Diurnal expression of circadian clock genes period 1 and period 3 in Pelteobagrus vachellii*

Chuanjie QIN, Ting SHAO , Xufeng LIAO , Yang HE , Jun WANG , Peng HU

1 Key Laboratory of Sichuan Province for Fishes Conservation and Utilization in the Upper Reaches of the Yangtze River,Neijiang Normal University, Neijiang 641112, China

2 College oflife Science, Neijiang Normal University, Neijiang 641112, China

Abstract Circadian clock genes are crucial for generating and sustaining most rhythmic daily functions in the animal kingdom, which entrain the rhythms of biochemical, physiological, and behavioural processes.To better understand the molecular oscillations of the circadian rhythms in darkbarbel catfish ( Pelteobagrus vachellii), we isolated and characterized two circadian clock genes in P. vachellii, period 1 ( per1), and period 3 ( per3). The circadian clock gene per1 was found to encode a 1 428-amino acid polypeptide, including PER-ARNT-SIM (PAS) dimerisation domains, a PAS-associated C-terminal motif (PAC), a short mutable domain (S/M), and a nuclear export signal (NES). The 4 902-bp per3 cDNA includes an open reading frame encoding a 1 292-amino acid residue polypeptide with a PER-ARNT-SIM (PAS) domain, cytoplasmic localisation domain (CLD), interaction site (TIS), and a nuclear localisation signal (NLS). The per1 and per3 gene was constitutively expressed in all examined tissues. Moreover, per1 expression within a light/dark cycles showed rhythmic expression in the diencephalon, brain, liver and intestine, with the acrophase at 15꞉15, 12꞉52, 7꞉51, and 12꞉55, respectively. Daily expression of per3 was rhythmic in the diencephalon,brain, liver and intestine, with the acrophase at 8꞉15, 9꞉54, 10꞉39, and 10꞉25 h, respectively. These findings expand our understanding of circadian mechanism at the molecular level in this species.

Keyword: cDNA; circadian rhythm; diurnal expression; Pelteobagrus vachellii; period 1; period 3

1 INTRODUCTION

According to the environmental periodicity induced by the Earth’s rotation, animal behavior and metabolism can be coordinated and anticipated through an autonomous endogenous clock (Bell-Pedersen et al., 2005). Circadian systems are remarkably well-conserved from insects to mammals(Panda et al., 2002). The bases of the circadian rhythm are genetic and molecular and are controlled by clock genes, which are highly conserved in the animal kingdom (Bell-Pedersen et al., 2005). In mammals,these rhythm genes are rhythmically expressed in the hypothalamic suprachiasmatic nucleus (SCN) and in all peripheral tissues (Balsalobre, 2002). Similarly,the rhythmic expression of clock genes also appears in various tissues (muscle, kidney, liver, intestine and heart) of non-mammalian vertebrates, such as fish,shrimp, flies and other insects (Green and Besharse,2004; Okamura, 2004). At present, many circadian clock genes have been identified in various organisms(Wang, 2008), including circadian locomotor output cycle kaput ( CLOCK), periods, timeless, cycle,cryptochrome ( CRY), vrille, and brain- muscle- Arntlike 1 ( BMAL1) (Bell-Pedersen et al., 2005). In mammals, it is certain that CLOCK and BMAL are positive elements of a transcription-translation positive feedback loop. Likewise, Periods and Cryptochromes are negative elements of this feedback mechanism (Hastings, 2000).

As a key component of the animal circadian system, the period gene was first identified in Drosophila (Reddy et al., 1984). Wang (2008)characterized the period homologs per1, per2, and per3 in teleost fish, which are widely studied as the circadian oscillators (Robinson and Reddy, 2014). In zebrafish ( Danio rerio), four period genes have been cloned and characterized (Delaunay et al., 2000; Ziv et al., 2005). In invertebrates, period has been cloned and characterized in oriental river prawn Macrobrachium nipponense (Chen et al., 2017).Wang (2008) reported that extra copies of teleost period genes were generated by fish-specific genome duplication, and divergent evolution following duplication has resulted in the retention of various duplicate forms in diff erent fish species. Moreover,various rhythm genes have been characterized from flatfish ( Solea senegalensis) (Martín-Robles et al.,2011, 2012), gilthead sea bream ( Sparus aurata)(Vera et al., 2013), goldfish ( Carassius auratus)(Velarde et al., 2009), and reef fish ( Siganus guttatus)(Park et al., 2007), as well as periods from the European sea bass ( Dicentrarchus labrax) (Sánchez et al., 2010) and cavefish ( Phreatichthys andruzzii)(Cavallari et al., 2011). However, little information on the molecular circadian oscillation in diff erent fish species is available.

In China, the total production of Pelteobagrus was close to 450 000 tons in 2018. Yang et al. (2006)indicated that juveniles of P. fulvidraco displayed nocturnal feeding behavior. Similarly, the CLOCK has been cloned and analyzed in P. vachellii, and its mRNA showed rhythmic expression in the tissues(Qin and Shao, 2015). However, the components of molecular clock and its function in Pelteobagrus have been little studied. This work aimed to knowledge the tissue distribution and diurnal expression of per1 and per3 in P. vachellii,

2 MATERIAL AND METHOD

2.1 Fish and rearing

Adult catfish P. vachellii individuals (15.26±3.67 g)was obtained from our lab and maintained in six 300/L tanks (30/fish tank) with water circulation. At the beginning, fish were fed a commercial diet at lights on (8꞉00) of each day with a 13-h꞉11-h natural light꞉dark cycle. After 30 days, fish were randomly collected at Zeitgeber Time (ZT) 0꞉00 (control), 4꞉00,8꞉00, 12꞉00, 16꞉00 and 20꞉00. At each ZT, fish were successively sampled from diff erent tanks, with a dim red light used at ZT0꞉00, 4꞉00 and 20꞉00. Nine fish per ZT were randomly taken from diff erent tanks,anesthetized with tricaine mesylate (MS-222), and sacrificed. Diencephalon, intestine, brain (without diencephalon), and liver tissues were harvested from the nine samples collected at each ZT. Moreover,diff erent tissues [diencephalon (control), gill, spleen,heart, retina, adipose tissues and muscle] from at least nine fish were collected at ZT0 for further tissues analysis. All samples were stored in liquid nitrogen.All experimental methods were performed following the guidelines for the care and use of experimental animals of China (China’s national standard: GB/T358922018).

2.2 RNA extraction and reverse transcription-PCR

The total RNA from the samples (Section 2.1) was extracted with RNA buff er (TaKaRa, Japan) with the manufacturer’s protocol. To identify period gene cDNA, per1 unigenes (c40089_g1, SRP108959) and per3 unigenes (c40089_g1) were selected to obtain the full-length period cDNA (Qin et al., 2017). Firststrand cDNA was synthetized using the protocol of MMLV reverse transcriptase (TaKaRa, Japan).

Following the methods for the rapid amplification of cDNA ends (RACE), primer sets consisting of PER1-5′ gene-specific primers (GSP), PER3-5′ GSP and the universal primer A mix (UPM) were used to obtain the 5′ cDNA of PER1 and PER3, respectively(Table 1). For 3′-RACE, the primer set consisted of PER1-3′ GSP, PER3-3′ GSP, and the UPM (Table 1).PCR amplification was carried out following the manufacturer’s instructions using the Advantage 2 PCR Kit (Clontech Lab., Inc., Mountain View, CA,USA).

2.3 Cloning, sequencing and analyses

Following the manufacturer’s protocol (Promega Corporation, Madison, WI, USA), gel-purification and sequencing of PCR fragments were carried out as described in Qin and Shao (2015). The open reading frame (ORF) was analyzed on http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi, and the protein sequence similarity analysis was performed in http://www.ch.embnet.org/software/BOX_form.html. Moreover,conserved domains identification was conducted in https://smart.embl-heidelberg.de/. The neighborjoining (NJ) algorithm in MEGA version 5.0 was used to construct the phylogenetic tree of period. The amino acid sequences and GenBank accession numbers are shown in Table 2.

Table 1 The sequences of primers

Table 2 The per1 and per3 proteins in Pelteobagrus vachellii used for multiple sequence alignments with other Period protein

2.4 Expression of per1 and per3 in tissue

The extraction of the total RNA and the first-strand cDNA synthesis were performed following the handbooks of the RNA extraction buff er and MMLV reverse transcriptase, respectively, and then the cDNA was diluted 10-fold for quantitative real-time PCR(qPCR). Two pairs of gene-specific primers (per1-s and per1-α, per3-s, and per3-α) were designed to amplify the 129-bp and 184-bp products of per1 and per3, respectively. Moreover, a 200-bp fragment, as an internal reference gene, was amplified by the primers of β- actin (Zheng et al., 2010).

Next, following the protocol of FastStart Essential DNA Green Master (Roche, USA), diluted cDNA from each sample was subjected to quantitative qPCR reaction on a Light Cycler Nano Real-Time PCR System (Roche, USA), and then, the 2-ΔΔCtmethod was used to calculate the expression levels of per1 and per3 (Livak and Schmittgen, 2001).

2.5 Statistical analysis

differences in mRNA expression were analyzed by one-way analysis of variance (ANOVA I) with SPSS 18.0 software (SPSS, Inc., an IBM Company),followed by Tukey’s tests. Values are presented as the mean±standard error (S.E.; n=9). The significance level was fixed at P ＜0.05 for all statistical analyses.Additionally, the rhythmic expression of the period gene was analyzed with the cosine function Y= M+ A×cos ( Ωt+ Φ) in the diencephalon, brain, liver and intestine, where M, A, Ω, t and Φ were the mesor,amplitude, angular frequency (2π-124 for circadian rhythms), time in hours, and acrophase, respectively(Del Pozo et al., 2012).

3 RESULT

3.1 Molecular sequences of the per1 and per3 genes

The cDNA sequence of Per1 comprised 5 872 bp with a 4 284-bp ORF. The sequence of per1 cDNA has been deposited to GenBank (Accession No.MH899118). This ORF encoded a protein of 1 428 amino acids that shared 74% and 76% sequence identity with its closest orthologs in Phreatichthys andruzzii and Ctenopharyngodon idella, respectively.The P. vachellii Period 1 protein contained two PERARNT-SIM (PAS) dimerization domains (residues 247-314 and 387-453), a PAS-associated C-terminal motif (PAC; residues 461-504), a short mutable domain (S/M; residues 653-675), a nuclear export signal (NES; residues 536-525), and carboxylterminal serine/threonine-glycine repeat region (SG;residues 1 173-1 234). Moreover, two casein kinase Iε (CKIε) phosphorylation regions were also found in amino acids 683 to 696 and 729 to 741 in the P.vachellii Period 1 protein (Suppl. Fig.S1).

The per3 cDNA of P. vachellii contained 4 201 bp,with a 3 882-bp ORF (Suppl. Fig.S2). The sequences of per3 cDNA have been submitted to GenBank(Accession No. MH920339). The Period 3 protein was composed of 1 293 amino acid residues and shared 74% and 59% sequence identity with its closest orthologs in Ictalurus punctatus and Astyanax mexicanus, respectively. Highly conserved domains were identified in the deduced amino acid sequences of Period 3, including PER-ARNT-SIM A (PAS A)(residues 219-286), PAS B (residues 359-425) and a cytoplasmic localization domain (residues 434-479.The motif scan analysis indicated the existence of highly conserved domains. Moreover, Period 3 protein also contained the conserved nuclear localization signal (NLS), NES, two CKIɛ phosphorylation sites, and a SG repeats domains(Suppl. Fig.S2).

3.2 Phylogenic analysis of periods

According to the pairwise alignment results of amino acid, Period 1 and Period 3 have 74% identity with P. andruzzii and 74% with I. punctatu s, which showed the highest identity. In the phylogenetic tree,the Period within the examined fish species was divided into two discrete clusters. In P. vachellii,Period 1 was sub-clustered with P. andruzzii, and Period 3 was sub-clustered with I. punctatu s.Furthermore, the two were clearly separated from the Period 2 genes (Fig.1).

3.3 The mRNA Expression of per1 and per3

The per1 and per3 mRNA expression profiles in tissues were detected by qPCR. The expression analysis indicated that per1 mRNA exhibited constitutive expression and was most abundant in retina and liver and least abundant in the head kidney and olfactory bulbs ( P＜ 0.05; Fig.2). Similarly, per3 exhibited constitutive expression in all tissues tested,with its presence remarkably high in the retina and liver and less apparent in the heart ( P＜ 0.05; Fig.2).

3.4 Diurnal expression of per1 and per3

Fig.1 Phylogenetic analysis of Periods constructed with amino acid sequences

The per1 mRNA expression varied within a light/dark cycle in the diencephalon, brain, liver, and intestine (Fig.3a, b, c & d). This rhythmic expression of per1 could be fitted to a cosinor curve and the acrophase at 15꞉15 in the diencephalon, at 12꞉52 in the brain, at 7꞉51 in the liver, and at 12꞉55 in the intestine. The expression of per1 mRNA at 0꞉00(dark) was obviously lower than that at 12꞉00 in diencephalon, liver and intestine (P＜0.05; Fig.3).

The level of per3 mRNA expression was significantly higher at 8꞉00 (light) in the diencephalon,liver and intestine and at 12꞉00 (light) in the brain than it was from 16꞉00 to 4꞉00 ( P ＜0.05; Fig.4). In addition, this rhythmic expression of per3 mRNA also fitted a cosinor curve (COSINOR, P＜ 0.05) with an acrophase at ZT8꞉15 in the diencephalon, at ZT9꞉54 in the brain, at ZT10꞉39 in the liver, and at ZT10꞉25 in the intestine.

The cosinor parameters of the per1 and per3 expression rhythms in P. vachellii in these four tissues are shown in Table 3.

4 DISCUSSION

Fig.2 Distribution analyses of per1 and per3 in P. vachellii

Period is a canonical circadian clock gene, which belongs to the PAS superfamily of cellular sensors(Kewley et al., 2004). In this study, the per1 and per3 genes were cloned from the darkbarbel catfish.Similar to Period 1 proteins from European sea bass and reef fish, typical Period domains were identified in Period 1 of P. vachellii, which was organized with a conserved PAS domain, S/M regions, and an SG repeat region present in mammals and Drosophila(Tei et al., 1997; Young, 1998). Structure prediction showed that Period 3 in P. vachellii shared conserved regions with mammalian proteins. Similar to other Period proteins, Period 1 and Period 3 in P. vachellii contained PAS domains in the N-terminal part of the protein. Ponting and Aravind (1997) indicated that the PAS domains functioned as dimerization domains to control their post-translational regulation. This region was critical for posttranslational modification that regulated Period activity (Lee et al., 2001). Moreover,casein kinase I epsilon (CKIε) was thought to be a phosphorylate Period protein, which thereby led to their instability and degradation. CKIε can also be phosphorylated and partially activate the transcription factor BMAL1 (Eide et al., 2005), which contributed to the establishment and maintenance of circadian rhythms (Lee et al., 2004), and this eff ect was also identified in Period 1 and Period 3 of P. vachellii. In addition, a CLD domain, which contributed to the proper structure of the PAS domain and to protein folding, was identified in Period 3 of P. vachellii(Ponting and Aravind, 1997). Structure prediction also revealed that the Period 1 and Period 3 proteins identified in this study belong to the Period protein family.

Fig.3 Relative level of per1 mRNA in the diencephalon (a), brain (b), liver (c), and intestine (d)

Clock genes, including CLOCK, BMAL1, periods and CRYs, exhibit wide tissue distribution in animals(Kamae et al., 2010; Del Pozo et al., 2012; Martín-Robles et al., 2012; Qin and Shao, 2015). In this study,qPCR results indicated that the per1 gene was distributed in all tissues tested and was most highly expressed in the retina and liver (Fig.2). Sánchez et al.(2010) showed that per1 mRNA was found in nine examined tissues. Similar observations of per1 expression have been described in most of the peripheral tissues of fish species, including in the flatfish and reef fish (Park et al., 2007; Martín-Robles et al., 2012). In addition, Martín-Robles et al. (2011)reported that per3 mRNA in the nocturnal flatfish S,senegalensis was distributed in all neural and peripheral tissues, particularly in the retina,cerebellum, diencephalon and optic tectum. Similarly,per3 in P. vachellii was detected in all examined tissues. These results suggested that the per1 and per3 genes are not only expressed in the diencephalon but also in peripheral tissues.

In mammals, the central circadian clock represents the ‘master’ pacemaker in the SCN, and all circadian clock gene expressions exhibit high-amplitude rhythms (Klein et al., 1991). Similarly, the diencephalon includes the retinorecipient suprachiasmatic nucleus of fish (Yáñez et al., 2009).Moreover, rhythmic circadian gene expression in tissues has been reported in various fish species including zebrafish, goldfish, flatfish, reef fish and Mexican blind cavefish (Kaneko et al., 2006; Park et al., 2007; Velarde et al., 2009; Martín-Robles et al.,2012; Beale et al., 2013). In European sea bass, the acrophase was close to lights-on (ZT0) (Sánchez et al., 2010), whereas Velarde et al. (2009) reported that the per1 expression achieved its peak at night in the retina of goldfish. In the brain and cultured pineal gland, Park et al. (2007) showed that per1 mRNA expression increased significantly at dawn. In the flatfish Senegalese sole, per1 expression reached maximum levels at ZT 18.12 (midnight), and minimum RNA expression were found at ZT4 to ZT8(daytime) (Martín-Robles et al., 2012). The per1 transcripts in the rainbow trout also achieved the peak value at ZT3 (daytime) (Patiño et al., 2011). In P.vachellii, the expression pattern of per1 has been analyzed over a 24 h period in the diencephalon, as well as in peripheral tissues (brain, liver and intestine),and all four tissues displayed an acrophase. The peak of mRNA level was close to lights-on (ZT12).Similarly, per3 of P. vachellii also displayed significant daily variation in the diencephalon, with an acrophase at ZT8. Martín-Robles et al. (2011)showed that per3 exhibited significant diurnal variation in the optic tectum and retina of S.senegalensis and was found in the brain (without the diencephalon), liver and intestine, in accordance with other clock genes in the oriental river prawn M.nipponense, European sea bass ( D. labrax), and S.senegalensis (Sánchez et al., 2010; Martín-Robles et al., 2011; Chen et al., 2017). However, no significant per3 rhythmic expression was observed in the diencephalon of S. senegalensis (Martín-Robles et al.,2012). The expression pattern of period varies in fish species, which may correlate with daily behavior, but this needs further investigation.

Fig.4 Relative level of per3 mRNA in the diencephalon (a), brain (b), liver (c), and intestine (d)

Table 3 The mRNA expression rhythms of per1 and per3 in various tissues

In this study, the acrophase of per1 and per3 was opposite to that of the CLOCK gene expression in P.vachellii. Qin and Shao (2015) showed that CLOCK gene expression in the brain, intestine and liver showed the acrophase at ZT 21꞉35, 23꞉23, and 23꞉00,respectively. Operating as negative regulators when produced in the cytoplasm, periods and cryptochromes are dimerized and translocated to the nucleus, where they inhibit CLOCK꞉BMAL1-mediated transcription(Froy et al., 2006). Therefore, periods and CLOCK have robust oscillations in opposite phases with their opposing functions.

5 CONCLUSION

In summary, full-length cDNA sequences of per1 and per3 were isolated from P. vachellii, and their expression patterns were analyzed in diff erent tissues to determine their diurnal expression patterns in the brain, intestine and liver. The expression of P.vachellii per1 and per3 exhibited diurnal variations in neural and peripheral tissues in light꞉dark cycle.

6 DATA AVAILABILITY STATEMENT

Data that support the findings of this study are available from the corresponding author upon reasonable request.