Chinese Journal of Oceanology and Limnology   2015, Vol. 33 Issue (3) : 597-603     PDF       
http://dx.doi.org/10.1007/s00343-015-4167-x
Shanghai University
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Article Information

QIN Chuanjie (覃川杰) , SHAO Ting (邵婷)
The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli
Chinese Journal of Oceanology and Limnology, 2015, 33 (3) : 597-603
http://dx.doi.org/10.1007/s00343-015-4167-x

Article History

Received Jul. 20, 2014;
accepted in principle Sep. 12, 2014;
accepted for publication Oct. 28, 2014
The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli
QIN Chuanjie (覃川杰) , SHAO Ting (邵婷)        
College of Life Sciences, Neijiang Normal University, Neijiang 641000, China
ABSTRACT:The Clock gene, a key molecule in circadian systems, is widely distributed in the animal kingdom. We isolated a 936-bp partial cDNA sequence of the Clock gene (Pva-clock) from the darkbarbel catfish Pelteobagrus vachelli that exhibited high identity with Clock genes of other species of fish and animals (65%-88%) . The putative domains included a basic helix-loop-helix (bHLH) domain and two period-ARNT-single-minded (PAS) domains, which were also similar to those in other species of fish and animals. Pva-Clock was primarily expressed in the brain, and was detected in all of the peripheral tissues sampled. Additionally, the pattern of Pva-Clock expression over a 24-h period exhibited a circadian rhythm in the brain, liver and intestine, with the acrophase at zeitgeber time 21:35, 23:00, and 23:23, respectively. Our results provide insight into the function of the molecular Clock of P. vachelli.
Key words: Clock gene     Pelteobagrus vachelli     circadian rhythms      circadian gene    
1 INTRODUCTION

Circadian rhythms,which exist widely in the animal kingdom,are self-sustained rhythms of physiology,biochemistry,or behavior generated according to the 24-h periodicity of day and night. This periodicity is auto-regulated by opposing positive and negative transcriptional-translational mechanisms involving a series of circadian genes and their proteins,such as Period,Clock,Cycle, and Cryptochrome(Bell-Pedersen et al., 2005; Hardin,2005; Yu and Hardin, 2006). The core molecular mechanism underlying the generation of sustained circadian rhythms involves dimerization of the transcription factor Clockwith Bmal1(Cerdá Reverter et al., 1998; Hardin,2009). Previous studies have demonstrated that circadian rhythms synchronized to the light/dark phase also exist in fish, and that the circadian rhythms of the gene(Period3,Dec,Cycle,Cryptochrome)cloud regulate the behavioral patterns of flatfish Solea senegalensis,European seabass Dicentrarchus labrax, and zebrafish Danio rerio. Thus,fish can display either diurnal and /or nocturnal behavioral patterns(Tamai et al., 2005; Martín-Robles et al., 2011; Del Pozo et al., 2012). However,the effects of the Clockgene on the physiological functions and molecular clock of fish remain unknown.

The Clockgene was first identified in mice in 1997(Antoch et al., 1997). Since then,Clockgenes have been isolated from mammals,insects,birds, and shrimp(Steeves et al., 1999; Yoshimura et al., 2000; Chang et al., 2003; Yang et al., 2006; Martín-Robles et al., 2011). Expression of the Clockgene drives the master Clockin the suprachiasmatic nucleus(SCN),which is light responsive and can be synchronized or reset by environmental cues such as the light/dark cycle. However,peripheral Clocks are not affected by light,although they are regulated by other external cues such as food,the availability of which is an important factor in the regulation of Clockgenes in the liver and colon(Whitmore et al., 1998; Stokkan et al., 2001; Montoya et al., 2010; Feliciano et al., 2011). A number of researchers have studied the circadian genes Periods and Cryptochromes, and havedocumented their molecular structure and daily rhythm in fish(Martín-Robles et al., 2011; Del Pozo et al., 2012). However,the components of the molecular Clock in fish are poorly understood,with the exception of the zebrafish Danio rerio.

The darkbarbel catfish Pelteobagrus vachelliis an important freshwater aquaculture species in China(Zheng et al., 2010). Because of its high market value,there has been increased interest in the culture and study of this species in recent years. To date,research has focused on growth,body lightness,lipid metabolism,immune responses, and feeding behavior(Xue et al., 2011; Li et al., 2013). Our objective was to clone the Clockgene and to analyze the circadian rhythms of its expression in P. vachelli. 2 MATERIAL AND METHOD 2.1 Animal and experimental design

One hundred and fifty adult darkbarbel catfish(15.26±2.58 g)were obtained from a commercial fish farm in Neijiang,China and r and omly divided into six groups. Fish were reared at the Key Laboratory of Sichuan Province for Fish Conservation and Utilization in the Upper Reaches of the Changjiang(Yangtze)River(Neijiang,China). Fish were acclimated in the laboratory for 2 weeks. They were held in two 500-L tanks with an open water system and constant water temperature(17°C). Fish were fed a commercial catfish diet for 2 weeks(Haida,Sichuan,China) and were maintained under the natural photoperiod.

The animals were starved for 3 d before experimentation to avoid the effects of feeding. Nine darkbarbel catfish were anesthetized and sacrificed every 4 h(zeitgeber time,ZT 00:00,04:00,08:00,12:00,16:00, and 20:00), and a sample of the intestine,brain(diencephalon), and liver was dissected from each of the nine fish at each sample point. The fish were taken from alternating tanks at each time-point to minimize stress. Additionally,tissue(heart,muscle,spleen,gill,adipose tissue)samples were taken from the nine fish at ZT 08:00 for tissue expression analysis. RNA extraction buffer(Aidlab Biotechnologies,Beijing,China)was immediately added to the samples,which were then stored at -80°C for future analysis. 2.2 RNA isolation and reverse transcriptionpolymerase chain reaction

Total RNA was isolated from the suprachiasmatic nuclei(SCN)of the brain and further purified using an RNA extraction buffer(TaKaRa,Dalian,China)according to the manufacturer’s protocol. First-str and cDNA was synthesized from 2 μg of total RNA using the SuperScript™/First-Str and cDNA Synthesis Kit(TaKaRa),following the manufacturer’s protocols. The first-str and cDNA was used as the template, and the cDNA fragment encoding the Clockgene was amplified by PCR using a pair of degenerate primers designed based on the cDNA sequences of Daniorerio(AAI63244.1),Gallus(AAD32860.1), and Homo sapiens(AAF13733.1). The sequences of the degenerate primer pair used to amplify the ClockcDNA fragments in darkbarbel catfish were CLO-R(5′-TCWGGYYTBGAATTSMACTG-3′) and CLO-F(5′-GARAAGAARMGWMGAGATCA-3′)(Yang et al., 2006). The PCR program was: 94°C for 5 min,followed by 35 cycles of 94°C for 30 s,54°C for 30 s, and 72°C for 1 min,with the addition of a final polymerization step at 72°C for 10 min. 2.3 Cloning,sequencing, and analyses

The PCR fragments were subjected to agarose gel(1.5%)electrophoresis. The amplified cDNA fragments were cloned into the pGEM-T Easy vector following the manufacturer’s instructions(Promega Corporation,Madison,WI,USA). Recombinant bacteria were identified by blue/white screening.

Plasmids containing the insert were purified using a mini-prep kit(Promega) and used as a template for DNA sequencing. The deduced amino acid sequence of the partial cDNA was analyzed with the ORF(Open Reading Frame)finder of http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi. Protein sequence similarity searches were conducted using the Clustal W Multiple Alignment program(http://www.ebi.ac.uk/clustalw/,http://www.ch.embnet.org/software/BOX_form.html). Domain identification was performed with the Motif scan program(http://hits.isb-sib.ch/cgi-bin/PFSCAN). 2.4 Tissue distribution of Pva- Clock and daily expression pattern

Total RNA was extracted as described in Section 2.1. The first-str and cDNA was synthesized using a cDNA first-str and synthesis kit with MMLV reverse transcriptase(TaKaRa)with approximately 5 μg total RNA, and diluted 10 times. Two gene-specific primers,CLO-GS(5′-CTTCCATTGGGCGAACACTCTGA-3′) and CLO-GA(5′-GTGGAGTTAGGCACGGTGTTGAG-3′),were designed to amplify the 218 bp product of the Clock gene.

The cDNA of all samples was used as a substrate for quantitative real-time PCR. Samples were analyzed on a LightCycler ®Nano Real-Time PCR System(Roche,Basel,Switzerl and )with FastStart Essential DNA Green Master(Roche); the PCR temperature profile and reaction conditions were the same as in the manufacturer’s instructions. The primers β-actin,F(5′-CACTGTGCCCATCTACGAG-3′) and β-actin R(5′-CCATCTCCTGCTCGAAGTC-3′)were used to amplify the 200-bp fragment as a reference gene(Zheng et al., 2010). The comparative C Tmethod was used to analyze the level of Clockgene expression. The ΔCt(difference in Ctbetween the target and internal control)for each sample was subtracted from that of the calibrator,denoted as ΔΔCt. The level of Clockgene expression was calculated using the 2 -ΔΔCtmethod(Livak and Schmittgen, 2001).

Fig. 1 The partial nucleotide sequence and deduced amino acid sequence of Pelteobagrus vachelli Clock cDNA

The conserved helix-loop-helix domain is shown with a single line. The two predicted period-ARNT-single-minded domains are shown with double lines.

2.5 Statistical analysis

Brain tissue was used as a control for Clock gene expression analyses. Samples obtained at ZT 08:00 were used as a control for analysis of the daily variation in Clockgene expression in the different tissues. Differences in the relative Clock gene expression within each tissue(brain,intestine, and liver)over 24 h were analyzed using one-way ANOVA(ANOVA I)followed by Tukey’s Test in SPSS 18.0. The cosine function(Y= M+ A×cos(Ωt+ Φ))of Microsoft Office Excel(2007)was used to analyze the rhythmic expression of the Clockgene in the three tissues where Mis mesor,Ais amplitude,Ωis angular frequency(2π/24 for the circadian rhythms), and Φ is acrophase(Del Pozo et al., 2012). The significance level was fixed at P<0.05 for all statistical analyses. 3 RESULT 3.1 Cloning of Pva- Clock and multiple alignment analysis

Comparison of the isolated partial cDNA sequences with those of other fish Clockgenes using BLAST(National Center for Biotechnology Information,NIH,USA)led to the identification of Clock gene sequences as a putative Clockgene. The partial cDNA(936 bp)encoded a protein consisting of 311 amino acid residues(Figs.1 and 2). Sequence comparisons showed that the deduced amino acid sequence of PvaClockhad an overall identity with the Clockgenes of Danio rerio(88%,AAI63244.1),Oncorhynchustshawytscha(88%,ABI34137.1),Haplochromisburtoni(80%,ABP97104.1),Sebastes schlegelii(76%,AAO15522.1), and Homo sapiens(65%,AAF13733.1)(Fig. 2). Multiple alignments of the amino acid sequences of P. vachelliusing the Clustal W program indicated that the Clockgene also shared common functional domains with the aforementioned species,including a basic helix-loop-helix(bHLH)domain,a period-ARNT-single-minded-A(PAS-A)(where ARNT is equivalent to the AhR(aryl hydrocarbon receptor)nuclear translocator)domain, and a PAS-B domain(Figs.1 and 2). The P. vachelliClockcDNA sequence and the deduced amino acid sequence have been submitted to the National Center for Biotechnical Information GenBank database(accession number KF742501).

Fig. 2 Multiple alignment of the P. vachelli Clockgene with other Clockgenes using the Clustal W Multiple Alignment program

The single and double lines are described in the legend for Fig. 1. The white letters and asterisks indicate identical residues. The black letters with a light background indicate semi-conserved amino acid substitutions.(-)represents a gap or deletion. The Clockof P. vachelliis aligned with the Clocks of Danio rerio(GenBank: AAI63244.1),Phreatichthys and ruzzii(ADL62686.1),Haplochromis burtoni(ACI46597.1),Sebastesschlegelii(AF448805_1),Oncorhynchus tshawytscha(ABI34137.1),Ovis aries(NP_001124404.1),Cricetulus griseus(ERE90739.1),Homo sapiens(AAF13733.1) and Gallus gallus(ABL75445.1).

3.2 Tissue distribution of Pva- Clock and its daily expression

The Pva- Clockwas detected in all the tissues we analyzed. ClockmRNA levels were significantly higher in the brain than in the liver,heart,muscle,spleen,gill,adipose tissue, and intestine at ZT08:00(P<0.05; Fig. 3). Additionally,the level of Pva- Clockexpression varied throughout the day in the brain,liver and intestine(Fig. 4). This rhythm could be fit to a cosinor curve(COSINOR,P<0.05),having an acrophase at approximately ZT21:35 in the brain,ZT23:00 in the liver, and ZT23:23 in the intestine. In these three tissues,the expression of Pva- ClockmRNA from 20:00 to 04:00 ZT(dark)was significantly higher than from 08:00 to 16:00(P<0.05; Fig. 4). The cosinor parameters(mesor,amplitude, and acrophase) and statistical significance of Pva- Clock expression rhythms in P. vachellibrain,liver, and intestine are shown in Table 1.

Fig. 3 Relative level of ClockmRNA expression in different tissues of P. vachelli

Values represent the mean±SE(n=9)relative to expression in the brain. Br: brain; Li: liver; In: intestine; Gi: gill; Mu: muscle; Sp: spleen; Ad: adipose tissue; He: heart. Different letters indicate significant differences among treatments.

Fig. 4 Relative expression of ClockmRNA in the brain(a),liver(b), and intestine(c)of P. vachelli

Values represent the mean±SE(n=9). The zeitgeber time(ZT,in hours)is represented on the horizontal axis and the relative expression as fold change(log10)is plotted on the vertical axis. Different letters indicate significant differences among treatments. The dotted line indicates the circadian rhythms fitted by the cosinor method.

Table 1 Cosinor analysis of Pva- Clock gene mRNA expression rhythms in the brain,liver, and intestine of P. vachelli
4 DISCUSSION

The Clockgene belongs to the bHLH/PAS superfamily of transcription factors. There is a growing interest in the study of circadian genes in teleosts such as Salmo salar,Dicentrarchus labrax,Carassiusauratus, and Solea senegalensis(Park et al., 2007; Davie et al., 2009; Velarde et al., 2009; Martín-Robles et al., 2011). In the present study,the partial sequence of the Clockgene was identified in the darkbarbel catfish P. vachelli. The bHLH domain spans amino acids 1–42 of the Clockgene and this region in P. vachelliis similar to that of other vertebrate and invertebrate Clocksin terms of both sequence and length. The bHLH domain is well documented and has been described in many proteins,having >64% identity among species, and functions as a DNA-binding domain. The PAS-A and -B domains,spanning amino acids 64–136 and 244–290,respectively,are also highly conserved and have >44% and 70% identity among species,respectively(Yang et al., 2006). The PAS-A and -B dimerization regions are involved in protein–protein interactions and mediate binding to the heterodimeric partner cycle. These domains may be an important structural feature of a subset of genes involved in photoreception and circadian rhythmicity(Tamai et al., 2005). All Clocksexhibit high overall identities, and even higher identities in their functional domains may partially explain why species in the animal kingdom share relatively conserved circadian systems(Young and Kay, 2001).

The master Clockis located in the SCN of the anterior hypothalamus in the brain of mammals,which forms multiple and single-cell circadian oscillators. These oscillator cells maintain the 24-h periodicity determined by light,even without light/dark cycles(Hirota and Fukada, 2004; Kohsaka and Bass, 2007). In the present study,real-time PCR analysis revealed that P. vachelli Clockwas expressed in all the tissues we tested(brain,liver,heart,muscle,spleen,gill,adipose tissue, and intestine)at ZT 08:00. However,the mRNA of M. rosenbergii Clockwas primarily expressed in central nervous tissues. This broad distribution is consistent with observations in mice,zebrafish,M. rosenbergii, and humans(Allada et al., 1998; Tamai et al., 2005; Hardin,2009),suggesting that,along with Drosophila and mammals,the Clockgene in P. vachelliis present throughout the body.

In mammals,daily cycles(dark and light)are coordinated by a master hypothalamic Clockthat is reset primarily by ambient light(Allada et al., 1998). In the present study,the expression of P. vachelliClockwas significantly upregulated at 16:00 ZT,with peak expression occurring after sunset(20:00 ZT). Following this,expression decreased significantly and returned to basal levels by the end of the night,with minimum values at 08:00 ZT. These results are similar to those found for Clockin the giant river prawn M. rosenbergii(Yang et al., 2006). Clock gene expression in M. rosenbergiitended to increase in the central nervous system(brain,thoracic, and abdominal ganglia)of eyestalk-ablated prawns or those held in constant dark. The pattern of P. vachelli Clock mRNA expression was also similar to that of the period gene in the nocturnal fish species Solea senegalensis(Martín-Robles et al., 2011). In S. senegalensis,the expression of periodwas upregulated before sunrise,with its acrophase at the end of the night(MartínRobles et al., 2011). The Clockgene in P. vachelli and periodin S. senegalensisexhibited the same daily variations. It is thought that the protein products of Clock and Bmal1form heterodimers that bind to E-box sequences,resulting in enhanced transcription of Period and Cryptochromegenes(Allada et al., 1998; Yu et al., 2006). The expression of Clockin the liver and intestine of P. vachelliexhibited a significant daily oscillation in keeping with that of Clock in the brain, and was characterized by upregulation at 16:00 ZT with a peak at 20:00 ZT. This observation suggests that the Clockgenes in the brain and peripheral tissues play an important role in forming circadian rhythms(Yang et al., 2006). 5 CONCLUSION

A partial coding region of the Clockgene from the darkbarbel catfish,P. vachelli,was cloned. This Clockgene included bHLH and two PAS domains, and ClockmRNA was widely distributed in the brain and peripheral tissues. Furthermore,the expression of Pva- Clockexhibited a circadian rhythm in the brain,liver, and intestine. Our results provide new avenues to analyze the function of the molecular Clockof P. vachelli.

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