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Agricultural and Environmental Chemistry

Biosynthesis of polyunsaturated fatty acids in the razor clam Sinonovacula constricta: Characterization of #5 and #6 fatty acid desaturases Zhaoshou Ran, Jilin Xu, Kai Liao, Shuang Li, shubing chen, and Xiaojun Yan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00968 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Journal of Agricultural and Food Chemistry

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Biosynthesis of polyunsaturated fatty acids in the razor clam Sinonovacula

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constricta: Characterization of ∆5 and ∆6 fatty acid desaturases

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Zhaoshou Ran1,2, Jilin Xu1*, Kai Liao1, Shuang Li3, Shubing Chen3, Xiaojun Yan2*

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1 Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ministry of

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Education of China, Ningbo, Zhejiang, 315211, China

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2 Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy

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Aquaculture, Ningbo University, Ningbo, Zhejiang, 315211, China

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3 Ningbo Entry-Exit Inspection and Quarantine Bureau Technology Center, Ningbo,

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Zhejiang, 315000, China

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*Corresponding author:

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Jilin Xu, Email: [email protected]; Phone: 86-0574-87609570; Fax:

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0574-87609570

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Xiaojun Yan, Email: [email protected]; Phone: 0574-87600738; Fax:

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0574-87600458

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ACS Paragon Plus Environment


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ABSTRACT: To investigate the endogenous LC-PUFA biosynthetic ability in

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Sinonovacula constricta, fatty acid desaturases (Fads) of this bivalve were cloned and

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characterized in current study, namely Scfad5a, Scfad5b and Scfad6. Meanwhile, the

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tissue distributions of S. constricta Fads and fatty acids (FAs) were examined.

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Heterologous expression in yeasts confirmed that Scfad5a and Scfad5b were both ∆5

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Fads, while Scfad6 was a ∆6 Fad. However, compared with Fads in other organisms,

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the desaturation activities of S. constricta Fads were relatively low (especially for

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Scfad6), indicating an adaptation to living conditions. S. constricta Fads were

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expressed in all tissues examined, and particularly high expressions were found in

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intestine and gonad. Moreover, FAs were differently distributed among tissues, which

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might be correlated with their corresponding physiological roles. Taken together, the

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results provided an insight into LC-PUFA biosynthesis in S. constricta. Notably,

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Scfad6 was the first functionally characterized ∆6 Fad in marine molluscs to date.

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KEYWORDS: Sinonovacula constricta; fatty acid desaturase; LC-PUFA; marine

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molluscs

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INTRODUCTION

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Long-chain polyunsaturated fatty acids (LC-PUFA), such as arachidonic acid

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(20:4n-6, ARA), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid

36

(22:6n-3, DHA), are essential nutrients for humans. They play critical roles in

37

membrane structure, neurological development, immune response and disease

38

treatments.1-5 However, LC-PUFA can not be sufficiently biosynthesized by human.

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In order to meet normal metabolic needs, additional LC-PUFA must be acquired from

40

diets. Marine molluscs are emerging as an excellent dietary resource of LC-PUFA for

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human. Because they not only contain high levels of LC-PUFA,6 but have potential

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ability to endogenously biosynthesize LC-PUFA.7-9

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Biosynthesis of LC-PUFA in vertebrate involves sequential desaturation and

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elongation of C18 precursors, which are mediated by fatty acid desaturases (Fads) and

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elongases of very long-chain fatty acid (Elovls) (Figure S1).10,11 Generally, 18:2n-6 is

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first desaturated to 18:3n-6 by ∆6 Fad, followed by elongation to 20:4n-6 by Elovl5,

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which is further desaturated by ∆5 Fad to give ARA. The synthesis from 18:3n-3 to

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EPA requires the same enzymes and pathway as for ARA. However, DHA synthesis

49

from EPA will require two further elongation steps by Elovl5/2/4 and Elovl2/4,

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another desaturation step by ∆6 Fad and a peroxisomal chain shortening step.

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Exceptionally, in organisms containing ∆8 Fad, the first two steps can be reversed,

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while in organisms containing ∆4 Fad, DHA can be directly produced from 22:5n-3.

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The extent to which a species biosynthesizes LC-PUFA greatly varies and depends on

54

the integrity of Fads and Elovls, especially for Fads. In marine animals, ∆5 Fad has 3



55

been acknowledged as a rate limiting step for the biosynthesis of LC-PUFA.12-15

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Meanwhile, ∆6 Fad is also considered as a key control point, which not only

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metabolizes 18:3n-3 but also desaturates 24:5n-3, a progenitor of DHA.16,17

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The razor clam, Sinonovacula constricta, is an economically important bivalve,

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which widely resides in the estuarine and intertidal zones along the coasts of China,

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Japan and Korea. This bivalve contains high levels of nutritional compounds,

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especially LC-PUFA.18 However, little is known regarding its endogenous ability of

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LC-PUFA biosynthesis. Importantly, our previous results have indicated that S.

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constricta may possess a certain capacity to produce LC-PUFA.19 In recent years, the

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biosynthesis of LC-PUFA has been well studied in some marine molluscs,12,20-26 but

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not in S. constricta.

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In the present study, we aimed to clone and characterize Fads from S. constricta.

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Meanwhile, we also examined tissue distributions of S. constricta Fads and fatty acids

68

(FAs) in order to better reveal their physiological roles in this organism. Our findings

69

provided valuable insights into the biosynthesis of LC-PUFA in S. constricta.

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MATERIALS AND METHODS

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Full-length cloning of S. constricta Fad cDNAs

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Fresh S. constricta tissues of foot muscle, gill and gonad were collected,

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homogenized (T10 basic ULTRA-TURRAX®, IKA, German) and used to extract total

74

RNA using MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan). RNA

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integrity was examined on 1% agarose gels. RNA concentration was determined by

76

NanoDrop® ND-1000 (NanoDrop, USA). Subsequently, 1 µg of purified RNA was 4


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reversely transcribed into cDNA using PrimeScriptTM RT-PCR Kit (TaKaRa, Japan).

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The resulting cDNA was used as templates for PCR analysis.

79

The partial fragments of S. constricta Fad cDNAs were obtained by searching

80

against its transcriptome data. To verify those fragments, specific primers (V-F, V-R,

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Table 1) were designed (Primer 5.0) and subjected to PCR using LA Taq® (TaKaRa,

82

Japan). Based on the verified fragments, gene-specific rapid amplification of cDNA

83

ends (RACE) primers (Table 1) were designed and used to produce full-length

84

cDNAs by two-round PCR using SMARTer® RACE 5´/3´ Kit (Clontech, USA).

85

Notably, the above-mentioned PCR amplicons were first purified from 2% agarose

86

gel, and then the target bands were excised, recycled, inserted into pMDTM18-T

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Vector (TaKaRa, Japan) and sequenced by BGI Tech Co., Ltd. (Shanghai, China).

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Full-length cDNAs of S. constricta Fads were finally obtained by aligning the 3´-end

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with 5´-end fragments. They were named as Scfad5a, Scfad5b and Scfad6,

90

respectively.

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Sequence and phylogenetic analysis

92

In order to provide important clues for predicting functions or evolution of the

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target genes, multiple sequence alignments were performed with ClustalX 2.1, and

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phylogenetic trees were constructed using the maximum-likelihood approach (MEGA

95

7 package), on the basis of deduced amino acid sequences of Fads from S. constricta

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and

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uncharacterized Fads from marine molluscs. Confidence in the resulting phylogenetic

98

tree branch topology was measured by bootstrapping through 1,000 iterations.

representative

mammals,

fish

and

all

functionally

5


characterized

or


99 100

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Functional characterization by heterologous expressions of S. constricta Fad open reading frame (ORF) in yeasts

101

PCR fragments corresponding to the ORFs of Scfad5a, Scfad5b and Scfad6 were

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amplified from cDNA template by High Fidelity PrimeScript® RT-PCR Kit (TaKaRa,

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Japan) using specific primers harboring restriction sites (underline) of KpnI and

104

EcoRI (Table 1). The resulting DNA products were purified, digested with the

105

corresponding restriction endonucleases (New England BioLabs, USA) and inserted

106

into similarly digested pYES2 vector (Invitrogen, USA) using DNA Ligation Kit

107

(TaKaRa, Japan). The resulting plasmid constructs, pYScfad5a, pYScfad5b and

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pYScfad6, were firstly transformed into Escherichia coil DH5α competent cells

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(TaKaRa, Japan). Then the correct recombinants and pYES2 (the control) were

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further transformed into Saccharomyces cerevisiae competent cells using the S.c.

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EasyComp

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corresponding recombinants were selected using S. cerevisiae minimal medium-uracil

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(2% glucose, 0.67% nitrogen base and 0.19% uracil dropout medium). The presence

114

of resulting plasmid constructs in S. cerevisiae was further confirmed by DNA

115

sequencing.

Transformation

Kit

(Invitrogen,

USA).

Yeasts

containing

the

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The successfully transformed yeasts with pYES2 (the control), pYScfad5a,

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pYScfad5b or pYScfad6 were first cultured in a transition medium (2% raffinose, 0.67%

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nitrogen base and 0.19% uracil dropout medium) for 24 h. Subsequently, the cell

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suspensions were centrifuged at 500 g for 2 min at room temperature. The precipitated

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yeasts were re-suspended with induction medium (2% galactose, 0.67% nitrogen base, 6


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1% tergitol type NP-40 and 0.19% uracil dropout medium) at OD600=0.4. Notably, at

122

this point, one type of potential FA substrates at various concentrations of 0.5 mM

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(C18), 0.75 mM (C20) and 1 mM (C22) was added to each culture. The FA substrates

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included 18:2n-6 and 18:3n-3 for ∆6 Fad activity characterization, 20:3n-6 and

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20:4n-3 for ∆5 Fad activity characterization, 20:2n-6 and 20:3n-3 for ∆8 Fad activity

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characterization, and 22:5n-3 and 22:4n-6 for ∆4 Fad activity characterization

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(Cayman Chemicals, USA; Figure S1). Each experiment was performed in triplicate.

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Subsequently, cell cultures were placed into a shaker and incubated at 30°C for 2 days

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at 250 rpm/min. After incubation, approximately equal amounts of yeasts were

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harvested (500 g for 2 min) and washed twice with 5 mL ice-cold Hanks’s Balanced

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Salt Solution (Invitrogen, USA). Finally, the obtained yeasts were freeze-dried for

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further FA analyses.

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FA analysis by GC-MS

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The FAs were extracted using a method described by Xu et al.27 Briefly, the crude

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lipid was extracted with CHCl3/CH3OH/H2O (1:2:0.8, v/v/v) containing 0.01%

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butylated hydroxytoluene (BHT) as antioxidant. Secondly, 0.2 mL of toluene, 1.5 mL

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of methanol and 0.3 mL of HCl (8%, w/v) in methanol/water (85:15, v/v) were

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sequentially added to the extracted lipid samples. Then the mixture (2 mL) was heated

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at 100°C for 1 h to obtain fatty acid methyl esters (FAMEs). Finally, FAMEs were

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extracted by hexane-chloroform (4:1, v/v), dried under nitrogen and stored at -20°C.

141

Prior

142

chromatography-grade hexane.

to

GC-MS

analyses,

FAMEs

were

re-dissolved

7


using

1

mL


143

FAMEs were analyzed by Agilent GC/MS (7890B/7000C) using CD-2560

144

capillary column (100 m × 250 µm × 0.2 µm, CNW, Germany) equipped with a

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sampling system of Gerstel MPS. Briefly, the GC oven was temperature-programmed

146

from 140°C (5 min) to 240°C (20 min) with an increment of 4°C/min. The injector

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temperature was maintained at 250°C with an initial pre-column pressure of 30.36 psi.

148

FAMEs in hexane were filtered through a 0.22-µm ultra filtration membrane

149

(Millipore, Billerica, MA, USA), and 1 µL sample was injected with splitless mode.

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The highly pure helium was provided as carrier gas, at a constant flow rate of 0.81

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mL/min. The temperatures of MS ion source, transmission line and quadrupole were

152

set at 230°C, 255°C and 150°C, respectively. The collision energy was 70 ev, and the

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scanning range of mass spectrometer was 40-600 m/z.

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FAs were identified by relative retention times of standards and mass spectral

155

databases (NIST 14.L). The conversion rate of FA substrate to desaturated product

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was determined by 100%*[product area/(product area + substrate area)].

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Tissue distributions of S. constricta Fads and FAs

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To minimize the influences of diets as much as possible, the animals (shell length

159

of 55.23±3.31 mm) were acclimated in a small simulated culture pond. During the

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acclimation, the microalgal diets were completely deprived. After 3 days, when the

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diets in intestine were disappeared, the tissues were isolated.

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S. constricta is a typical benthic bivalve distributed in intertidal and coastal areas,

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where confront with great ambient challenges. LC-PUFA are key compounds for cell

164

membranes and involved in many biological functions, such as osmoregulation and 8


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immune response. Therefore, the external tissues of mantle, inhalant siphon, exhalant

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siphon, labial palps and gill were selected. Meanwhile, the foot muscle, intestine,

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digestive glands, gonad and heart as important storage organ, metabolic organ or

168

reproductive organ were also isolated. A total of eighteen individuals were used for

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tissue isolation. Each tissue was sampled from six individuals and pooled together,

170

which was performed in triplicate. Subsequently, each sample was homogenized with

171

a blender (T10 basic ULTRA-TURRAX®, IKA, German) on ice and then divided into

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two parts. One part was used to study the tissue distribution of S. constricta Fads,

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while the other part was used to examine the tissue distribution of FAs.

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Tissue distributions of Scfad5a, Scfad5b and Scfad6 were examined by quantitative

175

real-time PCR (qRT-PCR). Total RNA was extracted from 10 types of tissues, and 1

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µg of purified RNA was reversely transcribed into cDNA using PrimeScriptTM RT

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Master Mix (Perfect Real Time, TaKaRa, Japan). Specific primers of qRT-PCR were

178

designed for Scfad6 and in the different regions of Scfad5a and Scfad5b, respectively

179

(Table 1). qRT-PCR was carried out in a quantitative thermal cycler (Mastercycler ep

180

realplex, Eppendorf, Germany) using SYBR® Premix Ex TaqTM (Tli RNaseH Plus)

181

(TaKaRa, Japan). Briefly, after an initial denaturation step at 95°C for 30 s,

182

amplifications were carried out with 35 cycles at a melting temperature of 95°C for 5

183

s, an annealing temperature of 55°C for 15 s and an extension temperature of 72°C for

184

20 s, followed by a melting curve from 58°C to 95°C with an increment of

185

1.85°C/min. Relative expressions of target genes were calculated by the 2-∆∆CT

186

method,28 and β-actin was selected as the housekeeping gene. The relative expression 9



187

of Scfad5a, Scfad5b and Scfad6 in tissues was calibrated by that of mantle,

188

respectively.

189

Tissue distributions of FAs were examined according to GC-MS method as

190

described above.

191

Statistical analysis

192

Statistical analyses were carried out with SPSS 22.0 software (SPSS, Inc.,

193

Chicago, IL, USA). Data were subjected to One-way ANOVA and Newman-Keuls

194

tests. A P value less than 0.05 was considered as statistically significant.

195

RESULTS

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Sequence and phylogenetic analyses of S. constricta Fads

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The full lengths of mRNA transcripts for Scfad5a and Scfad5b were both 2,700 bp

198

with a 5´ untranslated region (UTR) of 304 bp, an ORF of 1,308 bp and a 3´ UTR of

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1,088 bp. Their ORF similarity of cDNA sequence was 97.48%, while that for

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deduced 435 amino acids was 97.93%, and the main different region was highlighted

201

by a bold frame in Figure 1. Meanwhile, the deduced amino acid sequences of

202

Scfad5a and Scfad5b exhibited 61%-65% homology to ∆5 Fads of Octopus vulgaris,

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Haliotis discus hannai and Chlamys nobilis, and they shared approximately 51%

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homology to ∆5 Fads of Danio rerio, Mus musculus and Homo sapiens (Figure 1).

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The Scfad6 cDNA was 1,790 bp in length, consisting of an 80-bp 5´ UTR, a 1,305 bp

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ORF and a 405-bp 3´ UTR. The Scfad6 ORF encoded a sequence of 310 amino acids,

207

which shared 48%-51% homology to ∆6 Fads of D. rerio, Siganus canaliculatus, M.

208

musculus and H. sapiens (Figure 2). All S. constricta Fads contained three histidine 10


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boxes (H***H, H**HH and QIEHH) and an N-terminal cytochrome b5 domain with

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heme-binding motif of a conserved HPGG in typical front-end Fads (Figures 1 and 2).

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The detailed sequence information of Scfad5a, Scfad5b and Scfad6 was deposited in

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the GenBank database with accession number of MH220404, MH220405 and

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MH220406, respectively.

214

The phylogenetic tree was mainly clustered into four groups (Figure 3, I-IV). ∆5/6

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Fads of some teleosts and mammals were grouped together; Scfad5a, Scfad5b, ∆5

216

Fads and Fad-like genes (not functionally characterized, denoted with *) of some

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marine molluscs were grouped together; Scfad6 and Fad-like genes of some marine

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molluscs were grouped together; and ∆5/6 Fads of some lower eukaryotes were

219

grouped together. Particularly, Scfad5a and Scfad5b were clustered together, which

220

were most closely related to ∆5 Fad of C. nobilis. Scfad6 was most closely related to

221

Fad-like genes of C. gigas and Aplysia californica, but far from ∆6 Fads of both

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lower and higher organisms.

223

Functional characterization of S. constricta Fads

224

Figures 4 and 5 showed that yeasts harboring empty pYES2 only contained the

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endogenous FAs, including 16:0, 16:1n-7, 18:0, 18:1n-9 (peaks 1-4) and the

226

exogenously added PUFAs (denoted with *). However, additional peaks were

227

observed in the FAs of yeasts transformed with pYScfad5a, pYScfad5b or pYScfad6.

228

Additional FAs were identified as AA and EPA in yeasts transformed with

229

pYScfad5a or pYScfad5b when cultured with 20:3n-6 or 20:4n-3, respectively (Figure

230

4), indicating that Scfad5a and Scfad5b were both ∆5 Fad. By calculating the 11



231

conversion efficiency (Table 2), 13.39% and 10.95% of 20:3n-6 were desaturated to

232

AA by Scfad5a and Scfad5b, respectively, while 11.71% and 8.58% of 20:4n-3 were

233

desaturated to EPA by Scfad5a and Scfad5b, respectively. The result indicated that

234

Scfad5a had a higher catalytic activity towards ∆5 Fad substrates than Scfad5b, and

235

their catalytic activities towards 20:3n-6 were higher than those towards 20:4n-3.

236

Simultaneously, the endogenous 18:0 was converted to 18:1n-13 by yeasts

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transformed with pYScfad5a or pYScfad5b, indicating that Scfad5a and Scfad5b also

238

exhibited desaturation activities towards saturated FAs for 18:0 but not 16:0. The

239

desaturation activities towards 18:0 of Scfad5a and Scfad5b were 16.21% and 21.36%,

240

respectively. No activities of ∆4, ∆6 and ∆8 Fads were observed for Scfad5a and

241

Scfad5b.

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Additional FAs were identified as 18:3n-6 and 18:4n-3 in yeasts transformed with

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pYScfad6 when cultured with 18:2n-6 or 18:3n-3, respectively (Figure 5), indicating

244

that Scfad6 was ∆6 Fad. However, the enzymatic activity of Scfad6 was very low,

245

while only 1.56% of 18:2n-6 and 3.59% of 18:3n-3 were desaturated to 18:3n-6 and

246

18:4n-3, respectively. No activities of ∆4, ∆5 and ∆8 Fads were observed for Scfad6.

247

Tissue distributions of S. constricta Fads and FAs

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qRT-PCR results revealed that the transcripts of Scfad5a, Scfad5b and Scfad6 were

249

detected in all examined tissues (Figure 6A). Particularly, the Scfad5a expression in

250

intestine was significantly higher than that in gonad, which was further significantly

251

higher than that in other eight tissues. However, the expression of Scfad5a in those

252

eight tissues showed no significant differences. The Scfad5b expression was 12


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significant high in gonad, followed by exhalent siphon and foot muscle. While the

254

expression of Scfad5a in other seven tissues exhibited no significant differences. The

255

Scfad6 expression was highest in intestine, followed by labial palps, gill and gonad.

256

While the expression of Scfad6 in other tissues were relatively low.

257

Total of 39 FAs were identified in tissues examined (Figure 6B, Supporting

258

Information Table S1). In general, saturated FAs accounted for 33.08%-41.92%,

259

which mainly consisted of 14:0 (1.89%-7.57%), 16:0 (16.99%-26.88%) and 18:0

260

(5.26%-13.39%). Monounsaturated FAs accounted for 25.10%-31.26%, which were

261

dominated by 16:1n-7 (4.94%-13.65%), 18:1n-9 (3.25%-6.47%) and 20:1n-11

262

(2.56%-9.08%). PUFAs accounted for 26.44%-38.87%, which were represented by

263

AA (2.05%-7.41%), EPA (6.68%-12.59%) and DHA (1.99%-4.07%). Specifically, a

264

higher proportion (6.34%-7.57%) of 14:0 was found in intestine, gonad and digestive

265

gland, while a relatively lower proportion (1.89%-2.60%) was detected in other

266

tissues. Similar findings were also observed in 16:1n-7 and 18:1n-9. However,

267

compared with 14:0, 18:0 exhibited an opposite trend, showing a lower proportion

268

(5.26%-6.18%) in intestine, gonad and digestive gland, while a higher proportion

269

(9.87%-13.39%) in other tissues. Similar changing trends was observed with respect

270

to 20:1n-11. The proportion of AA was particularly high in heart, labial palps and gill,

271

which was 6.32%, 6.72% and 7.41%, respectively. The proportion of EPA was

272

particularly high in intestine, digestive gland and heart, which was 10.19%, 11.15%

273

and 12.59%, respectively. The proportion of DHA was particularly high in heart and

274

foot muscle, which was 4.07% and 4.02%, respectively. Meanwhile, two 13



275

non-methylene-interrupted (NMI)-FAs were identified, including 20:2(11,13) and

276

22:2(5,13), and they showed relatively higher proportions in labial palps and gill,

277

which were 2.23% and 2.24% or 1.92% and 2.01%, respectively.

278

DISCUSSION

279

It is well accepted that ∆5 and ∆6 Fads play crucial roles in the biosynthesis of

280

LC-PUFA,29 which are involved in complete desaturation steps in the ‘Sprecher’

281

pathway (Figure S1).11 However, in marine molluscs, only ∆5 Fad but not ∆6 Fad has

282

been functionally characterized to date.12,20,22,24 Though another ∆8 Fad has been

283

found in C. nobilis,26 DHA can not be biosynthesized without ∆6 or ∆4 Fad.

284

Excitingly, in the present study, not only ∆5 Fads (Scfad5a, Scfad5b) but also ∆6 Fad

285

(Scfad6) were characterized in S. constricta, providing compelling evidence that AA,

286

EPA and DHA could be simultaneously biosynthesized by this important bivalve

287

species.

288

The newly cloned Scfad5a, Scfad5b and Scfad6 all possessed typical features of

289

front-end Fads (Figure 2 and 3), indicating that Fads possessed highly conserved

290

functional domains during evolution.30 In the present study, though two transcripts of

291

∆5 Fads were characterized in S. constricta, Scfad5a exhibited a higher catalytic

292

activity towards ∆5 Fad substrates than Scfad5b (Table 2), which might be attributed

293

to the different amino acids between the first and second histidine boxes of the two

294

enzymes31,32 (Figure 2). Similar observation has also been found in the two ∆5 Fads

295

of H. discus hannai.12 The presence of multiple transcripts of ∆5 Fads in S. constricta

296

or H. discus hannai might be tightly related to their special physiological needs. At 14


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297

the molecular level, they might be caused by alternative splicing from one gene,

298

variations in chromosomal numbers or that they were basically different genes,33-35

299

and the exact reason could be clarified when their complete genomic information

300

become available.

301

The phylogenetic tree analysis revealed that Scfad5a, Scfad5b and ∆5 Fads or

302

Fad-like genes of other marine molluscs were clustered into one group (Figure 3).

303

Meanwhile, though with no strong bootstrap support, Scfad6 was clustered most close

304

with Fad-like genes of some marine molluscs (Figure 3), which was consistent with

305

the phylogenetic result obtained by Surm et al.8 It indicated that ∆5/6 Fads might also

306

exist in other marine molluscs, and this finding would undoubtedly deepen the

307

understanding of LC-PUFA biosynthesis in these organisms.

308

The desaturation efficiency towards 20:3n-6 and 20:4n-3 of Scfad5a and Scfad5b

309

ranged from 8.58% to 13.39% (Table 2), which was lower than that in H. discus

310

hannai and C. nobilis (14.9%-31.4%),12,24 as well as that in O. vulgaris and S.

311

officinalis (39%-48%).20,22 The significantly different ∆5 Fad activities among species

312

might be significantly correlated with their living conditions. S. constricta resides in

313

intertidal zones and estuarine waters along the West Pacific Ocean, where usually rich

314

in LC-PUFA-rich microalgae. The superior living conditions may weaken its ∆5 Fad

315

activity to some extent. Meanwhile, S. constricta is a typical benthic bivalve with

316

little mobility once settled, and the microalgal diets usually are seasonally dependent.

317

LC-PUFA in microalgae also greatly vary among species, and these features in turn

318

may force this bivalve to retain some ∆5 Fad activity. Besides, Scfad5a and Scfad5b 15



319

exhibited higher desaturation activities towards 18:0 (16.21% and 21.36%,

320

respectively) (Figure 4 and Table 2), indicating that 18:1n-13 or its following

321

products might have particular biological significance for S. constricta. In some

322

marine molluscs, ∆5 Fads have also been demonstrated to participate in the

323

biosynthesis of NMI-FAs,20,22,24 while no NMI-FA products were observed in yeasts

324

transformed with pYScfad5a or pYScfad5b in the present study. Interestingly, FA

325

analysis showed that two NMI-FAs, including 20:2(11,13) and 22:2(5,13), were

326

identified in S. constricta tissues, which exhibited higher proportions in labial palps

327

and gill. Therefore, it is necessary to investigate whether Scfad5a and Scfad5b were

328

involved in the biosynthesis of NMI-FAs. Though Scfad6 was the first functionally

329

characterized ∆6 Fad in marine molluscs, its desaturation activities towards 18:2n-6

330

(1.56%) and 18:3n-3 (3.59%) were extremely low compared with marine teleosts,

331

such as Rachycentron canadum (28.3%-32.0%), Lates calcarifer (36.5%-50.8%) and

332

Scatophagus Argus (61.18%-82.25%).36-38 The result might also be strongly

333

correlated with the living environment of S. constricta, and the weak ∆6 Fad activity

334

of Scfad6 might play a critical role in its normal development when LC-PUFA-rich

335

diets were not available. Importantly, further investigation is highly required to clarify

336

whether the desaturation activities towards 24:4n-6 and 24:5n-3 of Scfad6 were also

337

extremely low, since substrate specificity is a significant feature for Fads.9

338

Scfad5a and Scfad5b both exhibited high expression level in gonad, and

339

particularly high expression of Scfad5a was found in intestine. The result was

340

consistent with tissue distributions of ∆5 Fad in other marine molluscs,12,20,22,24 which 16


Page 16 of 34

Page 17 of 34


341

might be attributed to the distinct physiological roles of those tissues. For instance,

342

intestine has been considered as the significant site to metabolize FAs from diets, and

343

the gonad requires more LC-PUFA to satisfy its needs during development and

344

reproduction. Similar to Scfad5a and Scfad5b, the highest expression of Scfad6 was

345

found in intestine, followed by gonad. Notably, labial palps and gill also exhibited

346

high expressions of Scfad6, indicating that LC-PUFA (especially for desaturated

347

products of ∆6 Fad or their following products) might be fundamental for labial palps

348

and gill to execute their physiological functions.

349

Investigation on tissue distributions of FAs will not only facilitate to understand the

350

biosynthesis of LC-PUFA in S. constricta, but also reveal which FAs play critical

351

roles in corresponding tissues. As the desaturation products of ∆6 Fad, extremely low

352

proportions of both 18:3n-6 and 18:4n-3 were found in all tissues examined. The

353

proportion of 18:4n-3 was 0.15%-1.40%, while that of 18:3n-6 was 0.17%-0.30%.

354

The result indicated that 18:3n-6 and 18:4n-3 played negligible physiological roles in

355

S. constricta. As the desaturation products of ∆5 Fad, relatively higher proportions of

356

AA (6.32%-7.41%) were found in heart, labial palps and gill. Moreover, relatively

357

higher proportions of EPA (10.19%-12.59%) were found in intestine, digestive gland

358

and heart. The result indicated that AA and EPA were both important for heart, while

359

AA was particularly essential for both labial palps and gill, and EPA was critical for

360

intestine and digestive gland. The presence of 22:5n-3 implied the DHA biosynthesis

361

through the ‘Sprecher’ pathway (Figure S1), which exhibited high proportion in heart

362

(2.12%), followed by intestine (1.57%), mantle (1.55%) and labial palps (1.5%). 17



363

Taken together, we carried out a unique report of S. constricta Fads, including

364

Scfad5a, Scfad5b and Scfad6. The results suggested that S. constricta possessed the

365

ability to endogenously biosynthesize LC-PUFA to some extent, at least for ARA and

366

EPA. However, the ability appeared to be limited because of the relatively low

367

desaturation activities of S. constricta Fads (especially for Scfad6), implying an

368

adaptation to living marine environment. Tissue distributions of S. constricta Fads

369

and FAs provided important clues for further understanding the biosynthesis of

370

LC-PUFA in this important bivalve. Notably, Scfad6 was the first ∆6 Fad functionally

371

characterized in marine molluscs to our knowledge, which could be used as a valuable

372

candidate to explore the molecular evolution of whole Fad family.

373 374

ASSOCIATED CONTENT

375

Supporting Information

376

The Supporting Information is available free of charge on the ACS Publications

377

website.

378

Figure S1 presents the biosynthetic pathway of LC-PUFA.

379

Table S1 exhibits the tissue distributions of FAs in S. constricta presented as mean

380

±sd (%).

381 382

AUTHOR INFROMATION

383

Corresponding Author

384

*Tel.: 86-0574-87609570. Fax: 0574-87609570. Email: [email protected]. 18


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385

ORCID

386

Zhaoshou Ran: 0000-0002-1665-3116

387

JiLin Xu: 0000-0002-4496-6937

388

Funding

389

This research was supported by Ningbo Science and Technology Research Projects,

390

China (2017C110003); the Earmarked Fund for Modern Agro-industry Technology

391

Research System, China (CARS-49), and partly sponsored by K. C. Wong Magna

392

Fund in Ningbo University.

393

Notes

394

The authors declare no competing financial interest.

395 396

REFERENCES

397

(1) McMurchie, E. J. Dietary lipids and the regulation of membrane fluidity and

398

function. Physiol Regul. Membr. Fluidity 1988, 3, 189-237.

399

(2) Lauritzen, L.; Hansen, H. S.; Jørgensen, M. H.; Michaelsen, K. F. The essentiality

400

of long chain n-3 fatty acids in relation to development and function of the brain

401

and retina. Prog. lipid Res. 2001, 40, 1-94.

402 403

(3) Fritsche, K. Fatty acids as modulators of the immune response. Annu. Rev. Nutr. 2006, 26, 45-73.

404

(4) Russo, G. L. Dietary n-6 and n-3 polyunsaturated fatty acids: from biochemistry to

405

clinical implications in cardiovascular prevention. Biochem. Pharmacol. 2009, 77,

406

937-946. 19



407

(5) Moylan, S.; Maes, M.; Wray, N. R.; Berk, M. The neuroprogressive nature of

408

major depressive disorder: pathways to disease evolution and resistance, and

409

therapeutic implications. Mol. Psychiatr. 2013, 18, 595.

410 411

(6) Joseph, J. D. Lipid composition of marine and estuarine invertebrates. Part II: Mollusca. Prog. lipid Res. 1982, 21, 109-153.

412

(7) Monroig, Ó.; Tocher, D. R.; Navarro, J. C. Biosynthesis of polyunsaturated fatty

413

acids in marine invertebrates: recent advances in molecular mechanisms. Mar.

414

Drugs 2013, 11, 3998-4018.

415

(8) Surm, J. M.; Prentis, P. J.; Pavasovic, A. Comparative Analysis and Distribution of

416

Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs. PLoS One 2015, 10,

417

e0136301.

418

(9) Castro, L. F. C.; Tocher, D. R.; Monroig, O. Long-chain polyunsaturated fatty acid

419

biosynthesis in chordates: Insights into the evolution of Fads and Elovl gene

420

repertoire. Prog. lipid Res. 2016, 62, 25-40.

421 422 423 424

(10) Cook, H. W.; McMaster, R. C. R. Fatty acid desaturation and chain elongation in eukaryotes. New Compr. Biochem. 1996, 31, 129-152. (11) Sprecher, H. Metabolism of highly unsaturated n-3 and n-6 fatty acids. BBA-Mol. Cell Biol. L. 2000, 1486, 219-231.

425

(12) Li, M.; Mai, K.; He, G.; Ai, Q.; Zhang, W.; Xu, W.; Wang, J.; Liufu, Z.; Zhang,

426

Y.; Zhou, H. Characterization of two ∆5 fatty acyl desaturases in abalone (Haliotis

427

discus hannai Ino). Aquaculture 2013, 416, 48−56.

428

(13) Tocher, D. R. Fatty acid requirements in ontogeny of marine and freshwater fish. 20


Page 20 of 34

Page 21 of 34


429

Aquac. Res. 2010, 41, 717-732.

430

(14) Castro, L. F. C.; Monroig, O.; Leaver, M. J.; Wilson, J.; Cunha, I.; Tocher, D. R.

431

Functional desaturase Fads1 (∆5) and Fads2 (∆6) orthologues evolved before the

432

origin of jawed vertebrates. PloS one 2012, 7, e31950.

433

(15) Monroig, O.; Navarro, J. C.; Tocher, D. R. Long-chain polyunsaturated fatty

434

acids in fish: recent advances on desaturases and elongases involved in their

435

biosynthesis. Proceedings of the XI International Symposium on Aquaculture

436

Nutrition. Universidad Autonoma de Nuevo Leon Monterrey, Nuevo Leon, Mexico,

437

2011, 257-282.

438

(16) Li, Y.; Hu, C.; Zheng, Y.; Xia, X.; Xu, W.; Wang, S.; Chen, W.; Sun, Z.; Huang, J.

439

The effects of dietary fatty acids on liver fatty acid composition and ∆ 6-desaturase

440

expression differ with ambient salinities in Siganus canaliculatus. Comp. Biochem.

441

Phys. B 2008, 151, 183-190.

442

(17) Kabeya, N.; Yamamoto, Y.; Cummins, S. F.; Elizur, A.; Yazawa, R.; Takeuchi, Y.;

443

Haga, Y.; Satoh, S.; Yoshizaki G. Polyunsaturated fatty acid metabolism in a marine

444

teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2

445

desaturase and Elovl5 and Elovl4 elongases. Comp. Biochem. Phys. B, 2015, 188,

446

37-45.

447

(18) Ran, Z.; Li, S.; Zhang, R.; Xu, J.; Liao, K.; Yu, X.; Zhong, Y.; Ye, M.; Yu, S.; Ran,

448

Y.; Huang, W.; Yan, X. Proximate, amino acid and lipid compositions in

449

Sinonovacula constricta (Lamarck) reared at different salinities. J. Sci. Food Agr.

450

2017, 97, 4476-4483. 21



451

(19) Ran, Z.; Chen, H.; Ran, Y.; Yu, S.; Li, S.; Xu, J.; Liao, K.; Yu, X.; Zhong, Y.; Ye,

452

M.; Yan, X. Fatty acid and sterol changes in razor clam Sinonovacula constricta

453

(Lamarck 1818) reared at different salinities. Aquaculture 2017, 473, 493-500.

454

(20) Monroig, Ó.; Navarro, J. C.; Dick, J. R.; Alemnay, F.; Tocher, D. R. Identification

455

of a ∆5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier

456

1797) involved in the biosynthesis of essential fatty acids. Mar. biotechnol. 2012,

457

14, 411-422.

458

(21) Monroig, O.; Guinot, D.; Hontoria, F.; Tocher, D. R.; Navarro, J. C. Biosynthesis

459

of essential fatty acids in Octopus vulgaris (Cuvier, 1797): Molecular cloning,

460

functional characterisation and tissue distribution of a fatty acyl elongase.

461

Aquaculture 2012, 360, 45-53.

462

(22) Monroig, O.; Hontoria, F.; Varó, I.; Tocher, D. R.; Navarro, J. C. Investigating the

463

essential fatty acids in the common cuttlefish Sepia officinalis (Mollusca,

464

Cephalopoda): Molecular cloning and functional characterisation of fatty acyl

465

desaturase and elongase. Aquaculture 2016, 450, 38-47.

466

(23) Monroig, Ó.; de Llanos, R.; Varó, I.; Hontoria, F.; Tocher, D. R.; Puig, S.;

467

Navarro, J. C. Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris:

468

Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase

469

and an Elongation of Very Long-Chain Fatty Acid 4 Protein. Mar drugs 2017, 15,

470

82.

471

(24) Liu, H.; Guo, Z.; Zheng, H.; Wang, S.; Wang, Y.; Liu, W.; Zhang, G. Functional

472

characterization of a ∆5-like fatty acyl desaturase and its expression during early 22


Page 22 of 34

Page 23 of 34


473

embryogenesis in the noble scallop Chlamys nobilis Reeve. Mol. Biol. Rep. 2014,

474

41, 7437-7445.

475

(25) Liu H, Zheng H, Wang S, Wang, Y.; Li, S.; Liu, W.; Zhang, G. Cloning and

476

functional characterization of a polyunsaturated fatty acid elongase in a marine

477

bivalve noble scallop Chlamys nobilis Reeve. Aquaculture 2013, 416, 146-151.

478

(26) Liu H, Zhang H, Zheng H, Wang, S.; Guo, Z.; Zhang, G. PUFA biosynthesis

479

pathway in marine scallop Chlamys nobilis Reeve. J. Agr. Food chem. 2014, 62,

480

12384-12391.

481

(27) Xu, J.; Zhou, H.; Yan, X.; Zhou, C.; Zhu, P.; Ma, B. Effect of unialgal diets on

482

the composition of fatty acids and sterols in juvenile ark shell Tegillarca granosa

483

Linnaeus. J. Agr. Food Chem. 2012, 60, 3973-3980.

484 485 486 487 488 489 490 491 492 493 494

(28) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ∆∆CT method. Methods 2001, 25, 402-408. (29) Nakamura, M. T.; Nara, T. Y. Structure, function, and dietary regulation of ∆6, ∆5 and ∆9 desaturases. Annu. Rev. Nutr. 2004, 24, 345-376. (30) Sperling, P.; Ternes, P.; Zank, T. K.; Heinz, E. The evolution of desaturases. Prostag. Leukotr. Ess. 2003, 68, 73-95 (31) Meesapyodsuk, D.; Qiu, X. The front-end desaturase: structure, function, evolution and biotechnological use. Lipids 2012, 47, 227-237. (32) Sayanova, O.; Beaudoin, F.; Libisch, B.; Shewry, P.; Napier, J. Mutagenesis of the borage ∆6 fatty acid desaturase. Biochem. Soc. Trans. 2000, 28, 636-638. (33) Brenna, J. T.; Kothapalli, K. S.; Park, W. J. Alternative transcripts of fatty acid 23



495

desaturase (FADS) genes. Prostag. Leukotr. Ess. 2010, 82, 281-285.

496

(34) Ren, H.; Yu, J.; Xu, P.; Tang, Y. Influence of dietary fatty acids on muscle fatty

497

acid composition and expression levels of ∆6 desaturase-like and Elovl5-like

498

elongase in common carp (Cyprinus carpio var. Jian). Comp. Biochem. Physiol. B

499

2012, 163, 184-192.

500

(35) Monroig, Ó.; Zheng, X.; Morais, S.; Leaver, M. J.; Taggart, J. B.; Tocher, D. R.

501

Multiple genes for functional ∆6 fatty acyl desaturases (Fad) in Atlantic salmon

502

(Salmo salarL.): Gene and cDNA characterization, functional expression, tissue

503

distribution and nutritional regulation. Biochim. Biophys. Acta. 2010, 1801,

504

1072-1081.

505

(36) Zheng, X.; Ding, Z.; Xu, Y.; Monroig, Ó.; Morais, S.; Tocher, D. R.

506

Physiological roles of fatty acyl desaturases and elongases in marine fish:

507

characterisation of cDNAs of fatty acyl ∆6 desaturase and elovl5 elongase of cobia

508

(Rachycentron canadum). Aquaculture 2009, 290, 122-131.

509

(37) Mohd-Yusof, N. Y.; Monroig, O.; Mohd-Adnan, A.; Wan, K. L.; Tocher, D. R.

510

Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass,

511

Lates calcarifer. Fish Physiol Biochem. 2010, 36, 827-843.

512

(38) Xie, D.; Chen, F.; Lin, S.; Wang, S.; You, C.; Monroig, O.; Tocher, D. R.; Li, Y..

513

Cloning, functional characterization and nutritional regulation of ∆6 fatty acyl

514

desaturase in the herbivorous euryhaline teleost Scatophagus argus. PloS One 2014,

515

9, e90200.

516 24


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Figure captions Figure 1. Peptide sequence alignment of Scfad5a and Scfad5b with ∆5 Fads from representative mammals, fish and marine molluscs, including Homo sapiens (AAF29378), Mus musculus (BAB69894), Danio rerio (AAG25710), Octopus vulgaris (AEK20864), Chlamys nobilis (AIC34709) and Haliotis discus hannai (ADK38580, ADK12703) by using ClustalW. The cytochrome-b5 like domain is underlined with a solid line, the heme-binding motif of HPGG is highlighted with a short bold line, and the three histidine boxes are denoted with frames. Notably, the different domains between Scfad5a and Scfad5b are indicated by bold frame.

Figure 2. Peptide sequence alignment of Scfad6 with ∆6 Fads from representative mammals and fish, including H. sapiens (AAD20018), M. musculus (AAD20017), D. rerio (AAG25710) and Siganus canaliculatus (ABR12315) by using ClustalW. The cytochrome-b5 like domain is underlined with a solid line, the heme-binding motif of HPGG is highlighted with a short bold line, and the three histidine boxes are denoted with frames.

Figure 3. Phylogenetic tree comparing the deduced amino acid sequences of Scfad5a, Scfad5b and Scfad6 (bold fonts) with ∆5/6 Fad proteins from representative mammals, fish, marine molluscs and other lower eukaryotes. The tree was constructed using the maximum-likelihood approach with MEGA 7. The horizontal branch length is proportional to amino acid substitution rate per site. The numbers represent the 25



frequencies with which the tree topology presented was replicated after 1,000 iterations. An asterisk indicates Fad sequences of Crassostrea gigas, Aplysia californica and Lottia gigantea that have not been functionally characterized but their complete genomes are currently available.

Figure 4. Functional characterization of Scfad5a and Scfad5b. FAMEs were extracted from yeasts transformed with pYES2 alone (A and D), pYScfad5a (B and E) or pYScfad5b (C and F), which were cultured in the presence of FA substrates (highlighted with *). Peaks 1-4 are the main endogenous FAs of S. cerevisiae, namely 16:0, 16:1 isoforms, 18:0 and 18:1n-9, respectively. The additional peaks were identified as 18:1n-13 (B, C, E and F), 20:4n-6 (B and C) and 20:5n-3 (E and F). Vertical axis represents FID response, and horizontal axis means retention time.

Figure 5. Functional characterization of Scfad6. FAMEs were extracted from yeasts transformed with pYES2 alone (A and C) or pYScfad6 (B and D), which were cultured in the presence of FA substrates (highlighted with *). Peaks 1-4 are the main endogenous FAs of S. cerevisiae, namely 16:0, 16:1 isoforms, 18:0 and 18:1n-9, respectively. The additional peaks were identified as 18:3n-6 (B) and 18:4n-3 (D). Vertical axis represents FID response, and horizontal axis means retention time.

Figure 6. Tissue distributions of Scfad5a, Scfad5b and Scfad6 (A), and representative FAs (B). Relative expression of S. constricta Fad was measured by qRT-PCR, 26


Page 26 of 34

Page 27 of 34


normalized by β-actin, and calibrated by that of mantle, respectively. Letters on the top of columns sharing a common letter with same color are not significantly different (P ≥ 0.05).

Tables Table 1 Primers used for Scfad5a, Scfad5b and Scfad6 full length cloning, functional characterization and qRT-PCR.a Transcript

Aim

Primer

Sequence

Fragment PCR

V-F

ATGGGCAAAGGCGGTCAG

V-R

CTAGTCACTGTGAAACTCCCTGTA

RACE PCR

5’RACE-outer

CCATGTTGACATGACCACCAGTCCGAG

5’RACE-inner

TCTGCATGAACCGTGGACCGGCATAC

3’RACE-outer

CTGTTTCCCACCATGCCGCGACAT

3’RACE-inner

TGGCGTGTCGTACCAAGTGAAGCCG

Sc5-F

GGGGTACCATGGGCAAAGGCGGTCAG

Scfad5a/b

ORF cloning

qRT-PCR

Sc5-R

CGGAATTCCTAGTCACTGTGAAACTCCCTGTATGT

Scfad5a-F

ACATCCCAGGCCCAAGGC

Scfad5a-R

CCCTTGACAAACCCGGTCAA

Scfad5b-F

TTATTCCACATCCCAGGTACAGACT

Scfad5b-R

CCCTTTGTGAAGCCCATGGT

Scfad6 Fragment PCR

RACE PCR

ORF cloning

qRT-PCR

V-F

ATGCACAATGATCGGGAGTATG

V-R

TTACATTCCTTCATAGTATGCATTGTAC

5’RACE-outer

CGTAGAATAGTGGGTTGACACGCAGCAG

5’RACE-inner

CATGAACTTTCCGACATACTCCCGATCA

3’RACE-outer

GCATCATCTCTTCCCTACGATGCCTCG

3’RACE-inner

GGTCCTTGGAACACTCTGGGGAAATCTG

Sc6-F

GGGGTACCATGGGGAAAGGCGGACAGAA

Sc6-R

CGGAATTCTTACATTCCTTCATAGTATGCATTGTACC

Scfad6-F

CTAACGAGGTGGACTTTGATGG

Scfad6-R

AGAGTGTTCCAAGGACCTGACC

β-actin-F

CCATCTACGAAGGTTACGCCC

β-actin-R

TCGTAGTGAAGGAGTAGCCTCTTTC

β-actin qRT-PCR

a

Fragment PCR is used to verify the sequences obtained from S. constricta 27



Page 28 of 34

transcriptome data. RACE PCR is used to obtain S. constricta Fads’ full length. ORF cloning is used to construct recombinant plasmid. The qRT-PCR is used for tissue distribution analysis. Restriction sites of KpnI and EcoRI are underlined.

Table 2 Substrate conversion rates of yeasts transformed with pYScfad5a, pYScfad5b and pYScfad6.a

a

Substrate

Product

18:2n-6 18:3n-3

Conversion rate (%)

Activity

Scfad5a

Scfad5b

Scfad6

18:3n-6

-

-

1.56±0.17

∆6

18:4n-3

-

-

3.59±0.19

∆6

20:2n-6

20:3n-6

-

-

-

∆8

20:3n-3

20:4n-3

-

-

-

∆8

20:3n-6

20:4n-6

13.39±0.32 a

10.95±0.54 b

-

∆5

20:4n-3

20:5n-3

11.71±0.28 a

8.58±0.03 b

-

∆5

22:4n-6

22:5n-6

-

-

-

∆4

22:5n-3

22:6n-3

-

-

-

∆4

18:0

18:1n-13

16.21±1.54 a

21.36±0.93 b

-

∆5

Values within the same row sharing a common letter are not significantly different

(P ≥ 0.05). ‘-’ means not detected.

28


Page 29 of 34


Figure graphics Figure 1.

29



Figure 2.

30


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Page 31 of 34


Figure 3.

31



Figure 4.

Figure 5.

32


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Figure 6.

33



Graphic for table of contents

34


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Biosynthesis of polyunsaturated fatty acids in the ... - ACS Publications

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