Oreochromis Niloticus): Molecular Cloning, Tissue Distribution, and Transcriptional Changes in T Various Salinity of Seawater

Genomics 112 (2020) 2213–2222

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Genomics

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Characterization of two kcnk3 genes in Nile tilapia (Oreochromis niloticus): Molecular cloning, tissue distribution, and transcriptional changes in T various salinity of seawater

Zheng-Yong Wena,b, Chao Bianb, Xinxin Youa,b, Xinhui Zhangb, Jia Lib, Qiuyao Zhana,b, ⁎ ⁎ Yuxiang Penga,b, Yuan-You Lic, , Qiong Shia,b, a BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China b Shenzhen Key Lab of Marine Genomics, Guangdong Provincial Key Lab of Molecular Breeding in Marine Economic Animals, BGI Academy of Marine Sciences, BGI Marine, BGI, Shenzhen 518083, China c School of Marine Sciences, South China Agricultural University, Guangzhou 510642, China

ARTICLE INFO ABSTRACT

Keywords: As one important member of the two-pore-domain potassium channel (K2P) family, potassium channel subfamily kcnk3 gene K member 3 (KCNK3) has been reported for thermogenesis regulation, energy homeostasis, membrane potential Gene structure conduction, and pulmonary hypertension in mammals. However, its roles in fishes are far less examined and Genomic survey published. In the present study, we identified two kcnk3 genes (kcnk3a and kcnk3b) in an euryhaline fish, Nile Phylogenetic analysis tilapia (Oreochromis niloticus), by molecular cloning, genomic survey and laboratory experiments to investigate Tissue distribution their potential roles for osmoregulation. We obtained full-length coding sequences of the kcnk3a and kcnk3b kcnk3 cluster Osmoregulation genes (1209 and 1173 bp), which encode 402 and 390 amino acids, respectively. Subsequent multiple sequence Nile Tilapia (Oreochromis niloticus) alignments, putative 3D-structure model prediction, genomic survey and phylogenetic analysis confirmed that two kcnk3 paralogs are widely presented in fish genomes. Interestingly, a DNA fragment inversion of a kcnk3a cluster was found in Cypriniforme in comparison with other fishes. Quantitative real-time PCRs demonstrated that both the tilapia kcnk3 genes were detected in all the examined tissues with a similar distribution pattern, and the highest transcriptions were observed in the heart. Meanwhile, both kcnk3 genes in the gill were proved to have a similar transcriptional change pattern in response to various salinity of seawater, implying that they might be involved in osmoregulation. Furthermore, three predicted transcription factors (arid3a, arid3b, and arid5a) of both kcnk3 genes also showed a similar pattern as their target genes in response to the various salinity, suggesting their potential positive regulatory roles. In summary, we for the first time characterized the two kcnk3 genes in Nile tilapia, and demonstrated their potential involvement in osmoregulation for this economically important fish.

1. Introduction members were classiﬁed into six separate subfamilies according to their expression patterns, functions and electrophysiological or biophysical

Two-pore-domain potassium channel (K2P) family is one of the most properties [2,5]. These subfamilies include TWIK (TWIK1, TWIK2, important families of potassium channels with two typical pore (P) KCNK7), TASK (TASK1, TASK3, TASK5), TREK (TREK1, TREK2, domains and four transmembrane regions [1,2]. The K2P channels, TRAAK), TALK (TALK1, TALK2, TASK2), THIK (THIK1, THIK2), and distributing in many tissues such as brain, heart, adrenal gland and TRESK [3,6]. + kidney, are involved in controlling of the membrane potential and TWIK-related acid-sensitive K channel, TASK1 (K2P3.1, also therefore of cellular excitability [3]. TWIK1 (two P-domain in a weakly known as KCNK3), was first identified in 1997 with distribution in the + inward rectifying K channel), a prototypical member of the K2P central nervous system (CNS) and some peripheral tissues such as heart channel class, was identified from an expressed sequence tag database and adrenal gland [7,8]. It was reported that KCNK3 can be stimulated of human kidney in 1996 [4]. Since then, a total of 14 genes related to by certain natural and chemical effectors, such as extracellular pH [7], TWIK1 were cloned based on their high sequence similarity, and these volatile anesthetics [9,10], hypoxia [11], and heavy metal ions [11,12].

⁎ Corresponding authors. E-mail addresses: [email protected] (Y.-Y. Li), [email protected] (Q. Shi). https://doi.org/10.1016/j.ygeno.2019.12.017 Received 10 July 2019; Received in revised form 23 December 2019; Accepted 23 December 2019 Available online 24 December 2019 0888-7543/ © 2019 Elsevier Inc. All rights reserved. Z.-Y. Wen, et al. Genomics 112 (2020) 2213–2222

These functional properties suggest that KCNK3 might play an im- was set as the control, and the other three groups were designed as portant role in chemosensory function [8], adrenal gland zonation [13], experiment groups. Subsequently, five fishes from each tank were modulation of auto-immune inflammation [14], aldosterone secretion randomly selected and their gills were collected. All samples were im- [15], pulmonary arterial hypertension [16], and regulation of breathing mediately frozen in liquid nitrogen and then kept at −80 °C until use. [17]. Recently, a study revealed that KCNK3 also transcribed in brown All the animal experiments were conducted in accordance with the and beige adipose tissues, and functional experiments indicated that Chinese Ministry of Science and Technology for Humane Treatment of KCNK3 plays as a negative regulator in thermogenesis process by Laboratory Animals, and approved by the Animal Care and Use dampening cAMP-PKA signaling [18]. Committee of BGI (approval ID: FT18134). In contrast to mammals, related studies on fish kcnk3 genes were rarely reported. The first two orthologs of human KCNK3 were identi- 2.2. Molecular cloning of two kcnk3 genes fied in zebrafish in 2014 [19]. Whole-mount in situ hybridization suggested that zebrafish kcnk3a and kcnk3b genes were highly distributed Based on our previous experience [22], we isolated total RNA from in the central nervous system with a relatively weaker transcription in each brain sample with the Trizol reagent (Invitrogen, Carlsbad, CA, the heart; knockdown of zebrafish kcnk3a and kcnk3b resulted in lower USA) according to manufacturer's protocol. Subsequently, the extracted heart rate and higher atrial and ventricular diameters [19]. A sub- RNA was reversely transcribed to cDNA using Super ScriptTM II RT sequent study revealed that kcnk3 had a relative higher transcription reverse transcriptase (Thermo Fisher Scientific, Shanghai, China). Ac- value than kcnk9 in zebrafish heart, and it was higher in atrium as cording to the genomic DNA sequences identified from the Nile tilapia compared to ventricle [20]. These results suggest that KCNK3 may play genome database (Orenil1.0, Ensembl), we designed four pairs of pri- important roles in maintenance of heart beating in fishes, which is mers (Supplementary Table 1) to amplify the full-length cDNA se- consistent with the fact that atrial fibrillation decreases with upregu- quences of tilapia kcnk3a and kcnk3b genes. The basic cycling condi- lated KCNK3 in human [21]. Recently, two kcnk2 genes were identified tions of the PCRs were set as follows: a denaturing stage at 94 °C for from zebrafish, and they distributed in heart with similar functional 30 s, gene-specific annealing for 45 s and elongation stage at 72 °C for properties to their counterparts from mammals [6]. However, the exact 60 s, a total of 34 cycles. The target products were purified from roles of these channels in fish hearts are still largely unknown and in- agarose gels using the Universal DNA Purification Kit (Tiangen, Beijing, depth investigations are needed. China), and then sequenced at BGI-Wuhan (Wuhan, China). In our recent report of Northern snakehead (Channa argus), we also identified two kcnk3 genes and showed their wide distribution while 2.3. Sequence analysis and data processing with different patterns [22]. Our findings suggested that fish KCNK3s might play important roles not only in heart and CNS but also in per- The protein coding sequences were identified using the online ipheral tissues including gill. Moreover, a transcriptome study in Mo- software ORF finder as reported previously [22], and the protein se- zambique tilapia (Oreochromis mossambicus) revealed that kcnk3s could quences were predicted with Primer Premier 5.0 software (Primer be involved in osmoregulation [23]. However, information about mo- Biosoft International, Palo Alto, CA, USA). Moreover, the isoelectric lecular mechanisms of the osmoregulation is still limited. point (PI) and multiple sequence alignments were determined using the In order to resolve this issue, we attempt to identify two orthologs of public Bioedit software as described in two previous works [24,25]. kcnk3 genes from another economically important fish, Nile tilapia In addition, the online SWISS-MODEL tool was used to predict the (Oreochromis niloticus), which can survive in various low-salinity of 3D-structure models of our selected KCNK3 proteins [22]. Furthermore, seawater. Subsequently, we tried to explore tissue distribution of both DISPHOS 1.3 (http://www.dabi.temple.edu/disphos/) was used to kcnk3 genes, and quantify their transcriptional changes in response to predict the putative C-terminal phosphorylation site and NetNGlyc 1.0 different salinity. Meanwhile, we also want to measure the transcription (http://www.cbs.dtu.dk/services/NetNGlyc/) was employed to predict of three predicted transcription factors (TFs) of kcnk3a and kcnk3b the putative N-terminal glycosylation site. Meanwhile, the promotor genes. Our data will undoubtedly expand our knowledge about fish regions and the putative transcription factors (TFs) were predicted by KCNK3s, which may provide some novel genetic loci for marker-as- online Promoter 2.0 Prediction Server (http://www.cbs.dtu.dk/ sisted fish breeding. services/Promoter/) and JASPAR 2018 (http://jaspar.genereg.net/), respectively. Finally, synteny and gene structures of fish kcnk3 genes 2. Materials and methods were compared on the basis of a comparative genomic survey [22], and the zebrafish genome assembly (GRCz11, Ensembl) and the snakehead 2.1. Fish sampling genome assembly [26] were used as the reference.

Juvenile Nile tilapia (O. niloticus; average weight of 2.4. Phylogenetic analysis 166.8 ± 14.7 g) were obtained from our Baguang fishery base of BGI Marine at Shenzhen, Guangdong, China. They were transported to our A phylogenetic tree was reconstructed based on a dataset of KCNK3 local laboratory at BGI (Shenzhen) for further experiments. These fishes protein sequences, which were downloaded from NCBI or Ensembl. were kept in 50-L tanks (50 cm × 35 cm × 30 cm) under a natural Subsequently, these KCNK3 protein sequences were aligned by light-dark condition (12 L/12 D) with aeration of freshwater at CLUSTAL X2.1 [22]. Next, the aligned amino acid data sets were used 17.9 ± 0.3 °C. They were fed with commercial fish diet once a day at to reconstruct a phylogenetic tree with the maximum likelihood (ML) 19:00. approach using Mega 6.0 software [27,28]. The evolutionary model After acclimation for 2 weeks, six fishes were randomly selected for was evaluated by ProtTest 2.4 [29], and finally the JTT + G model was cloning and tissue distribution studies. Fishes were anesthetized on ice selected as the best. Moreover, a nonparametric bootstrap analysis with before sacrificing by decapitation. Twelve tissue samples, including 1000 resampling replicates was used to assess the robustness of the tree brain, eye, gill, gonad (ovary), heart, intestine, kidney, liver, muscle, topology. Spotted gar was used as the outgroup. spleen, skin, and stomach, were collected and immediately frozen in liquid nitrogen before use. 2.5. Quantitative real-time PCRs For the salinity tolerance experiment, fishes were assigned to four groups, and they were cultured at four different salinity of seawater (0, Total RNAs isolated from all the examined tissues were reverse 5, 10 and 20 psu) for two weeks. Before sample collection, the fishes transcribed to cDNA as described before [22,24]. Subsequently, quan- were allowed to adapt the environments for one week. The 0-psu group titative real-time PCRs were performed to measure mRNA levels of the

2214 Z.-Y. Wen, et al. Genomics 112 (2020) 2213–2222 gill kcnk3 genes and three transcription factors (arid3a, arid3b, and 2.6. Statistical analysis arid5a) with a Light Cycler Real-Time system (Applied Biosystems Inc., Waltham, MA, USA). The reversely transcribed products were used as Statistical analysis was performed with SPSS 22.0 (IBM, Armonk, templates, and the final volume of the PCRs was 20 μL. Melting curves NY, USA). Significant differences were calculated using one-way ana- with single peaks were used to verify the uniqueness of the target genes. lysis of variance (ANOVA) followed by Tukey's and Duncan's multiple A set of five housekeeping genes (β-actin, GAPDH, EF1α, Tubα1 and 18S) range test, after confirming data normality and homogeneity of var- were selected to test their transcription stability, and finally the 18S iances. All data were expressed as mean ± SEM (standard error of the was used for calculation. Relative transcription levels were calculated mean). Differences were considered to be significant if P < .05. using the Pfaffl method [30,31]. Quantification primers for amplifica- tion of the target genes were listed in Supplementary Table 1.

Fig. 1. Complete coding sequences and deduced protein sequences of the kcnk3a gene (A) and kcnk3b gene (B) in the Nile tilapia. Six putative transmembrane conserved domains are labeled in rectangular boxes. Positions of nucleotides and amino acids are labeled on both sides. The predicted N-terminal glycosylation site and C-terminal phosphorylation site are marked in the green and blue oval boxes, respectively. Underlines represent the initiation codon and termination codon respectively. The stop codon is marked by an asterisk (*). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

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3. Results tilapia and largemouth bass), Tetraodontiformes (Fugu and Tetraodon), Cyprinodontiformes (Amazon molly and platyfish), Salmoniformes 3.1. Molecular cloning of two kcnk3 genes from Nile tilapia (rainbow trout and Atlantic salmon), Beloniformes (medaka), Gaster- osteiformes (three-spined stickleback) and Gadiformes (Atlantic cod). PCRs with specific primer pairs (the top eight sequences in We proved that two kcnk3 genes are presented in all the fifteen fish Supplementary Table 1) were performed to obtain fragments for se- genomes, and observed that the kcnk3a genes had a conserved location quencing and subsequent assembly of open reading frame (ORF) se- between ppp2r5a and enpp1 or between enpp1 and slc35f6 (Fig. 3A), quences of the two kcnk3 genes. The ORF of kcnk3a was 1209 bp in while the kcnk3b genes showed a conserved location between hhipl2 length, encoding a protein of 402 amino acid (aa) residues (Fig. 1A). and ezra (Fig. 3B). However, the kcnk3b was shorter, with 1173 bp in length for a 390-aa Interestingly, a special kcnk3a cluster, opn8c-tagap-rsph3-ezrb-sytl3- protein (Fig. 1B). Sequence analysis showed that both deduced protein enpp1-kcnk3a was identified in Perciforms (including the Nile tilapia), sequences possess six transmembrane α-helix domains (marked rec- Tetraodontiformes, and so on; however, a reversal as kcnk3a-enpp1- tangular boxes in Fig. 1A, B), which are highly conserved in vertebrates sytl3-ezrb-rsph3-tagap-opn8c was identified in Cypriniformes (such as including fishes [22]. Moreover, a common N-terminal glycosylation zebrafish and common carp; Fig. 3A). In all examined fish genomes, site and a C-terminal phosphorylation site were predicted in the two these clusters are fitted similarly between ppp2r5a and slc35f6. genes (green and blue oval boxes in Fig. 1), respectively. These two It seems that there is a kcnk3b cluster, hhipl2-kcnk3b-ezra, in the sequences were deposited to GenBank with accession numbers examined fish genomes (Fig. 3B). This cluster can be extended as hhipl2- MN047291 and MN047292. kcnk3b-ezra-nenf (Perciformes, Tetraodontiformes, and etc.)orhhipl2- kcnk3b-ezra-tagapa-nenf (in Cypriniformes). In addition, two copies of 3.2. Multiple sequence alignments and predicted 3D structures of the kcnk3a and kcnk3b genes, along with their surrounding genes, were deduced KCNK3 proteins identified in Salmoniformes. We also comparatively analyzed gene structures of both kcnk3 genes We performed multiple alignments of protein sequences and con- (Fig. 4)infive representative fish genomes, which are of high quality. firmed that the two KCNK3 proteins in Nile tilapia are similar, with We observed that these kcnk3a genes share a similar gene structure with conserved sequences for the six α-helix domains, the N-terminal gly- two exons and one intron each, while the tilapia kcnk3a gene has the cosylation site and the C-terminal phosphorylation site (see more de- longest intron (approximately 45.60 kb) and the fugu kcnk3a has the tails in Fig. 2A). Meanwhile, we also noticed variable sequences be- shortest intron (approximately for 17.15 kb; Fig. 4A). Similarly, the five tween the two KCNK3 proteins at the positions of 265–310 and kcnk3b genes have two exons and one intron each, and the introns are 345–370, in which two remarkable variances are located at 290–305 ranging from 1.05 kb to 2.67 kb (Fig. 4B). Obviously, the introns of fish and 360–303 (dotted boxes in Fig. 2A). The tilapia KCNK3a is 12-aa kcnk3a genes are significantly longer than those of fish kcnk3b genes. longer than KCNK3b. Sequence identity analysis showed that the tilapia KCNK3a shared 3.4. Phylogenetic analysis of the two kcnk3 genes 50.7–72.2% identical to its homologs from human (69.4%), mouse (67.2%), chicken (72.2%), platypus (52.9%), lizard (55.7%) and frog For a better understanding of the evolutionary relationships among (50.7%); it shared a higher identity with its counterparts from fish various vertebrate kcnk3 genes, we constructed a phylogenetic tree species (such as Stickleback: 89.8%, snakehead: 91%, fugu: 90.2%, using the maximum likelihood method. A total of 45 KCNK3 protein medaka: 82.5% and zebrafish: 73.5%; see Supplemental Table 2). A sequences from various vertebrate species, from fishes to mammals, similar pattern was observed for the tilapia KCNK3b, which shared were collected for the phylogenetic analysis. As shown in Fig. 5, the lower identity with its orthologs from tetrapods (human: 62.4%, mouse: phylogenetic topology can be divided into two groups of tetrapods and 60.7%, chicken: 65%, platypus: 46.3%, lizard: 47%, and frog: 50%) teleosts, and the teleost group can be further clustered into two clades than those of that from fish species (such as Stickleback: 85.4%, sna- of kcnk3a and kcnk3b subtypes. This tree is highly in line with the kehead: 88.7%, fugu: 84.9%, medaka: 80.5% and zebrafish: 67.5%; see morphological classification, although the tilapia kcnk3a is close to more details in Supplemental Table 2). However, tilapia KCNK3a seabass kcnk3a while the tilapia kcnk3b locates closely with medaka shared only 62% identity with its paralog KCNK3b. This is similar to the kcnk3b. corresponding identities of KCNK3s in other fishes (Supplemental Table 2), such as in the Northern snakehead (64.3%) and zebrafish 3.5. Tissue distribution of the transcripts of the tilapia kcnk3 genes (65.4%) [22]. In addition, 3D-structure models of five selected KCNK3 proteins Tissue transcriptional patterns of both kcnk3a and kcnk3b genes were predicted. It appears that two significantly different models be- were determined by quantitative real-time PCRs. Twelve selected tis- tween KNCK3s were observed in zebrafish but not in tilapia (Fig. 2B). sues, including brain, eye, gill, gonad (ovary), heart, intestine, kidney, That is to say, the tilapia KCNK3 proteins shared a similar 3D structure liver, muscle, spleen, skin and stomach, were checked. We observed despite they had a lower identity in comparison with that in zebrafish. that the tilapia kcnk3a was widely transcribed in all the examined tis- We noticed that the tilapia KCNK3 proteins are much similar to the sues (Fig. 6A), with the following order of heart > (gonad, muscle) > human KCNK3 and zebrafish KCNK3b. Moreover, the electronic points (brain, eye, gill) > (intestine, liver, stomach) > (kidney, spleen, of the tilapia KCNK3a and KCNK3b were calculated to be 8.60 and 8.38; skin). A similar pattern was observed for the tilapia kcnk3b transcrip- their molecular weights were predicted to be 44.47 and 43.96 KD, re- tion, with the highest in the heart (Fig. 6B). spectively. 3.6. Effects of salinity variances on the transcriptions of kcnk3 genes and 3.3. A comparative analysis of kcnk3a and kcnk3b genes in fifteen fish three putative TF genes genomes As mentioned in the Introduction, the main goal of this section was As proposed in our previous study [22], two kcnk3 genes (a and b) to investigate the potential osmoregulatory roles of tilapia kcnk3 genes. might be widely existent in fishes. In order to further verify this hy- Transcriptions of kcnk3 genes in gills were quantified by quantitative pothesis, we performed a comparative analysis of both genes in fifteen real-time PCRs when in response to various salinity (0, 5, 10, and 20 representative fish genomes (Fig. 3), including Cypriniformes (zebra- practical salinity unit (psu)) of seawater for two weeks. Compared with fish, Mexican tetra, common carp and grass carp), Perciformes (Nile the control group (0 psu), the kcnk3a mRNA level was slightly increased

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Fig. 2. Sequence alignment (A) and 3D-structural model comparisons (B) among vertebrate KCNK3s. Dashes represent gaps in the protein sequences after the comparative alignment. Six transmembrane α-helix domains are highlighted in grey and labeled with I to VI respectively. Predicted N-terminal glycosylation site and C-terminal phosphorylation site are marked in green and blue boxes, respectively. Two dotted boxes represent the variable regions between KCNK3a and KCNK3b. Asterisks (*) indicate conservation of the amino acids among the examined sequences. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) at 5 psu, dramatically decreased at 10 psu, while significantly increased 4. Discussion at 20 psu (Fig. 7A). For the gill kcnk3b, a similar pattern was observed (Fig. 7B). KCNK3, a two-pore-domain potassium channel protein, has been For a better understanding of the potential regulatory mechanisms, well investigated in mammals. However, the physiological roles of this we identified three putative transcription factors (arid3a, arid3b and protein are still largely unknown in fishes. Here, we identified two arid5a) from the promotor regions of tilapia kcnk3 genes. Subsequently, kcnk3 genes from the Nile tilapia, and their potential roles involved in their transcriptional changes in response to various salinity were also osmoregulation were also investigated. examined (Fig. 8). Compared with the control group (0 psu), the gill arid3a mRNA level was significantly decreased at 5 and 10 psu (with 4.1. Species-specific relationships between the two KCNK3 proteins the lowest value in the latter group), while dramatically increased at 20 psu (Fig. 8A). As to the gill arid3b, its transcription was slightly Our phylogenetic analysis was performed to explore the evolu- decreased at 5 psu while dramatically decreased to the lowest level at tionary relationships among vertebrate kcnk3 genes (Fig. 5). We ob- 10 psu, and almost the same at 20 psu in comparison with the control served that the phylogenetic tree was divided into two groups of tet- group (Fig. 8B). For the gill arid5a, we detected a different pattern, rapods and teleosts; the tetrapod group was further divided into four fi which showed signi cantly decrease at 5, 10 (with the lowest value), clads of mammals, avians, repetiles, and frogs, while the teleost group and 20 psu (Fig. 8C). was clustered into two subgroups of kcnk3a and kcnk3b, respectively. These findings are consistent with our previous report [22], suggesting widespread existence of two kcnk3 genes in fishes. This phenomenon

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Fig. 3. Synteny comparisons of the kcnk3a (A) and kcnk3b (B) genes in fifteen fish genomes. The colorful blocks represent different genes, and the solid and dotted lines represent intergenic regions without genes and with genes, respectively. As shown in the figure, both kcnk3 genes are identified from the fifteen fish genomes, and the reversal region (A) and the insertion (B) are marked within dotted boxes. might be generated by the fish specific whole genome duplication event and 391 aa respectively. In addition, the tilapia KCNK3b was shorter occurred in the evolutionary process [32,33]. than KCNK3a, and it shared more close relationship with human The full-length coding sequences of tilapia kcnk3a and kcnk3b genes KCNK3, suggesting that mammal KCNK3 might inherit the original were 1209 and 1173 bp, encoding 402 and 390 aa, respectively. These roles of fish KCNK3b [4,22]. data are similar to our recent work in Northern snakehead (Channa Multiple sequence alignments indicated that the two kcnk3 genes argus)[22], in which snakehead KCNK3a and KCNK3b consist of 400 are widely presented in fish genomes, and fish KCNK3a proteins are

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A 01020304050(kb) 283 bp 926 bp Tilapia kcnk3a 45.6 kb

283 bp 890 bp Zebrafish kcnk3a 32.95 kb

283 bp 914 bp Medaka kcnk3a 30.35 kb

283 bp 920 bp Snakehead kcnk3a 26.35 kb

283 bp 860 bp Fugu kcnk3a 17.15 kb

0 2 4 6 8 10 (kb) B 283 bp 890 bp 1.17 kb Tilapia kcnk3b

283 bp 869 bp 2.67 kb Zebrafish kcnk3b

283 bp 875 bp 1.75 kb Medaka kcnk3b

283 bp 893 bp 2.42 kb Snakehead kcnk3b

283 bp 893 bp 1.05 kb Fugu kcnk3b

Fig. 4. Gene structural comparisons of the kcnk3a (A) and kcnk3b (B) genes in five representative fish species. The blocks and solid lines stand for coding sequences and introns, respectively. Rulers are shown upon the diagram for quantification of length. Please note that numbers above the boxes and lines represent the length of coding sequences and introns, respectively. approximatively 10-aa longer than fish KCNK3b. Meanwhile, six occurred in Cypriniformes species (as kcnk3a-enpp1-sytl3-ezrb-rsph3- transmembrane domains, a putative N-terminal glycosylation site and a tagap-opn8c; Fig. 3A). An insertion of tagapa gene also happened in C-terminal phosphorylation site were identified in both KCNK3 pro- Cypriniformes species (as hhipl2-kcnk3b-ezra-tagapa-nenf) for the teins, implying that these proteins may play some similar roles [19,22]. common knck3b cluster, hhipl2-kcnk3b-ezra-nenf, which has been pop- However, these proteins were predicted in the Nile tilapia with a similar ular in the examined Perciformes (including the Nile tilapia), Tetra- 3D model, and both were close to human KCNK3, indicated their si- odontiformes, and so on (Fig. 3B). That is to say, Cypriniformes had milar roles with tiny difference in tilapia. Interestingly, these findings experienced special evolutionary events, such as fragment insertion and were different from the results in zebrafish (Fig. 2B) and snakehead reversal, which may happen during their special genome duplication. [22], which showed significantly different models between their Additionally, two copies of kcnk3a and kcnk3b genes, along with KCNK3a and KCNK3b proteins, suggesting that fish KCNK3 proteins are their surrounding genes, were identified in Salmoniformes (Fig. 3), species-specific with independent evolutions. which might be due to their extra genome duplication event [34]. A recent phylogeny study based on genomic and transcriptomic data revealed the close relationship of Cypriniformes and Salmoniformes [35], 4.2. Existence of the knck3a and knck3b gene clusters in fishes: which might provide a reasonable explanation for the identification of conservation and variations tagapa around the Salmonid kcnk3b genes (Fig. 3B).

In order to verify the widespread existence of two typical kcnk3 genes in fishes, we examined the gene synteny and structures by per- 4.3. Potential roles of the knck3 genes in tilapia osmoregulation forming a genomic survey. Consistent with our previous findings in snakehead [22] but not zebrafish (Fig. 3), tilapia kcnk3a and kcnk3b Tissue distribution patterns of tilapia kcnk3 genes were determined genes are located in different scaffolds with different gene orders. by using quantitative real-time PCRs (Fig. 6). Both the kcnk3a and As the first time to report existence of two knck3 clusters, we de- kcnk3b genes were extensively presented in all the examined tissues termined a knck3a cluster between ppp2r5a and slc35f6 (Fig. 3A) and a with the highest values in the heart, which is similar to the results in knck3b cluster between hhipl2 and ezra (Fig. 3B). However, it appears snakehead [22]. The high expression of tilapia kcnk3 genes in the heart that a genomic fragment reversal for the popular knck3a cluster (opn8c- and CNS is consistent with their important roles in heart beating, ap- tagap-rsph3-ezrb-sytl3-enpp1-kcnk3a in the Nile tilapia and other fishes) petite regulation and cell excitability maintenance [19,22,36].

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Chimpanzee kcnk3 (ENSPTRP00000020172) Human kcnk3 (ENSP00000306275) Orangutan kcnk3 (ENSPPYP00000014042) Mouse kcnk3 (ENSMUSP00000098987) Rat kcnk3 (ENSRNOP00000063456) Cow kcnk3 (ENSBTAP00000024214) Sheep kcnk3 (ENSOARP00000020392) Mammals Rabbit kcnk3 (ENSOCUP00000018143) Opossum kcnk3 (ENSMODP00000019671) Squirrel kcnk3 (ENSSTOP00000000032) Tetrapods Horse kcnk3 (ENSECAP00000016698) Platypus kcnk3 (ENSOANP0000000082) Marmoset kcnk3 (ENSCJAP0000000244) Lizard kcnk3 (ENSACAP00000006437) Chinese turtle kcnk3 (ENSPSIP00000010109) Reptiles Chicken kcnk3 (ENSGALP00000041576) Turkey kcnk3 (ENSMGAP00000017751) Avians Frog kcnk3 (ENSXETP00000061322) Amphibian Rice ield eel kcnk3b (XP 020459868.1) Snakehead ish kcnk3b (MK214759) Seabass kcnk3b (XP 018521764.1) Amazon molly kcnk3b (ENSPFOP00000007260) Platyish kcnk3b (ENSXMAP00000002489) Stickleback kcnk3b (ENSGACP00000006823) Rabbitish kcnk3b (unpublished) Fugu kcnk3b (ENSTRUP00000021757) kcnk3b Tetraodon kcnk3b (ENSTNIP00000013446) Tilapia kcnk3b (MN047292) Medaka kcnk3b (ENSORLP00000022723) Cod kcnk3b (ENSGMOP00000014008) Northern pike kcnk3 (XP 010877659.1) Caveish kcnk3b (unpublished) Teleosts Hirame kcnk3 (XP 019953092.1) Amberjack kcnk3 (XP 022593617.1) Seabass kcnk3a (XP 018517262.1) Tilapia kcnk3a (MN047291) Amazon molly kcnk3a (ENSPFOP00000019046) Platyish kcnk3a (ENSXMAP00000017437) Stickleback kcnk3a (ENSGACP00000013257) Cod kcnk3a (ENSGMOP00000001816) kcnk3a Tetraodon kcnk3a (ENSTNIP00000019499) Fugu kcnk3a (ENSTRUP00000045401) Rabbitish kcnk3a (unpublished) Medaka kcnk3a (ENSORLP00000015116) Spotted gar kcnk3 (ENSLOCP00000020233) Semionotiformes Outgroup

Fig. 5. A phylogenetic tree of vertebrate kcnk3 genes. It was reconstructed based on 45 protein sequences using the maximum likelihood (ML) method. Spotted gar kcnk3 was used as the outgroup. Values at the nodes represent bootstrap percentages from 1000 replicates. Protein sequence IDs are provided in the brackets after the species and gene names.

Meanwhile, both kcnk3 genes had relatively high transcriptions in the A gill, suggesting their potential involvement in osmo-regulation. 600 To explore their potential roles in osmoregulation, we measured noisserpx their transcriptions in response to different salinity of seawater (Fig. 7). 500 Compared with the control group (0 psu), the two genes were increased 400 at 5 psu, while significantly decreased to much low levels at 10 psu eANR fi ff 300 although there was no signi cant di erence at 20 psu. These data 50 suggest that water salinity has a critical impact on the expression of

m 40 tilapia kcnk3 genes, which is consistent with the physiological perfor- a3k mance of the Nile tilapia. This euryhaline ﬁsh tolerates brackish water,

nck 30 but it is usually cultured in fresh water. It seems that 5 psu is possible, 20 evitaleR but 10 psu might be too much high, for the practical aquaculture of our 10 tilapia strain.

0 The potential osmoregulatory roles of kcnk3 genes were reported Br Ey Gi Go He In Ki Li Mu Sp Sk St previously in Mozambique tilapia [23], although related kcnk3 ex- B pression was signiﬁcantly lower in seawater as composed to freshwater. The lowest transcriptions of kcnk3 genes in Nile tilapia were found in 350

noisser the groups at 10 psu, possibly due to the lower salinity tolerance of the 300 Nile tilapia, although its various strains were reported to have good 250 performances when cultured at a salinity of 8 to 12 psu [37,38]. Ad- pxeANRm 200 ditionally, the transcriptions of three predicted transcription factors, 150 including arid3a, arid3b, and arid5a, were aﬀected in a similar pattern 100 when the examined ﬁshes were treated with various salinity of seawater 80

b3knck (Fig. 8). It seems that these transcription factors might be involved in

60 osmoregulation through regulation of the two kcnk3 genes in Nile tilapia. 40 evitaleR

0 5. Conclusions Br Ey Gi Go He In Ki Li Mu Sp Sk St fi fi Fig. 6. Tissue distribution of the kcnk3a (A) and kcnk3b (B) genes in the Nile We identi ed two kcnk3 genes from Nile tilapia for the rst time by tilapia. Twelve tissues were collected and examined in the present study. molecular cloning. Multiple alignments of protein sequences, 3D- Relative values were normalized with the 18S RNA gene. Each error bar re- structure model prediction, genomic synteny and gene structure pre- presents a standard error of the mean (n = 6). Various tissues: Br, Brain; Ey, diction, and phylogenetic analysis confirmed that two kcnk3 genes Eye; Gi, Gill; Go, Gonad; He, Heart; In, Intestine; Ki, Kidney; Li, Liver; Mu, (kcnk3a and kcnk3b) are widely presented in fishes. Quantitative PCRs Muscle; Sp, Spleen; Sk, Skin; St, Stomach. revealed that tilapia kcnk3 genes showed a similar distribution pattern with the highest transcription in the heart, suggesting their similar roles

2220 Z.-Y. Wen, et al. Genomics 112 (2020) 2213–2222

Fig. 7. Effects of various salinity on the transcriptions of A B kcnk3a (A) and kcnk3b (B) genes in the gill. 18S RNA was fi ff 2.0 2.0 used to normalize gene transcriptions. Signi cant di erences noisserpxeANRm (P < .05) between groups were analyzed using a one-way ANOVA. Groups with significant differences are marked by different letters above the bars. Data are presented as 1.5 1.5 b mean ± SEM (n = 5). a ac a 1.0 c 1.0 a mRNA expression mRNA a3knck

0.5 kcnk3b 0.5 ev

i b c taleR

0.0 Relative 0.0 0 5 10 20 0 5 10 20

A B C 2.0 2.0 2.0 noisserpxeANRm

1.5 1.5 d 1.5 a

1.0 a a a a 1.0 1.0 mRNA expression mRNA expression

a3dira 0.5 0.10 arid5a 0.5 arid3b 0.5

evitaleR b b 0.05 b b c b Relative 0.0 Relative 0.0 0.00 0 5 10 20 0 5 10 20 0 5 10 20

Fig. 8. Effects of various salinity on the transcriptions of arid3a (A), arid3b (B) and arid5a (C) genes in the gill. 18S RNA was used to normalize gene transcriptions. Significant differences (P < .05) between groups were analyzed using a one-way ANOVA. Groups with significant differences are marked by different letters above the bars. Data are presented as mean ± SEM (n = 5). in tilapia. Various salinity of seawater was proved to induce similar Program for Upgrading Key Links to Strategies for the Emerging and transcriptional changes of tilapia kcnk3 genes and related transcription Future Industries (no. 20170428173357698). factor genes in the gill, suggesting that kcnk3 genes are involved in osmo-regulation in Nile tilapia. References Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ygeno.2019.12.017. [1] D. Kim, Physiology and pharmacology of two-pore domain potassium channels, Curr. Pharm. Des. 11 (2005) 2717–2736. [2] C. Schmidt, F. Wiedmann, P.A. Schweizer, H.A. Katus, D. Thomas, Cardiac two- ’ Authors contributions pore-domain potassium channels (K2P): physiology, pharmacology, and therapeutic potential, Dtsch. Med. Wochenschr. 137 (2012) 1654–1658.

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