Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor

Accepted Manuscript Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor

Zhideng Lin, Yisheng Huang, Weiguang Zou, Hua Rong, Meiling Hao, Xiaobo Wen PII: DOI: Reference:

S0378-1119(18)30284-1 doi:10.1016/j.gene.2018.03.046 GENE 42668

To appear in:

Gene

Received date: Revised date: Accepted date:

12 September 2017 8 March 2018 15 March 2018

Please cite this article as: Zhideng Lin, Yisheng Huang, Weiguang Zou, Hua Rong, Meiling Hao, Xiaobo Wen , Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2018.03.046

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ACCEPTED MANUSCRIPT Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu’s croaker Nibea coibor

Zhideng Lin

a, 1

, Yisheng Huang

a, 1

, Weiguang Zou a, Hua Rong a, Meiling Hao b, Xiaobo

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Wen a, **

Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, Shantou 515063,

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a

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Running title: Cloning and expression analysis of Elovl5 elongase in Nibea coibor

College of Marine Science, South China Agriculture University, Guangzhou 510642, China

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b

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China

Shantou

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** Corresponding author: Xiaobo Wen, Guangdong Provincial Key Laboratory of Marine Biology, University,

243

Daxue

Road,

Shantou,

Guangdong

515063,

(+86)-754-86503435; Fax: (+86)-754-82903473; E-mails: [email protected]. 1

Joint first authorship

1

China.

Tel:

ACCEPTED MANUSCRIPT Abstract: Enzymes that lengthen the carbon chain of polyunsaturated fatty acids (PUFA) are key to the biosynthesis of the long-chain polyunsaturated fatty acids (LC-PUFA). Here we report on the molecular cloning, tissue distribution, functional characterization and nutritional regulation of a elovl5 gene from Nibea coibor. The full-length cDNA was 1315 bp, including a 5-untranslated region (UTR) of 134 bp, a 3-UTR of 296 bp and an open reading frame of 885 bp, which specified

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a peptide of 294 amino acids. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of microsomal fatty acyl elongases, including the so-called

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histidine box (HXXHH), the canonical C-terminal endoplasmic reticulum retention signal, several

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predicted transmembrane regions and other highly conserved motifs. Expression of elovl5 was strongly observed in stomach, and more weakly in kidney, spleen, intestine, brain, eye, liver, gill,

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muscle and heart. Functional characterization revealed that the chu’s croaker Elovl5 was able to elongate both C18 and C20 PUFA substrates. Nutritional study indicated that the hepatic

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expression of elovl5 could be up-regulated by low dietary n-3 LC-PUFA. These results may contribute to better understanding the LC-PUFA biosynthetic pathway and regulation mechanism

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in chu’s croaker.

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Keywords: Cloning; Functional characterization; elovl5; Nibea coibor; Nutritional study; LC-PUFA 1. Introduction

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Long-chain polyunsaturated fatty acids (LC-PUFA) are a group of polyunsaturated fatty acids (PUFA) with more than three double-bonds and twenty carbons. Physiologically active LC-PUFA

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mainly includes arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), which are proved to have pivotal roles in animal’s growth, development and immune function (Nænss and Lie, 1998; Masuda et al., 1998; Benítez-Santana et al., 2007; Vivianeverlhac, 2010; Imanpoor et al., 2011; Zakeri et al., 2011; Campoy et al., 2012; Delgado-Lista et al., 2012; Gil et al., 2012; Zuo et al., 2012; Ma et al., 2013; Tacon and Metian, 2013; Tocher, 2015). At present, fish are an important source of n-3 LC-PUFA for human. With global fisheries generally in decline (Worms et al., 2006), aquaculture produces an escalating proportion of fish/seafood in the human food basket amounting to around 50 % in 2008 to till recent year (FAO, 2012). However, the high levels use of vegetable oils (VO) in aquafeeds has 2

ACCEPTED MANUSCRIPT resulted in reduced deposition of n-3 LC-PUFA in farmed fish, especially marine teleosts deficient or weak in synthesizing LC-PUFA, and decreased the nutritional value for human consumers (Bell et al., 2001; Lee and Cho, 2009; Turchini et al., 2010; Duan et al., 2014; Sprague et al., 2016). Hence, there is great interest in elucidating the endogenous LC-PUFA biosynthesis pathway and potential regulation mechanism, which will facilitate the efficient and effective utilization of sustainable VO while maintaining the nutritional quality of farmed fish.

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As with all vertebrates, fish lack Δ12 and Δ15 desaturases so that they cannot synthesize the

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linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3) from oleic acid (18:1n-9). LA and ALA therefore are essential fatty acids (EFAs) for fish. These EFAs can be further desaturated and

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elongated to form biologically active compounds, such as DHA, EPA and ARA. However, the extent to which fish can transform the C18 PUFA (LA and ALA) into LC-PUFA depends upon the

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presence and expression of two categories of enzymes, fatty acid desaturase (Fads) and elongase of very long-chain fatty acids (Elovls) (Hastings et al., 2001; Miyazaki and Ntambi, 2008). Elovls

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are responsible for the condensation reaction in the elongation step resulting in the addition of a two-carbon unit to the carboxyl end of the fatty acid (Guillou et al., 2010). Seven family members

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termed Elovl1 to Elovl7, with differing fatty acid substrate specificities, have been identified in

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vertebrates (Leonard et al., 2000; Leonard et al., 2002; Agaba et al., 2004; Leonard et al., 2004; Zheng et al., 2005; Tamura et al., 2009; Guillou et al., 2010; Castro et al., 2016). Generally, Elovl2, Elovl4 and Elovl5 are regarded as critical enzymes in the elongation of PUFA (Jakobsson et al.,

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2006). Previous studies have indicated that marine fish species lack Elovl2, which is predominantly active on C20 to C22 PUFA, and is regarded as an essential enzyme in DHA

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biosynthesis (Agaba et al., 2004; Morais et al., 2009; Gregory and James, 2014; Oboh et al., 2016). Elovl4 has been isolated from several marine and freshwater fish species (Carmona-Antoñanzas et al., 2011; Monroig et al., 2011; Kabeya et al., 2015; Monroig et al., 2012; Li et al., 2015, 2017; Jin et al., 2017), and could elongate C20 fatty acids to longer-chain fatty acids (up to C36). The lack of Elovl2 in marine fish has been considered as one possible reason for their low LC-PUFA biosynthetic capability. However, Elovl4 can also elongate 22:5n-3 to 24:5n-3 in fish, suggesting that this enzymes has the potential to participate in the production of DHA, and thus partly compensate for the above mentioned absence of Elovl2 in marine species (Morais et al., 2009; Jin et al., 2017). To date, elovl5 have been cloned and functionally characterized from numerous fish 3

ACCEPTED MANUSCRIPT species (Agaba et al., 2004; Hastings et al., 2004; Morais et al., 2009; Monroig et al., 2012; Monroig et al., 2013; Kabeya et al., 2015; Li et al., 2017). These studies confirmed that fish Elovl5, similar to mammalian homologues (Jakobsson et al., 2006), showed high activity towards C18 (C18:4n-3 and C18:3n-6) and C20 (C20:4n-6 and C20:5n-3) PUFA, with a low activity towards C22 PUFA (C22:4n-6 and C22:5n-3).

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The Nibea coibor is an important carnivorous marine fish species and have a wide distribution covering India, the Philippines, and the South and East China Sea (Shen et al., 2004).

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Due to their high tolerance and viability, large size, high meat yield, delicious meat and high market price, Nibea coibor has been identified as a potential candidate for aquaculture in China

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(Shao, 2006; Chai et al., 2013; Huang et al., 2016a, 2016b, 2016c). In recent years, numerous studies have been carried out to investigate culture technique of N. coibor in cages, reproduction

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in captivity, nutrient requirements and sequence analysis of mitochondrial 16S rRNA (Shen et al., 2004; Xin et al., 2007; Huang et al., 2016a, 2016b, 2016c). However, to the best of our knowledge,

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little information was available about the molecular basis of LC-PUFA biosynthesis in chu's croaker (Huang et al., 2017). In the present study, we describe the cDNA cloning, tissue

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distributions, functional characterization and nutritional regulation a Elovl5 elongase from chu's

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croaker under different palm oil (PO) substitution levels. These results may contribute to better understanding of the LC-PUFA biosynthetic pathway and regulation mechanism in this species. 2. Materials and methods

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2.1. Experimental diets

Diets were formulated to be isolipidic and isonitrogenous (45.0 % protein and 12.5 % lipid)

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with different levels of fish oil (FO) and PO, named 100 % FO (FO), 80 % FO and 20 % PO (2PO), 60 % FO and 40 % PO (4PO), 40 % FO and 60 % PO (6PO), 20 % FO and 80 % PO (8PO) and 100 % PO (10PO) (Table 1). Dietary protein in the diet derived from defatted white fish meal (Coastal Villages Pollock, Anchorage, AK), casein (Hualing Casein Co. Ltd., Gansu, China) and soy protein concentrate (Wangdefu Group Co. Ltd., Shandong, China), while lipids were provided by FO (Improved Variety Introduction Co. Ltd., Guangdong, China) and crude PO (Yihai Food Marketing Co. Ltd., Shanghai, China). The fat-free white fish meal was defatted by using ethanol (1:1, w/v) with five successive treatments until (Sheen, 2000). All of the ingredients were finely ground and passed through a 120-mesh sieve, mixed with oil and water until a stiff dough was 4

ACCEPTED MANUSCRIPT obtained. Pellets were made by using a laboratory feed-pelletizer equipped with a 2-mm die (SLP-45; Fishery Mechanical Facility Research Institute, Shanghai, China) at 45 ± 5 C (Wang et al., 2014). Diets were dried at 16 C overnight in air conditioner room, sealed in vacuum-packed bags, and stored frozen at -20 C until used. About 20 g of each diet were sampled for further analysis. Main fatty acids (% area) of the experimental diets are shown in Table 1.

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2.2. Growth experiment and sampling The growth experiment was held at NanAo Marine Biology Station (NAMBS), Shantou

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University, Shantou, China. The juveniles purchased from a local hatchery were reared in culture cages (2.8 m × 2.8 m × 2.2 m) and fed on a commercial diet (45.0 % protein and 12.5 % lipid) for

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20 days to acclimate to the experimental conditions. The juveniles were fasted for 48 h before the start of the experiment, and weighed under anesthesia (40 mg L-1 eugenol) to minimize stress

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(Eugenol, Shanghai Reagent, Shanghai, China) (lversen et al., 2003). First, six fish were randomly collected and stored at -20 °C for initial body composition analysis. Second, a total of 450 healthy

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juveniles (initial body weight 76.0 ± 0.8 g) were randomly distributed into 18 experimental culture cages (25 fish a cage, cage size: 1 m × 1 m × 1.5 m). Fish were fed twice daily until apparent

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satiation (07:30 and 16:30 h). The experimental conditions were recorded daily from May to July,

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2015 (56 days), including temperature 24.5 to 30.5 °C, pH 7.8 to 8.1, salinity 31 to 33 g L-1, ammonia nitrogen ≤ 0.05 mg L-1, and dissolved oxygen 5.2 to 5.5 mg L-1. Before the final sampling, the juveniles were anaesthetized with eugenol as described above.

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Livers were dissected from six fish per treatment (two fish from each replicate cage) and stored in liquid nitrogen until further analysis. In order to determine the tissue distribution of chu’s croaker

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putative elongase, tissues including heart, liver, gills, stomach, spleen, kidney, muscle, skin, brain and eye were also collected from six chu’s croaker fed with diet (45.0 % protein and 12.5 % lipid) and frozen in liquid nitrogen until further analysis. 2.3. RNA isolation and full length cDNA cloning Total RNA was extracted from liver of chu’s croaker following the manufacturer’s instructions of Trizol® Reagent (Invitrogen, USA). Potential genomic DNA contamination was eliminated by DNase I digestion (Takara, Dalian, China). The quality and quantity of purified RNA were detected by measuring the absorbance at 260/280 nm with Thermo Scientific NanoDrop 2000 spectrophotometer (Thermo Scientific Nanodrop, USA), the integrity was 5

ACCEPTED MANUSCRIPT confirmed by using 1 % (w/v) agarose gel. First strand cDNA was synthesized from 4 μg total RNA with PrimeScriptTM II 1st Strand cDNA Synthesis Kit following the manufacturer’s instructions (Takara, Dalian, China). The primers Elovl5F and Elovl5R (Table 2) were designed, based on conserved regions from the alignment of Argyrosomus regius (KC261977.1), Nibea mitsukurii (FJ952143.1),

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Rachycentron canadum (FJ440239.1) and Larimichthys crocea (NP_001290303) Elovl5 sequences. The first elovl5 gene fragment was amplified using liver cDNA as template for

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polymerase chain reaction (PCR) with 2 × Taq PCR MasterMix (Tiangen, Beijing, China). The process included an initial denaturation of 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 59 °C for

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30 s and 72 °C for 1 min, and with a final extension step at 72 °C for 5 min. The final products were separated using a 1 % agarose gel, and then purified with TIANgel Midi Purification Kit

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following the manufacturer’s instruction (Tiangen, Beijing, China). The purified PCR product was then ligated with pMD19-T vector (Takara, Dalian, China), and transformed into competent

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Escherichia coli cells. Positive recombinant clones were identified by PCR screening with M13R and M13F primers (Table 2), and subsequently sequenced by a commercial company (BGI,

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Shenzhen, China).

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Based on the obtained elovl5 partial fragment, four gene-specific primers, Elovl5 3-1, Elovl5 3-2, Elovl5 5-1 and Elovl5 5-2 (Table 2), were designed. The full-length elovl5 cDNA was amplified through 5’ and 3’ rapid amplification of cDNA ends (RACE) PCR with the SMARTerTM

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RACE cDNA Amplification Kit (Clontech, USA) using touch-down PCR (first round PCR) and nested PCR (second round PCR) strategies. The first round PCR was performed using the gene

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specific sense primers Elovl5 3-1 or Elovl5 5-1 and the adapter specific primer UPM (0.4 mM Long Universal Primer and 2 mM Short Universal Primer). The PCR reactions were in a 50 µl volume containing 5 µl of 10 × PCR buffer, 8 µl of dNTPs (2.5 mM), 2.0 µl of Elovl5 3-1 or Elovl5 5-1 primer (10 µM), 5 µl of UPM, 0.5 µl of Taq polymerase (5 U µl-1) (Takara, Dalian, China), 2 µl of cDNA and 27.5 µl PCR-grade water. PCR consisted of an initial denaturation at 95 °C for 3 min, followed by 5 cycles at 95 °C for 30 s, 72 °C for 2 min 30 s; 5 cycles at 95 °C for 30 s, 68 °C for 30 s, 72 °C for 2 min; 25 cycles at 95 °C for 30 s, 64 °C (3’RACE) or 63 °C (5’RACE) for 30 s, 72 °C for 2 min; and a final elongation step at 72 °C for 10 min. The product of first round PCR was used as template for the subsequent nested PCR, which was also 6

ACCEPTED MANUSCRIPT performed in a 50 µl reaction volume including 25 µl of 2 × Taq PCR MasterMix (Tiangen, Beijing, China), 1 µl of Elovl5 3-2 or Elovl5 5-2 primers, 1 µl of NUP primer, 0.5 µl of template, and 22.5 µl PCR-grade water. The nested PCR conditions consisted of an initial denaturing step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 63 °C (3’RACE) or 64 °C (5’RACE) for 30 s, extension at 72 °C for 2 min, followed by a final extension

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at 72 °C for 5 min. The PCR products were sequenced according to the method described above. The sequences of all PCR primers used in this study are shown in Table 2.

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generated

sequences

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2.4. Sequence and phylogenetic analyses of chu’s croaker elovl5

similarity

by

using

BLASTx

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(http://blast.ncbi.nlm.nih.gov/). The partial fragment, as well as 3’- and 5’- end sequences, were assembled in DNAMAN to obtain the complete elovl5 cDNA. The deduced amino acid sequence

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was obtained with the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi). The molecular mass and the theoretical isoelectric point (pI) of the Elovl5 protein was predicted using Compute

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pI/Mw tool (http://cn.expasy.org/tools/pi_tool.html). Multiple protein sequences alignment was performed using the ClustalX2. Phylogenetic analysis of elongase polypeptides was performed by

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constructing a tree using the neighbor-joining method (Saitou and Nei, 1987). Confidence in the

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resulting phylogenetic tree branch topology was measured through bootstrapping through 10000 iterations.

2.5. Functional characterization of putative Elovl5 by heterologous expression in yeast

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Functional characterization of the putative Elovl5 was conducted by expressing the PCR fragment corresponding to the open reading frame (ORF) in the yeast Saccharomyces cerevisiae.

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Primers, named ORF Elovl5F and ORF Elovl5R containing restriction enzyme sites for HindIII and XbaI, respectively (underlined in Table 2), were designed for amplification of ORF from liver cDNA using the Pfu PCR MasterMix (Tiangen, Beijing, China). PCR consisted of an initial denaturing step at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 1 min 30 s, followed by a final extension at 72 °C for 5 min. The DNA fragments were then digested with the corresponding restriction endonucleases (Takara, Dalian, China) and ligated into a similarly restricted pYES2 yeast expression vector (Invitrogen, USA). The recombinant plasmids were used to transform S. cerevisiae (strain INVSc1, Invitrogen, USA) using the S.C. EasyCompTM Transformation kit 7

ACCEPTED MANUSCRIPT (Invitrogen, USA). Selection of yeast containing the pYES2-Elovl constructs was on S. cerevisiae minimal medium minus uracil (SCMM−uracil). Culture of the recombinant yeast was carried out in SCMM−uracil broth as described previously (Hastings et al., 2001), using galactose induction of gene expression. Each culture was supplemented with one of the following fatty acid substrates: linoleic acid (C18:2n-6), α-linolenic acid (C18:3n-3), γ-linolenic acid (C18:3n-6), arachidonic acid

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(C20:4n-6), eicosapentaenoic acid (C20:5n-3), adrenic acid (C22:4n-6) and docosapentaenoic acid (C22:5n-3). The PUFA substrates were added to the yeast cultures at final concentrations of

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0.5(C18), 0.75 (C20) and 1.0 (C22) mM as PUFA substrates, uptake efficiency has been shown to decrease with increasing chain length (Zheng et al., 2009). After 2 days, yeast were harvested and

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washed with Hank’s balanced salt solution containing 1 % fatty acid-free albumin for further analysis. Yeast transformed with pYES2 containing no insert were cultured under the same

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conditions as a control treatment. 2.6. Fatty acid analysis of yeast

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Yeast samples were homogenized in chloroform/methanol (2:1, v/v) containing 0.01 % (w/v) butylated hydroxytoluene (BHT) as antioxidant (Folch et al., 1957). Fatty acid methyl esters

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(FAME) were prepared, extracted, purified by thin layer chromatography (TLC), and analysed by

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gas chromatography (GC2010-plus, Shimadzu, Japan), all as described previously (Hastings et al., 2001). Conversions from PUFA substrates were calculated as the proportion of exogenously added fatty acid substrates converted to elongated fatty acid products as [individual product area/

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(all products areas + substrate area)] × 100 (Li et al., 2010). 2.7. Materials (chemicals)

obtained

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All fatty acid substrates (all ≥ 98% pure) used for the functional characterization assays, were from

Cayman

Chemical

Company.

BHT,

fatty acid-free

BSA,

galactose,

3-(hydroxymethyl) pyridine, nitrogen base, raffinose, tergitol NP-40 and uracil dropout medium were purchased from Sigma Chemical Co. 2.8. Tissue distribution of elovl5 mRNA Tissue distribution of elovl5 in chu’s croaker were determined by quantitative real-time PCR (qRT-PCR). Total RNA from heart, liver, stomach, intestine, spleen, muscle, liver, kidney, brain, eye and gill were extracted as described above. A total of 500 ng RNA of each tissue was reverse-transcribed into cDNA using PrimeScript® RT reagent Kit with gDNA Eraser (Takara, 8

ACCEPTED MANUSCRIPT Dalian, China) according to the manufacturer’s instruction. The specific primers of elovl5 named Q-Elovl5F/R5 were used to amplify the target gene (117bp), and β-actin named β-actin F/R was used as an internal control (Table 2). The relative expression of target gene was normalized with β-actin expression calculated by the 2−ΔΔCt algorithm, qRT-PCR was conducted using the SYBR® Premix Ex TaqTM II Kit (Takara, Dalian, China) in a LightCycler® 480 (Roche, USA). The total

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reaction volume was 20 µl containing 10 µl of SYBR® Premix Ex TaqTM II, 2 µl of the three-fold diluted cDNA, 0.8 µl (10 mM) each of forward and reverse primer, and 6.4 µl of sterilized

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ultra-pured water. The amplification procedure included a denaturation step of 95 °C for 30 s, and 40 cycles of 95 °C for 5 s and 60 °C for 20 s. At the end, a dissociation curve of 0.5 °C increments

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from 65 °C to 95 °C was performed to confirm the single amplification in each reaction. Each sample was run in triplicate, and reactions without templates were used as negative controls. For

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normalization purposes, the stability of potential references genes including β-actin and 18S rRNA was tested using BestKeeper (Pfaffl et al., 2004). The results confirmed that β-actin was very

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stable (stability value was 0.29) and was subsequently used as a reference gene to normalise the expression levels of the candidate genes. Standard curves were generated using five different

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dilutions (in triplicate) of the cDNA samples, and the amplification efficiency was analysed

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according to the following equation E=10(−1/Slope)–1. The primer amplification efficiency was 1.031 for Q-Elovl5, 1.012 for β-actin. 2.9. Statistical analysis

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Results were presented as per means ± standard error (n = 6). All data were subjected to one-way analysis of variance (ANOVA). When there were significant differences (P < 0.05), the

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group means were further compared with Duncan’s multiple range tests. Statistical analyses were performed using SPSS 20.0 (SPSS, Chicago, IL, USA). 3. Results

3.1. Sequence analysis of chu’s croaker elovl5 cDNA An Elovl5 elongase cDNA was isolated from the liver of chu’s croaker by using a combination of RT-PCR and RACE technology. The complete cDNA sequence of this gene and the deduced amino acids are shown in Figure 1. The full-length cDNA is 1315 bp and contains an 885 bp ORF, a 134 bp 5’-untranslated region (UTR) and a 296 bp 3’-UTR with a predicted polyadeylation signal site (AATAAA) and a poly (A) tail (GenBank accession no. KX158842). 9

ACCEPTED MANUSCRIPT Sequence analysis revealed that the elovl5 cDNA encodes a polypeptide of 294 amino acids with predicted theoretical isoelectric point of 9.33 and molecular weight of 35.08 kDa. The polypeptide sequence exhibited all the structural characteristic of microsomal fatty acid elongase including a single histidine box redox center motif (HXXHH), a putative endoplasmic reticulum (ER) retention signal at its carboxyl end, and multiple transmembrane-spanning domains. The

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KXXEXXDT, QXXFLHXYHH (which contains the histidine box), NXXXHXXMYXYY and TXXQXXQ motifs are highly conserved in all PUFA elongases cloned up to now (Figure 2)

3.2. Multiple sequences alignment and phylogenetic analysis

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(Meyer et al., 2004).

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The deduced polypeptide sequences displayed high percentage of identity with those of other fish, such as A.regius (99.7 %), L.crocea (99.0 %), N.mitsukurii (99.0 %), Epinephelus coioides

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(95.6 %), Dicentrarchus labrax (95.2 %), Thunnus thynnus (94.2 %), R.canadum (93.2 %) and Sparus aurata (92.5 %) (Table 3).

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To reveal the relationship between chu’s croaker Elovl5 protein with other vertebrate members of the Elovl family, the ClustalX2 software was used for multiple sequence comparison

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of amino acid sequences of elongases from Elovl1 to Elovl7 and a phylogenetic tree was then

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constructed using the neighbor-joining (NJ) methods on MEGA 4.0 program. The phylogenetic analysis showed that the chu’s croaker elovl5 gene grouped together with other teleost orthologues, and separately from other members of the Elovl family clusters. Moreover, the chu’s croaker

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Elovl5 clustered most closely to Elovl5 from N.mitsukurii, A.regius and L.crocea (Figure 3). 3.3. Tissue distribution of elovl5 transcripts

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Tissue distribution of the chu’s croaker elovl5 gene was determined by qRT-PCR on cDNA samples obtained from a range of tissues including brain, eye, spleen, intestine, heart, gill, liver, stomach, muscle, liver and kidney. β-actin was used as the reference gene for internal controls. The expression pattern of the chu’s croaker elovl5 exhibited a widespread distribution in all tissues tested, with highest expression in stomach and moderate expression in the kidney and spleen. And low levels of expression were detected in the intestine, brain, eye, liver, gill, muscle and heart. 3.4. Functional characterization in yeast The functional characterization of chu’s croaker Elovl5 was determined by the fatty acid profiles of transformed S.cerevisiae with either empty pYES2 vector (control) or the vector 10

ACCEPTED MANUSCRIPT containing elovl5 ORF inserts and grown in the presence of potential fatty acid substrates (C18:2n-6, C18:3n-3, C18:3n-6, C20:4n-6, C20:5n-3, C22:4n-6 and C22:5n-3). The yeast transformed with vector alone only contained the four most abundant yeast endogenous fatty acids (C16:0, C16:1n-7, C18:0 and C18:1n-9), in agreement with earlier observations (Hastings et al., 2001; Agaba et al., 2004). Functional characterization of the chu’s croaker elongase confirmed the

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isolated cDNA encoded a protein with Elovl5 activity, which exhibited high conversion towards C18 PUFA substrates (18:3n-3, 18:2n-6 and 18:3n-6) and C20 PUFA substrates (20:5n-3 and

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20:4n-6), but no activity towards C22 PUFA substrates (Figure 5; Table 4).

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3.5. Nutritional regulation of chu’s croaker elovl5

The effects of different dietary formulations on elovl5 mRNA expression in the liver were

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determined by qRT-PCR. Relative mRNA expression of elovl5 in liver increased proportional to dietary PO content. The replacement ratio of FO by PO up to 20 %, 40 %, 60 %, 80 %, and 100 %

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were significantly up-regulated by about 1.42-fold, 1.68-fold, 2.27-fold, 2.20-fold and 2.52-fold compared with that in the control group (FO) respectively (P < 0.05) (Figure 6).4. Discussion Elovl are responsible for the condensation reaction, which is the rate-limiting step in the two

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carbon elongation of pre-existing fatty acyl chains (Nugteren, 1965). To date, elovl5 cDNAs have

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been identified in numerous fish species and demonstrated it could effectively converted C18 and C20 PUFA to elongated polyenoic products (Zheng et al., 2009; Monroig et al., 2012; Monroig et al., 2013; Wang et al., 2014; Kabeya et al., 2015; Kuah et al., 2015; Li et al., 2016; Li et al., 2017).

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In this study, a full-length elovl5 cDNA sequence was isolated from the liver of chu’s croaker

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based on the PCR technology. The deduced 294 amino acids possessed all the main structural features common for Elovl protein family members, including the so-called histidine box (HXXHH), the canonical C-terminal ER retention signal, several predicted transmembrane regions and other highly conserved motifs (Meyer et al., 2004). The putative protein also showed high identity with elongases of some marine fish species, particularly A.regius (99.7 %), L.crocea (99.0 %), N.mitsukurii (99.0 %), E.coioides (95.6 %), D.labrax (95.2 %), T.thynnus (94.2 %), R.canadum (93.2 %) and S.aurata (92.5 %). The phylogenetic analysis indicated that the chu’s croaker Elovl5 clustered most closely with orthologues from teleosts and mammals while more distantly with other members of the Elovl family (Elovl1, Elovl2, Elovl3, Elovl4, Elovl6 and 11

ACCEPTED MANUSCRIPT Elovl7). Functional characterization exhibited that the chu’s croaker Elovl5 could efficiently elongated C18 and C20 PUFA. All the above sequence similarity, conserved domains, phylogenetic analysis and functional characterization support that the cloned gene is a elovl5 cDNA. According to previous studies, the tissue distributions of elovl5 transcripts were mainly

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expressed in the brain and liver, but expression of this gene in other tissues (skin, spleen, gill, stomach, liver, heart, kidney, muscle and intestine) varied among different fish species (Zheng et

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al., 2009; Monroig et al., 2012; Monroig et al., 2013; Wang et al., 2014; Kuah et al., 2015; Li et al., 2016; Zuo et al., 2016). In large yellow croaker, expression of elovl5 was strongly observed in

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liver, brain and gill, and weaker in spleen, intestine, heart and stomach (Zuo et al., 2016). In eel, elovl5 was primarily expressed in the brain, with the liver and intestine showing the next highest

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expression signals (Wang et al., 2014). In salmon and striped snakehead, the elovl5 was highly expressed in the intestine and liver (Zheng et al., 2005; Kuah et al., 2015). In this study, tissue

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expression study showed that chu’s croaker elovl5 was mainly expressed in the stomach, while moderately expressed in the kidney and spleen, and barely detected in the intestine, brain, eye,

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liver, gill, muscle and heart. Intestine is now acknowledged as a site of significant fatty acid, at

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least in salmonids (Bell et al., 2003). Similarly, stomach may also be a significant metabolic site for fatty acid, involving in the process of converting the short chain fatty acids to the long chain fatty acids.

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The functional characterization of chu’s croaker Elovl5, similar to mammalian homologues (Leonard et al., 2000), showed high activity towards C18 (C18:2n-6, C18:3n-3 and C18:3n-6) and

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C20 (C20:4n-6 and C20:5n-3) PUFA substrates, but was unable to elongate C22 (C22:4n-6 and C22:5n-3) PUFA substrates. Similar results were also observed in some teleost fishes and mollusks such as Anguilla japonica (Wang et al., 2014), Acanthopagrus schlegelii (Kim et al., 2012), E.coioides (Li et al., 2016), Sepia officinalis (Monroig et al., 2016) and Chlamys nobilis (Liu et al., 2013), which found the Elovl5 had the ability to elongate both C18 and C20 PUFA substrates, but no activity towards C22 PUFA substrates. In contrast, other fish species Elovl5 exhibited wider substrates specificities, with a low activity towards C22 PUFA (Agaba et al., 2004; Hastings et al., 2004; Morais et al., 2009; Monroig et al., 2012; Monroig et al., 2013; Kabeya et al., 2015; Li et al., 2017). In addition, the chu’s croaker Elovl5 has a preference for n-3 over n-6 12

ACCEPTED MANUSCRIPT PUFA substrates, in agreement with results obtained for most species studied previously, including both marine and freshwater fish (Hastings et al., 2004; Morais et al., 2011; Monroig et al., 2013; Li et al., 2016; Xie et al., 2016; Castro et al., 2016; Li et al., 2017). In this study, the chu’s croaker elovl5 was also nutritionally regulated, with transcription enhanced by low dietary n-3 LC-PUFA. Similar results were also observed in other teleost fishes

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such as orange-spotted grouper (Li et al., 2016), large yellow croaker (Zuo et al., 2016), striped snakehead (Kuah et al., 2015), common crap (Ren et al., 2012), which found high n-3 LC-PUFA

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supplement down-regulated the transcription of elovl5 significantly. These observations reflect the existence of negative feedback regulation in the LC-PUFA synthetic pathway. The feedback

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suppression of elovl5 expression may primarily involve two transcription factors, namely, sterol regulatory element binding protein-1 (SREBP-1) and liver X receptor (LXRα) (Minghetti et al.,

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2011). In mouse, it has been reported that the expression of elovl5 is directly regulated by SREBP-1 (Qin et al., 2009). In contrast, large yellow croaker, Atlantic salmon and orange-spotted

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grouper elovl5 reporter activities were induced by over-expression of LXRα, but not by over-expression of SREBP-1 (Minghetti et al., 2011; Li et al., 2016; Li et al., 2017). However, for

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chu’s croaker elovl5, further investigation is required to identify the mechanisms involved.

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To conclude, a elovl5 gene from chu’s croaker was cloned and investigated in the present study. The chu’s croaker elovl5 was broadly expressed in most tissues with the highest level in stomach, followed by kidney and spleen, and lowest level in intestine, brain, eye, liver, gill,

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muscle and heart. Functional characterization revealed that the chu’s croaker Elovl5 was able to elongate both C18 and C20 PUFA substrates, but no activity towards C22 PUFA substrates.. The

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hepatic expression of elovl5 could be up-regulated by low dietary n-3 LC-PUFA. These results may contribute to better understanding the LC-PUFA biosynthetic pathway and regulation mechanism in this species, which will help us optimize the efficient use of sustainable plant-based alternatives in aquafeed. Acknowledgement The research was supported by the National Natural Science Foundation of China (No. 31172424), Guangdong Oceanic and Fishery Science and Technology Foundation (No. A201005D06-1) and Young Creative Talents, Major Scientific Research Projects of Guangdong University Foundation (No. 2016KQNCX058). 13

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Figure 1. Nucleotide and deduced amino acid sequences of elovl5 from Nibea coibor. Total number of nucleotides: 1315; 5’UTR: 1-134 nt; open reading frame: 135-1019 nt; 3’UTR: 1020-1315 nt. Start codon (ATG) and stop codon (TGA) are marked in bold. The polyadenylation (AATAAA) is underline. GenBank accession no.

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KX158842.

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Figure 2. A deduced amino acid sequence comparison of the Elovl5 from Nibea coibor (KX158842) with fatty

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acyl elongases from Epinephelus coioides (AGT80149.1), Larimichthys crocea (NP_001290303.1), Argyrosomus regius (AGG69479.1), Lates calcarifer (ACS91459.1) and Nibea mitsukurii (ACR47973.1). The threshold for similarity shading was set at 75 %. Identical residues are shaded black, and similar residues are shaded pink. Highly conserved motifs are boxed, the ER retention signal is dash-underlined and seven putative transmembrane

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Figure 3. Phylogenetic relationship between the amino acid sequences of Elovl5 and 39 available Elovl sequences. 21

ACCEPTED MANUSCRIPT The amino acid sequences were derived from the GenBank under the following accession numbers (in parentheses): Homo sapiens: Elovl1 (NP_073732), Elovl2 (NP_060240), Elovl3 (NP_689523), Elovl4 (NP_073563), Elovl5 (NP_068586), Elovl6 (NP_076995), Elovl7 (NP_079206); Danio rerio: Elovl1 (NP_001005989), Elovl5 (NP_956747), Elovl6 (NP_955826), Elovl7 (NP_956169); Nibea mitsukurii: Elovl5 (ACR47973); Argyrosomus regius: Elovl5 (AGG69479); Larimichthys crocea: Elovl5 (NP_001290303.1); Epinephelus coioides: Elovl5 (AGT80149); Dicentrarchus labrax: Elovl5 (CBX53576); Lates calcarifer: Elovl5 (ACS91459); Rachycentron canadum: Elovl4 (ADG59898), Elovl5 (ACJ65150); Acanthopagrus schlegelii: Elovl5 (ANJ04908); Labrus bergylta: Elovl5 (XP_020510530); Salmo salar:Elovl2 (ACI62500), Elovl4 (NP_001182481), Elovl5 (NP_001005989); Xenopus laevis: Elovl2 (NP_001087564), Elovl5 (NP_001089883);

PT

Xenopus tropicalis: Elovl1 (NP_001016644), Elovl3 (XP_002935809), Elovl4 (XP_002936253), Elovl5 (NP_001011248), Elovl6 (NP_001017257), Elovl7 (NP_001005456); Patagioenas fasciata monills: Elovl5

Elovl2

(AGR34076);

Taeniopygia guttata:

Elovl2

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(OPJ73463), Elovl6 (OPJ75728), Elovl7 (OPJ87106); Mus musculus: Elovl5 (NP_599016); Oncorhynchus masou; (NP_001232477);

Cyprinodon variegatus:

Elovl3

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(XP_015233000). The tree was constructed using the neighbor joining method with MEGA 4.0. The horizontal branch length is proportional to amino acid substitution rate per site. Numbers represent the frequencies with

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which the tree topology presented was replicated after 1000 bootstrap iterations.

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ACCEPTED MANUSCRIPT

Figure 4. The relative expression level of elovl5 mRNA normalized to β-actin in the stomach (St), kidney (K), spleen (Sp), intestine (I), brain (B), eye (E), liver (L), gill (G), muscle (M) and heart (H) of Nibea coibor. Values

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D

are means ± SE (n = 5). Bars with different superscripts are significantly different (P < 0.05).

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Figure 5. Functional characterization of Nibea coibor putative Elovl5 in yeast Saccharomyces cerevisiae. Fatty

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acids were extracted from yeast transformed with the pYES2 vector alone or containing the ORF of the putative elovl5 as an insert, growing in the presence of potential fatty acids substrates. A represent control group; B, C, D, E, F, G and H represent group that adds substrate of C18:2n-6, C18:3n-3, C18:3n-6, C20:4n-6, C20:5n-3, C22:4n-6

AC

and C22:5n-3. Peaks 1-4 represent four endogenous fatty acids of S.cerevisiae, including C16:0 (1), C16:1n-7 (2), C18:0 (3) and C18:1n-9 (4). Additionally, peaks derived from exogenously added substrates or elongation products are indicated accordingly.

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ACCEPTED MANUSCRIPT

D

Figure 6. Relative elovl5 mRNA expressions in livers of Nibea coibor under six dietary treatments. Values are normalized to those of β-actin. Data are presented as means ± SE (n=6). Diets are blended with palm oil to make a

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decreased dietary n-3 LC-PUFA level of 23.3 %, 20.0 %, 16.5 %, 12.2 %, 10.1 % and 7.4 %, respectively.

Table 1 25

ACCEPTED MANUSCRIPT Ingredients and proximate composition of experimental diets.

Ingredient (g/kg diet)

FO a

2PO

4PO

6PO

8PO

10PO

Common ingredients b, c

875

875

875

875

875

875

Fish oil d

125

100

75

50

25

0

Palm oil e

0

25

50

75

100

125

Moisture (%)

15.82

13.56

14.00

15.24

14.75

14.02

Crude protein (%)

44.58

45.68

45.91

45.78

45.24

45.82

Crude lipid (%)

12.74

12.45

11.97

12.20

12.18

12.90

Ash (%)

8.10

7.49

7.92

7.66

8.09

7.38

α-tocopherol (mg/kg)

20.03

27.75

35.52

43.25

58.75

18:2n-6

3.4

5.6

7.0

SC

51.10

7.9

8.6

10.1

20:2n-6

0.9

0.7

0.6

0.5

0.3

0.3

20:4n-6

1.8

1.5

1.2

0.8

0.8

0.4

n-6 PUFA f

7.9

18:3n-3

1.0

20:5n-3

9.3

MA

Experimental diets

22:5n-3

1.7

22:6n-3

10.4

10.1

10.4

10.6

11.5

1.0

1.3

1.5

1.7

1.9

8.5

6.2

4.0

3.4

2.0

D

23.3

RI

9.1

1.4

1.3

1.0

0.8

0.5

8.4

7.1

4.9

3.7

2.3

20.0

16.5

12.2

10.1

7.4

PT E

n-3 PUFA g

NU

Main fatty acids (% area)

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Proximate composition

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Common ingredients (g/kg diet): White fish meal (Coastal Villages Pollock LLC., Anchorage, AK, USA), 200; Soy protein concentrate (Shandong Wangdefu Group Co. Ltd., China), 240; Casein (Gansu Hualing casein Co. Ltd., China), 160; Starch, 220; Cellulose, 20; Mineral mix, 10; Vitamin mix, 10; Monocalcium phosphate, 10; Choline chloride, 5. a FO = control; 2PO = 20 % palm oil; 4PO = 40 % palm oil; 6PO = 60 % palm oil; 8PO = 80 % palm oil; 10PO = 100 % palm oil. b Mineral mixture (g/kg): NaF, 2 mg; KI, 0.8 mg; CuSO4·5H2O, 10 mg; ZnSO4.H2O, 50 mg; CoCl2.6H2O (1 %), 50 mg; NaCl, 100 mg; FeSO4.H2O, 80 mg; MnSO4.H2O, 60 mg; MgSO4.7H2O, 1200 mg; Zeolite, 15.45 g; formulated with cellulose as filling. c Vitamin mixture (g/kg): A, 4×106 IU; D3, 2×106 IU; E, 20 g; K3, 6 g; B1, 7.5 g; B2, 16 g; B6, 12 g; B12, 100 mg; folic acid, 2 g; pantothenic acid, 36 g; nicotinic acid, 88 g; inositol, 100 mg; biotin, 100 mg and Vitamin C-monophosphate compound, 200 mg. d Fish oil, Guangzhou Yongxing concentrated feed Co. Ltd., China. e Palm oil, Shanghai Yihai Food Marketing Co. Ltd., China. f n-6 PUFA: 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6. g n-3 PUFA: 18:3n-3, 20:4n-3, 20:5n-3, 22:5n-3, 22:6n-3. 26

ACCEPTED MANUSCRIPT

Table 2 Primer used in this study. Sequence (5’— 3’)

Aim

Oligo-

AAGCAGTGGTATCAACGCAGAGTACXXXXX

First-strand cDNA synthesis

UPM (short)

CTAATACGACTCACTATAGGGC

RACE-PCR

UPM (long)

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT

RACE-PCR

NUP

AAGCAGTGGTATCAACGCAGAGT

RACE-PCR

Elovl5F

TCGACAACTACCCACCAACC

Elovl5R

GCAGCGTGCTCCAAAGAT

Elovl5 3-1

ACCAGAACGGCTCTCCCGTATC

Elovl5 3-2

GGAACGCCATCTTTGGAGCACGC

Elovl5 5-1

GACTGTGAGTGCAAAGGTTGGTGGG

Elovl5 5-2

GCAAAGGTTGGTGGGTAGTTGTCG

5’ RACE

ORF Elovl5F

CCCAAGCTTCAAATGGAGACCTTCAATCA

Expression

ORF Elovl5R

TGCTCTAGAAATGTCAATCCACCCTCAGT

Expression

Q-Elovl5F

CGAAAGAATAATCACCAGATCACC

qRT-PCR

Q-Elovl5R

GGAGGCACCGAAGTACGAAT

qRT-PCR

M13F

CGCCAGGGTTTTCCCAGTCACGAC

PCR screening

M13R

AGCGGATAACAATTTCACACAGGA

PCR screening

β-actin F

GGTTACTCCTTCACCACCACAG

qRT-PCR

β-actin R

TCCGTCGGGCAGCTCATA

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D

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Primer

Partial cDNA cloning Partial cDNA cloning 3’ RACE 3’ RACE 5’ RACE

qRT-PCR

AC

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Restriction sites for HindIII and XbaI are underlined in primer ORF Elovl5F and ORF Elovl5R sequences. X =undisclosed base in the proprietary SMARTer oligo sequence.

27

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Table 3 elovl5 sequences identities between Nibea coibor and 14 other organisms. Accession No.

% Identity

Argyrosomusregius

AGG69479.1

99.7

Larimichthyscrocea

NP_001290303.1

99.0

Nibeamitsukurii

ACR47973.1

99.0

Epinepheluscoioides

AGT80149.1

95.6

Dicentrarchuslabrax

CBX53576.1

95.2

Thunnusthynnus

ADX62355.1

Rachycentroncanadum

ACJ65150.1

Sparusaurata

AAT81404.1

Soleasenegalensis

AER58183.1

Siganuscanaliculatus

ADE34561.1

Danio rerio

NP_956747.1

Mus musculus

NP_599016.2

Homo sapiens

NP_068586.1

Pongo abelii

NP_001127147.1

RI

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Matched species

94.2

SC

93.2 92.5 85.4

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D

MA

NU

82.0

28

76.2 71.0 69.2 68.6

ACCEPTED MANUSCRIPT

Table 4 Functional characterization of Nibea coibor Elovl5 in yeast Saccharomyces cerevisiae. Results are expressed as a percentage of total fatty acid substrate converted to elongated products. Product

% Conversion

C18:2n-6

C20:2n-6

25.8

C18:3n-3

C20:3n-3

41.6

C18:3n-6

C20:3n-6

64.5

C20:4n-6

C22:4n-6

58.9

C20:5n-3

C22:5n-3

71.3

C22:4n-6

C24:4n-6

0

C22:5n-3

C24:5n-3

0

C18-20

RI

C18-20 C18-20

SC

C20-22

NU MA D PT E CE AC

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Activity

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Fatty acid substrate

C20-22 C22-24 C22-24

ACCEPTED MANUSCRIPT abb

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Long-chain polyunsaturated fatty acids: LC-PUFA Polyunsaturated fatty acids: PUFA Untranslated region: UTR Arachidonic acid: ARA Eicosapentaenoic acid: EPA Docosahexaenoic acid: DHA Linoleic: LA α-linolenic: ALA Essential fatty acids: EFAs

30

ACCEPTED MANUSCRIPT Highlights of the manuscript: 1. The full-length cDNA of fatty acyl Elovl5 elongase was isolated from the liver of Nibea coibor. 2. Tissue distribution analysis revealed that the Elovl5 transcripts are widely distributed in various tissues, with high expression levels in the stomach. 3. Functional characterization revealed that the chu’s croaker Elovl5 was able to elongate both

PT

C18 and C20 PUFA substrates. 4. Nutritional study indicated that the hepatic expression of Elovl5 could be up-regulated by low

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dietary n-3 LC-PUFA.

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