Genomics 112 (2020) 2213–2222 Contents lists available at ScienceDirect Genomics journal homepage: www.elsevier.com/locate/ygeno 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 sug- gested 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