manipulation. The sequence orientation and identity were confirmed by sequencing and the resulted plasmid was designated pYFAD2. Plasmids pYES2.0 and pYFAD2 were introduced into the S. cerevisiae INVSc1 using electroporation. Yeast cultures were grown to logarithmic phase at 28°C in synthetic minimal medium (SC-Ura). The cells were incubated at 15°C for 48 h. The cells were harvested, and fatty acids were assayed.
2.7 Fatty acid analysis
Fatty acids were extracted and determined according to the method of Yang et al. (2013). In brief, fatty acid methyl esters (FAME) were identified and quantified after splitless injection and run in temperature programming by using an Agilent 7890A GC instrument equipped with a HP-5MS capillary column (30 m×0.25 mm×0.25 µm) coupled to an Agilent 5975C mass spectrometer.
2.8 Statistical analysis
Analysis of variance (ANOVA) and t–test were conducted with SPSS version 16.0. Observations were expressed as mean±standard error. The probability of <0.05 was used in judging significant difference.
3 Results
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3.1 Cloning and characterization of the IgFAD2 gene from I. galbana
The cDNA of IgFAD2 contained a 1 158 bp ORF which encoded a protein of 386 amino acid residues with a deduced molecular mass of 42.8 kDa and the theoretical pI of 9.2. The sequence was deposited in GenBank with an accession number of AFB82638. The assembled full-length cDNA of IgFAD2 had 81% and 57% similarity to delta-12 fatty acid desaturases of Emiliania huxleyi CCMP1516 (XP005759480) and Chrysochromulina sp. CCMP291 (KOO28400), respectively. The deduced IgFAD2 was clustered within microsomal delta-12 fatty acid desaturase (FAD2) group. All of the plastidial delta-12 fatty acid desaturases (FAD6) are grouped. Based on the sequence similarity and phylogenetic analysis, it implies that IgFAD2 encodes a microsomal delta-12 fatty acid desaturase (Fig. 1). The deduced IgFAD2 was highly conserved; it contained three conserved motifs (HECGH, HAKHH and HVVHH), which is a characteristic feature of membrane-bound desaturases (Fig. 2). It also presented four strong hydrophobic transmembrane domains (66–88, 116–135, 174–195 and 230–257) (Fig. 3), which is common to most membrane-bound desaturases and presented well-conserved domains between algae and high plants (Fig. 2).
3.2 Effects of temperature, salinity and nitrogen concentration stress on the IgFAD2 expression of I. galbana
In temperature treatments, the abundance of IgFAD2 transcript increased by 8.6-fold when the alga was cultured at 15°C for 12 h compared with the control (Fig. 4). In salt stress treatments, the IgFAD2 transcript reached the maximum abundance (5.9-fold of the control) when the alga was cultured at a salinity of 62 for 24 h compared with the control (Fig. 5). In nitrogen concentration treatment, I. galbana was cultured at 880 μmol/L treatment (nitrogen concentration in f/2 medium) as control. The qRT-PCR analysis results showed that the maximum IgFAD2 mRNA expression level was reached (2.6-fold of the control) at 220 μmol/L treatment for 12 h (Fig. 6).
3.3 Functional analysis in S. cerevisiae
Heterologous expression in yeast was used to confirm delta-12 fatty acid desaturase regioselectivity and function. To validate the protein activity, both pYFAD2 and pYES2.0 (control) were transformed into S. cerevisiae INVSc1. The total lipids of the transformants were subjected to GC-MS analysis. The results showed that a novel peak, corresponding to LA (18:2Δ9, 12) methyl ester standards, were detected in the transgenic S. cerevisiae expressing the IgFAD2 gene (Table 2). In contrast, the peak normally was not present in the wild-type yeast cells. Four fatty acids were mainly found in S. cerevisiae, namely, C16:0, C16:1, C18:0, and C18:1. The finding indicates that pYFAD2 encodes a delta-12 fatty acid desaturase, which can convert C18:1 into C18:2 in yeast.
4 Discussion
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delta-12 fatty acid desaturase emerges to be the key enzyme in the synthesis of LA (18:2Δ9, 12), a crucial precursor for producing subsequent PUFA production. Isochrysis galbana is known to contain an essential amount of LA. Although several desaturases and elongases have been elucidated from I. galbana (Qi et al., 2002; Wang et al., 2016), the delta-12 fatty acid desaturase associated with LA biosynthesis has not been functionally identified. Currently, delta-12 fatty acid desaturase genes have been isolated from microalgae, including Phaeodactylum tricornutum (Domergue et al., 2003), Chlorella vulgaris (Lu et al., 2009), Parietochloris incisa (Iskandarov et al., 2010), and Chlamydomonas sp. (Zhang et al., 2011). In this research, we identified a delta-12 (∆12) fatty acid desaturase, IgFAD2, from I. galbana using RACE method. IgFAD2 contains three histidine boxes (HXXXH, HXXHH and HXXHH) that have similar characteristics to all membrane-bound desaturases, and FAD2s that are also similar to other plant species. The histidine-rich motifs in the sequence are thought to be involved in the oxygen activation and substrate activation process through formation of a di-iron center part (Khadake et al., 2009). Nevertheless, IgFAD2 did not contain a cytochromeb5-like domain including the HPGG motif in the heme-binding region, which is normally present in front-end desaturases.
Changes in temperature can affect the biomass of microalgae, which associates with gene transcription and related enzyme activity. It has been confirmed that the cell activity of microalgae will decline when the temperature decreases due to the reduction of enzyme activity (Chong et al., 2011). However, the expression of fatty acid desaturases involved in lipid biosynthesis will be upregulated to increase membrane fluidity at low temperature (Zhang et al., 2011; Han et al., 2013). The findings on the mechanisms of temperature-dependent fatty acid composition alterations in plant membrane lipids have provided evidence of control in both the transcriptional and translational levels for delta-12 fatty acid desaturase genes (Chinnusamy et al., 2007). Based on the qRT-PCR analysis results, IgFAD2 mRNA transcript level was higher at 15°C compared with the algae at 20°C (Fig. 4). The IgFAD2 mRNA expression levels increase to 8.6-fold at 15°C for 12 h. The IgFAD2 mRNA transcript expression is consistent with the delta-12 fatty acid desaturase, which was isolated from the Antarctic microalgae C. vulgaris NJ-7. The accumulation of delta-12 fatty acid desaturase gene transcripts increased by 2.2-fold at 15°C compared with the algae at 25°C (Lu et al., 2009). Miyasaka et al. (2000) reported that the level of transcript of delta-12 fatty acid desaturase gene of Chlamydomonas sp. increased by 2.3-fold at 4°C for 6 h, whereas the transcript level under heat stress (38°C) for 6 h was only 87% of the control.
Salt stress is among the main environmental factors that limit the growth and productivity of plants and microorganisms. Several reports have suggested that lipids might be involved in the protection against salt stress (Turk et al., 2004; Gostinčar et al., 2009). Salt stress induced increases in the unsaturated fatty acids of membrane lipids for the sake of adaptation to a wide range of NaCl concentrations (Lu et al., 2009). This phenomenon has been observed in yeasts (Gostinčar et al., 2009), fungi (Turk et al., 2007; Lin et al., 2017) and cyanobacteria (Allakhverdiev et al., 2001; Kumar et al., 2015). Furthermore, the delta-12 fatty acid desaturase gene is isolated from the Antarctic ice algae C. vulgaris NJ-7, and the mRNA accumulation of gene transcripts increased up to 8.5-fold at a salinity of 62 compared with the algae at a salinity of 31 (Lu et al., 2009). Similar results were obtained for the delta-12 fatty acid desaturase gene is isolated from Antarctic microalgae Chlamydomonas sp. ICE-L, in which the expression level increased by 3.8-fold at a salinity level of 62 for 2 h (Zhang et al., 2011). In this study, IgFAD2 was involved in the adaptation to high salinity stress and reached the maximum expression level of 5.9-fold after 62‰ NaCl treatment for 24 h compared with the control (Fig. 5). The unsaturated fatty acid (UFA) content in I. galbana increased at a salinity of 62 (data not shown).
Limited nitrogen concentration of the medium prompts fatty acid accumulation in a wide range of microalgal species (Jiang et al., 2012; Griffiths et al., 2012). Lipid content has been documented to increase when I. galbana is cultured under nitrogen starvation (Mairet et al., 2011), as it does in C. Reinhardtii (Miller et al., 2010). Wang et al. (2016) reported that the level of transcript of delta –6 fatty acid desaturase gene of Isochrysis sp. increased by 4.5-fold in nitrogen-deplete medium for 6 h. The results are consistent with our experiments. In nitrogen deficiency, IgFAD2 mRNA expression level increases by 2.6-fold at 220 μmol/L treatment for 12 h (Fig. 6).
Heterologous expression in yeast was used to confirm delta-12 regioselectivity and function of IgFAD2. Both pYFAD2 and empty vector, pYES2.0 (control), were transformed into the S. cerevisiae INVSc1. The total lipids of the transformants were determined through GC-MS analysis. The findings demonstrate that a novel peak was present in pYFAD2, which was absent from the control (Table 2). The novel peak was LA (18:2∆9, 12) by comparison of the retention time to FAME standard mixtures (Sigma). The result indicated that IgFAD2 encodes a delta-12 fatty acid desaturase, which can convert C18:1 into C18:2 in yeast. This finding is consistent with the recent reports on delta-12 fatty acid desaturases from the microalgae C. vulgaris (Lu et al., 2009), the fungus Lentinula edodes (Sakai and Kajiwara, 2005), Rhizopus arrhizus (Wei et al., 2004) and the higher plants Olea europaea (Hernández et al., 2005), Gossypium hirsutum (Zhang et al., 2009), Linum usitatissimum L. (Khadake et al., 2009). In contrast, delta-12 fatty acid desaturases from the higher plants Gossypium hirsutum (Pirtle et al., 2001), Tropaeolum majus (Mietkiewska et al., 2006), Physcomitrella patens (Chodok et al., 2013), Physaria fendleri (Lozinsky et al., 2014), and the microalgae P. tricornutum (Domergue et al., 2003) have been shown to have specific activity for C16:1 and C18:1 fatty acid substrates. Although the reason remain far to be elucidated, the different hydrophobic profiles of FAD2s that indicate diverse transmembrane topologies among various organisms may be useful to clarify this phenomenon (Wei et al., 2004; Chodok et al., 2013).
5 Conclusions
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In conclusion, a cDNA of the delta-12 fatty acid desaturase gene (IgFAD2) was isolated from I. galbana. The full-length cDNA of IgFAD2 contained a 1 158 bp ORF, which encodes a fatty acid desaturase protein comprising 386 amino acids with a deduced molecular mass of 42.8 kDa and the theoretical pI of 9.2. The computational analysis of the protein sequence of IgFAD2 revealed three conserved histidine motifs. Under different stress conditions, the results of qRT-PCR showed that the IgFAD2 expression was upregulated by low temperature (15°C), high salinity (62 and 93), and nitrogen starvation (220 μmol/L). Heterologous expression indicated that the cDNA encoded a delta-12 fatty acid desaturase from I. galbana was able to convert C18:1 into C18:2. These findings may clarify the importance of delta-12 desaturase activity in polyunsaturated fatty acids biosynthesis.
Funding
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  • The Basic Scientific Fund for National Public Research Institutes of China under contract No. 2017Q09; the Aoshan Science and Technology Innovation Project of Pilot National Laboratory for Marine Science and Technology (Qingdao) under contract No. 2016ASKJ02; the National Natural Science Foundation of China-Shandong Joint Funded Project under contract No. U1606404; the 973 Project from Chinese Ministry of Science and Technology under contract No. 2015CB755904; the National Natural Science Foundation of China under contract Nos 41776176 and 41806201; the Shandong Provincial Natural Science Foundation under contract No. ZR2015PD003.
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Article Info
doi: 10.1007/s13131-019-1354-1
  • Receive Date：2017-11-23
  • Online Date：2026-03-31
  • Published：2019-02-25
Article Data
Affiliations
History
  • Received：2017-11-23
  • Accepted：2018-05-10
Funding
The Basic Scientific Fund for National Public Research Institutes of China under contract No. 2017Q09; the Aoshan Science and Technology Innovation Project of Pilot National Laboratory for Marine Science and Technology (Qingdao) under contract No. 2016ASKJ02; the National Natural Science Foundation of China-Shandong Joint Funded Project under contract No. U1606404; the 973 Project from Chinese Ministry of Science and Technology under contract No. 2015CB755904; the National Natural Science Foundation of China under contract Nos 41776176 and 41806201; the Shandong Provincial Natural Science Foundation under contract No. ZR2015PD003.
Affiliations
    1 College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
    2 Changjiang Estuary Marine Ecosystem Research Station, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
    3 Marine Ecology and Environmental Science Laboratory, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
    4 Key Laboratory for Marine Bioactive Substances and Modern Analytical Technology, First Institute of Oceanography, Ministry of Natural Resources, Qingdao 266061, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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