Background

Na+/H+ exchanger (NHE) is a transmembrane protein that exists in all eukaryotic cells. Nine NHEs have been identified since human NHE1 cDNA was successfully cloned [1]. Based on subcellular localization and phylogenic analysis, NHEs can be classified into plasmalemmal subgroup (NHE1–5, SLC9A1–5) and intracellular subgroup (NHE6–9, SLC9A6–9) in fish [2]. Plasmalemmal NHEs usually cooperate with bicarbonate transporter to regulate cytoplasmic pH, cell volume, and intracellular fluid secretion, thereby maintaining the balance of acid-base, electrolyte, and cell volume in the entire life system [3, 4]. Intracellular NHEs can not only transport Na+, Li+, and K+, but can also limit the excess acidification of organelles caused by vacuolar H+-ATPase (HA) [4, 5]. NHE1 has been demonstrated to be involved in cardiac remodeling and myocardial fibrosis [6]. NHEs, such as NHE6 and NHE9, can inhibit proliferation and migration in a variety of tumors [7].

In aquatic environments, pH, salinity, and ammonia concentration are extremely important for aquatic animals. NHE is a two-way ion exchange carrier protein, which can transport Na+ into cells and exchange out H+, and also plays key roles in regulating intracellular pH, osmotic pressure, and ammonia concentration [8,9,10,11]. In fish, the plasma pH cannot be regulated via excreting CO2. Thus, H+ and HCO3− transport in fish gills plays a critical role in acid-base regulation [10, 12]. In hypertonic environments, fish excrete metabolic acids through the apical NHE, which is generally believed to play a critical role in ionocytes [13]. NHE can also coordinate with carbonic anhydrase and bicarbonate transporter in Tribolodon hakonensis and medaka (Oryzias latipes) at acidic environment [14, 15]. Salinity in aquatic environment is an important environmental factor for survival of aquatic animals. The expression level of NHE3 was higher in brackish water than seawater in Atlantic stingray (Dasyatis Sabina) and bull shark (Carcharhinus leucas) [16, 17]. Besides gills in elasmobranch species, in banded hound shark (Triakis scyllium), the expression changes of NHE3 were also detected in the kidney and intestine at different salinity environments [9]. At present, mechanisms of the ammonia nitrogen tolerance have been studied in fish, including inhibiting protein and amino acid catabolism, reducing environmental pH, NH4+ and NH3 continuous excretion, synthesis of non-toxic glutamine, and synthesis of urea, etc. [18,19,20]. Ammonia is mainly excreted as NH3 in zebrafish (Danio rerio) and medaka embryos [21, 22]. It is essential for NH3 excretion to form NH4+ via combine H+ [21]. Based on the acid-trapping hypothesis of ammonia excretion [23], NH3 excretion was promoted in acidic environment which would increase the conversion of NH3 to NH4+. NHE proteins are essential in the process of ammonia excretion [22] and has a combined effect with carbonic anhydrase (CA) and Rhesus-type ammonia transporter (Rh) [2, 24].

The Chinese tapertail anchovy (Coilia nasus) is an economically valuable fish widely distributed in China, Japan, and Korea. The sexually mature fish run thousands of kilometers from marine to river [25]. Ion uptake is activated in C. nasus during hypotonic environment, and ion excretion and water conservation are promoted in C. nasus during hypertonic environment [26]. Moreover, excessive ammonia nitrogen could lead to mass death of C. nasus larvae and juvenile during artificial breeding [27]. Based on previous studies, NHEs are involved in osmoregulation [26] and ammonia stress [27] in gills of C. nasus. To localize NHE genes in the C. nasus genome and their functions on salinity challenge and ammonia stress, we identified NHE gene family, and detected their expression levels under salinity and ammonia stress. Our findings will provide insight into their effects on ion transport and ammonia tolerance and be beneficial for healthy aquaculture of C. nasus.

Results

Identification of NHE genes

The entire NHE gene family, 10 NHE genes, were identified in C. nasus genome, including NHE1, NHEβ, NHE2, NHE2-like, NHE3, NHE5, NHE6a, NHE6b, NHE7, and NHE8. The detail information of C. nasus NHE genes were displayed in Table 1.

Table 1 The sequence information of C. nasus NHE gene family
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Chromosomal distribution of NHE genes

NHE1, NHE2, NHE2-like, NHE5, NHE6a, NHE6b, NHE7, and NHE8 were located on chromosome 19 (LG 19), LG 12, LG 17, LG 18, LG 20, LG 1, LG 14, and LG 21, respectively (Fig. 1). NHEβ and NHE3 were located on LG 6 (Fig. 1).

Fig. 1
figure 1

Chromosomal location of NHE gene family. The chromosomes were characterized by yellow bars. Chromosome numbers are shown on the left of the chromosomes. NHE genes are marked in red on the right of the chromosomes. LG:the name of chromosomes in C. nasus genome

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Phylogenetic analysis

NHEs can be classified into plasmalemmal subgroup (NHE1–5) and intracellular subgroup (NHE6–9) based on subcellular localization and phylogenic analysis [4]. Our phylogenic analysis showed that C. nasus NHEs were categorized into plasmalemmal subgroup (NHE1, NHEβ, NHE2, NHE2-like, NHE3, NHE5) and intracellular subgroup (NHE6a, NHE6b, NHE7, and NHE8) (Fig. 2). Moreover, phylogenic analysis cannot distinguish C. nasus NHE1 and NHEβ.

Fig. 2
figure 2

Phylogenetic analyses of NHE proteins from representative vertebrates. The tree was contributed by the neighbor-joining (NJ) method in MEGA X with 1000 bootstrap replications. Bootstrapping values were showed via circles on each branch. C. nasus NHEs were highlighted by the red star. The plasmalemmal subgroup (NHEβ, NHE1–5) and the intracellular subgroup (NHE6–9) were differentiated by red and green branches

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Structural analysis of the NHE genes

To further explore the characteristics of C. nasus NHE genes, analysis of gene structure, conserved domains and motif was performed. The exon numbers of NHE1 were the minimum (6 exons), and the exon numbers of NHE7 owned maximum exons (19 exons) (Table 1, Fig. 3A). All of C. nasus NHEs contained the Na_H_Exchanger domain, expect NHE8 which contained the Na_H_Exchanger superfamily domain (Fig. 3B). NHEβ, NHE3, NHE6a, NHE6b, and NHE8 contained only one domain, while others contained two or more domains. Motif analysis showed that motif 3, 4, and 6 existed in all of C. nasus NHEs. Noticeably, motif 5 only existed in plasmalemmal subgroup (NHE1, NHEβ, NHE2, NHE2-like, NHE3, NHE5), and motif 10 only existed in intracellular subgroup (NHE6a, NHE6b, and NHE7) (Fig. 3C). The sequences of each motif were shown in Supplementary material: Fig. S1.

Fig. 3
figure 3

Structural analysis of C. nasus NHE genes. A gene structure, (B) conversed domains and (C) motifs. The plasmalemmal subgroup (NHEβ, NHE1–5), and the intracellular subgroup (NHE6–8) were differentiated by pink and green

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Synteny analysis

The syntenic analysis was performed to further explore the evolutionary relationship of NHE genes between C. nasus and other fish species. The number of homolog pairs between C. nasus NHEs and other fish species, including channel catfish, Nile tilapia, common carp, Atlantic salmon, and Atlantic herring, were 7, 8, 2, 7, and 10, respectively (Fig. 4A-E).

Fig. 4
figure 4

Syntenic analysis of NHE genes between C. nasus and five other fish species, including Ictalurus Punetaus (A), Oreochromis niloticus (B), Cyprinus carpio (C), Salmo salar (D), and Clupea harengus (E). The gray lines indicate the collinear blocks within C. nasus and other fish genomes. The red lines indicate the pairs of NHE genes

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Expression profiles NHEs of multiple tissues and embryonic development stages

Expression profiles of NHEs was detected via qRT-PCR in C. nasus brain, eye, gill, heart, head kidney, kidney, intestine, liver, muscle, and spleen (Fig. 5A). NHE2 displayed higher expression in brain, gill, and heart. NHE3 displayed higher expression in brain, gill, and kidney. High expression level of NHE2-like was displayed in brain, eye, gill, heart, head kidney, kidney, liver, and muscle. High expression level of NHE5 was displayed in eye, heart, head kidney, kidney, intestine, and muscle. High expression level of NHE6a was displayed in eye, heart, and heart. NHE6b displayed higher expression in gill, liver, and spleen. NHE7 displayed higher expression in head kidney, intestine, liver, muscle, and spleen. NHEβ displayed higher expression in brain, eye, intestine, and liver. The expression of NHE1 and NHE8 were almost undetectable in all tissues used in our study.

Fig. 5
figure 5

Expression profiles of multiple tissues (A) and embryonic development stages (B). B: brain, E: eye, G: gill, H: heart, HK: head kidney, K: kidney, I: intestine, L: liver, M: muscle, S: spleen. The expression data were processed by log scale. The branches on the left represent the cluster analysis of different genes from different samples based on their expression patterns. The HK sample of NHE2 was used as the reference sample in tissues expression, and the 2-cell sample of NHE1 was used as the reference sample in embryonic development expression

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Expression profiles of NHEs was detected via qRT-PCR at fertilized stage, 2-cell stage, multi-cell stage, midgastrula stage, neural stage, muscle burl stage, prehatching, post-hatching, before first feeding, and after first feeding (Fig. 5B). NHEβ displayed higher expression at fertilized stage and before first feeding. NHE6a displayed higher expression at 2-cell stage and before first feeding. NHE8 displayed highest expression at post-hatching stage and before first feeding. High expression level of NHE7 was displayed in fertilized stage and midgastrula stage. High expression level of NHE3 was displayed in 2-cell stage. High expression level of NHE5 was displayed in 2-cell stage, multi-cell stage, midgastrula stage, and neural stage. High expression level of NHE2-like was displayed in midgastrula stage, neural stage, and muscle burl stage. NHE2 displayed highest expression at neural stage, and prehatching stage. NHE1 displayed higher expression at midgastrula stage, neural stage, and prehatching stage. NHE6b displayed higher expression at 2-cell stage, neural stage, muscle burl stage, prehatching.

Expression of NHEs in response to salinity challenge and ammonia stress

To explore C. nasus NHEs in response to hypotonic stress and hypertonic stress, their expression profiles were detected under hypotonic stress (F vs C) and hypertonic stress (S vs C) in the gill via qRT-PCR (Fig. 6). Overall, expression profiles of all NHEs were significantly upregulated at first, and then significantly downregulated under hypotonic stress. The converse trend was displayed in hypertonic stress. NHEs were significantly downregulated at first, and then significantly upregulated. During hypotonic stress, NHE1, NHE2-like, and NHE3 showed highest expression at 6 h. NHE6a and NHE7 showed highest expression at 12 h. NHEβ, NHE2, NHE5, NHE6b, and NHE8 showed highest expression at 24 h. During hypertonic stress, NHE1, NHE2, NHE7 showed lowest expression at 6 h. NHE2-like, NHE3, NHE6a, and NHE8 showed lowest expression at 12 h. NHEβ, NHE5, and NHE6b showed lowest expression at 24 h.

Fig. 6
figure 6

Expression profiles of NHE genes in C. nasus gills in response to hypotonic (FW vs Control) and hypertonic (SW vs Control) stress at multiple time points. Different capital letters indicate significant difference among different time points in the same groups at P < 0.05. Different lower-case letters indicate significant difference between different groups at the same time point at P < 0.05. FW: salinity ~ 1 ppt, Control: salinity 10 ppt, SW: salinity 30 ppt. The 0 h sample of Control group was used as the reference sample

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To explore the potential functions of NHEs of C. nasus larvae and juveniles in response to ammonia stress, their expression patterns were detected in the gill via qRT-PCR (Fig. 7). In C. nasus larvae, the expression of NHE2, NHE2-like, NHE3, and NHE6a were significantly enhanced under ammonia stress, while other NHEs displayed no significant difference. In juveniles, the expression of NHE2 and NHE3 were significantly enhanced under ammonia stress, while other NHEs displayed no significant difference.

Fig. 7
figure 7

Expression patterns of NHE genes in C. nasus larvae and juvenile gills responding to ammonia stress. Significant differences were noted by asterisk (P < 0.05). The samples of larvae and juveniles in control group were used as reference sample, respectively

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Discussion

Ten NHE genes were identified in C. nasus. Based on analysis of other vertebrates NHE gene family, NHEβ was only identified in teleost species, and NHE4 is generally missing in teleost fishes [28,29,30], which was generally identified in mammals [2]. In the present study, NHEβ was identified, and NHE4 missed in C. nasus NHE gene family. Furthermore, C. nasus NHE9 was not identified. As the oldest NHE gene, NHE9 lost in some fish [31]. Moreover, the syntenic analysis showed that the number of homolog pairs between C. nasus NHEs and other fish species, including channel catfish, Nile tilapia, Atlantic salmon, and Atlantic herring, were 7, 8, 7, and 10, respectively, which indicated that the genetic relationship between C. nasus and these fish species was close. The genetic relationship between C. nasus and common carp is closer than channel catfish and Nile tilapia based on traditional fish taxonomy, while homolog pairs of NHEs between C. nasus and common carp is only 2. Common carp is stenohaline fish, while other fish species are euryhaline fishes. These results suggested that most of NHEs between stenohaline fish and euryhaline fish are not conserved, or most of NHEs has been lost in stenohaline fish genome.

Based on tissue-specific expression patterns, NHE2 and NHE3 displayed higher expression levels in C. nasus gills. NHE2 and NHE3 are the primary isoforms expressed in gills of multiple fishes, including zebrafish [32], Pacific dogfish (Squalus suckleyi) [33], rainbow trout (Oncorhynchus mykiss) [34, 35], and Fundulus heteroclitus [36], which was similar to our results. In winter flounder (Pseudopleuronectes americanus) and zebrafish, NHE1 displayed high expression levels in red blood cells [32, 37]. Therefore, the expression level of NHE1 was almost undetectable in all tissues used in our study. However, few research focused on tissue distribution other NHEs. NHE5 and NHE6 in the brain, and NHE7 in the testis were three NHEs with higher expression levels in zebrafish [32]. NHE6b and NHE9 showed higher expression levels in the spleen, and NHE8 higher expression levels in the liver of European sea bass (Lateolabrax maculatus) [2]. These results were different from our results, which implied that the tissue distributions of NHEs were different from different fish species considering the habitats and lifestyle. C. nasus NHEs displayed different expression patterns during multiple embryonic development stages. Yolk proteins and amino acids are the main energy source in most teleost fish during embryonic development [38,39,40]. Their metabolism can continuously produce a waste product, ammonia. NHEs involved in ammonia excretion and Na+ uptake [41, 42]. NHE3 can induce Rhesus glycoprotein (Rh) proteins to involve in ammonia excretion during embryonic development stage in Coryphaena hippurus [43]. Similar results have been reported in medaka, zebrafish, and rainbow trout [42, 44]. Moreover, as a consequence, embryos of oviparous organisms are exposed to high respiratory CO2 within the egg capsule due to their increasing metabolic rate and the egg capsule wall acting as a diffusion barrier [45, 46]. High respiratory CO2 can decrease pH in the embryo and larvae. In acid-secreting ionocytes, NHEs are believed to be specialized in the secretion of acid equivalents [47]. It is proposed that ammonia transporters from the Rh family in combination with NHE3, expressed in HR cells are key players in mediating the active secretion of ammonia and protons in seawater teleost [22, 48]. A previous study demonstrated that NHE3 expressing epidermal ionocytes of cephalopod embryos are also involved in active secretion of acid equivalents [49]. Together, different expression patterns of C. nasus NHEs during multiple embryonic development stages seem to involve in ammonia excretion, Na+ uptake, and maintaining cellular pH homeostasis, or other physiological processes.

Gill is an indispensable tissue in fish, playing critical roles in osmoregulation and ammonia excretion. NHE is a two-way ion exchange carrier protein, which can transport Na+ into cells and exchange out H+ (or NH4+) [50, 51]. The expression of NHE3 was increased in the apical membrane of mitochondria-rich cells of Dasyatis sabina under low-salinity stress, thereby promoting the absorption of Na+ [16]. The mRNA expression level of NHE3 in Mozambique tilapia (Oreochromis mossambicus) gills in freshwater environment is higher than that in seawater environment [52]. Similar results have been reported in other fish, including D. sabina [16], zebrafish [32], banded hound shark [9], and Pacific dogfish [33]. In the present study, NHE2, NHE2-like and NHE3 with higher expression in the gills were displayed in C. nasus under hypotonic stress, which was consistent with other fish species. However, they were downregulated in the gills of C. nasus under hypertonic stress. This finding was also occurred in European sea bass [2]. Besides NHE2 and NHE3, expression levels of other NHE genes were also detected, and they showed higher expression levels in the gills of C. nasus under hypotonic stress and lower expression levels in hypertonic stress, which was similar to European sea bass [2]. These results indicated that C. nasus NHEs appear to have differing functions in hypotonic and hypertonic regulation via exchanging extracellular Na+ for intracellular H+.

Besides osmoregulation, NHEs are also essential for ammonia excretion. In the present study, the expression levels of NHE2, NHE2-like, NHE3, and NHE6a were significantly upregulated in C. nasus larvae gills under ammonia stress for 24 h. And the mRNA expression of NHE2 and NHE3 were significantly upregulated in C. nasus juvenile gills. Similarly, the mRNA expression of NHE2 and NHE3 were upregulated in Boleophthalmus pectinirostris under ammonia stress [41]. The mRNA expression level of NHE was upregulated more significantly in B. pectinirostris than Periophthalmus magnuspinnatus subjected to treatment with high environmental ammonia for 72 h [53]. Based on transport physiology, it is generally believed that NH3 and CO2 move across biological membranes to a much higher degree via membrane channels than simple, passive diffusion [54]. Ammonia excretion in fishes occurs via a “Na+/NH4+-exchange metabolon” which involves NHEs [22, 41, 55]. NH3 can diffuse from cells into water via Rh glycoproteins. As soon as it enters the water, NH3 combines with H+ which is pumped from the gill cell by H+-ATPase and/or by one or more NHE proteins, to form NH4+ [41]. There is indirect coupling of NH4+ efflux to Na+ uptake by either of these H+ efflux mechanisms. These results implied that significant up-regulation of NHE in C. nasus larvae and juveniles promoted NH3 to form NH4+ via pumping H+ into water to reduce ammonia toxicity. Moreover, the expression levels of Rhcg1, H+-ATPase, NHE, Na+/Ka+-ATPase (NKA), and Na+/Ka+/Cl− cotransporter (NKCC) were upregulated in Takifugu rubripes exposed to high external ammonia, and they showed combined effects on ammonia excretion [48]. The combined effects between NHEs and other proteins are required to further study in C. nasus under ammonia stress.

Conclusions

In the present study, 10 NHE genes were systematically identified from C. nasus genome. Phylogenetic analysis showed that NHE4 and NHE9 were lost in C. nasus genome. Syntenic analysis showed significant difference between stenohaline fish and euryhaline fishes. Different expression patterns of C. nasus NHE genes were displayed in multiple tissues. Different expression patterns of C. nasus NHE genes during multiple embryonic development stages were related to ammonia excretion. During hypotonic stress, C. nasus NHE genes were significantly upregulated. During hypertonic stress, they were significantly downregulated. During ammonia stress, NHE2 and NHE3 were significantly upregulated in C. nasus larvae and juveniles. These studies will provide insights into molecular mechanism of osmoregulation and ammonia tolerance in teleost.

Methods and materials

Identification of NHE gene family in C. nasus

To identify NHE gene family in C. nasus, the whole genome databases (GenBank GCA_007927625.1) were searched using BLAST GUI Wrapper on TBtools (v1.0692) according to amino sequences of human (Homo sapiens), zebrafish, and Atlantic herring (Clupea harengus) downloaded from Ensembl (http://www.ensembl.org) and NCBI (http://www.ncbi.nlm.nih.gov/) databases (cutoff value <1e-5). After removing repeated sequences, the unique sequences were validated via BLASTN against NCBI non-redundant protein database. Moreover, the Na_H_Exchanger domain (PF00999.21) was downloaded on Pfam database (http://pfam.xfam.org/). The Simple HMM search on TBtools (v1.0692) was used for seeking C. nasus NHE proteins with E-value < 0.01. The generated NHE proteins were verified on the Pfam databases. After removing the repeated sequences sought from the two methods above, the unique sequences were retained for further analysis.

The chromosomal location was performed based on their locations on C. nasus genome via Gene Location Visualize (Advanced) on the TBtools (v1.0692). the molecular weight and pI of NHE family were detected on ExPASy-Compute pI/Mw tool (https://web.expasy.org/compute_pi/).

Phylogenetic analysis of NHEs

The phylogenetic analysis of NHE sequences between C. nasus and several vertebrates were performed by MEGA X software. The accession numbers used for phylogenetic analysis were listed on Supplementary Table S1. The phylogenetic tree beautification was performed beautified on Interactive Tree of Life (iTOL, http://itol.embl.de/).

Structure analysis of NHEs

The gene structure of NHE genes were analyzed based on C. nasus genome database (gff3 file). Conserved domains of NHEs were analyzed via Conserved Domain Search Service (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Motifs of NHEs were analyzed via Multiple Em for Motif Elicitation (MEME) (https://meme-suite.org/meme/tools/meme). Gene Structure View (Advanced) on TBtools was used for the visualization of gene structure, conversed domains, and motifs.

Synteny analysis of NHEs

One Step MCScanX on TBtools was used to examine gene duplication [56, 57]. Homology of NHE genes was analyzed between C. nasus and other five fishes, including channel catfish (Ictalurus Punetaus), Nile tilapia (Oreochromis niloticus), common carp (Cyprinus carpio), Atlantic salmon (Salmo salar), and Atlantic herring. Dual Systeny Plot for MCScanX on TBtools was used for the visualization of synteny analysis.

Salinity challenge, ammonia stress, and samples collection

Healthy C. nasus (5.54 ± 0.63 cm, 2.25 ± 0.83 g) used in the present study were from Jiangzhiyuan Fishery Technology Co., Ltd. (Yangzhong, China). Before treatments, the salinity of aquatic environment was kept at 10 ppt for two weeks until salinity challenge experiment begins using NaCl. After acclimation, 180 C. nasus were randomly allocated into three groups (in triplicates): control group (C, salinity 10 ppt), hypotonicity group (F, salinity ~ 1 ppt), and hypertonicity group (S, salinity 30 ppt). After exposure for 0 hour (h), 3 h, 6 h, 12 h, 24 h, 24 h, 48 h, and 72 h, the treated fish were anesthetized via 70 mg/L buffered tricaine methanesulfonate (MS-222) (Greenhengxing, Beijing, China), and their gills were immediately collected and then stored at − 80 °C for further molecular assays.

Healthy C. nasus larvae (2.33 ± 0.25 cm, 1.11 ± 0.21 g) and juveniles (5.12 ± 0.45 cm, 2.35 ± 0.47 g) were reared for 10 days (27 ± 1.5 °C, pH 8.0 ± 0.4, salinity < 1.7, ammonia nitrogen < 0.005 mg/L). After acclimation, larvae and juveniles were randomly allocated into control group (ammonia nitrogen < 0.005 mg/L) and ammonia stress group (concentration of ammonia ~ 280 umole/L) (in triplicates), respectively. Stock solution (1.0 mol/L) of high purity NH4Cl was used as the source of the total ammonia. After exposed for 24 hours (h) (28 ± 2.1 °C, pH 7.5 ± 0.3, salinity < 2.3), gills were immediately collected and then stored at − 80 °C for further molecular assays.

For analysis of tissue distribution, three C. nasus (24.7 ± 1.68 cm, 11.9 ± 0.62 g) were anesthetized via 70 mg/L buffered MS-222, and then eye, gill, brain, liver, spleen, intestines, heart, head-kidney, kidney, and muscle were immediately collected and then stored at − 80 °C for further molecular assays. Tissue samples from all 3 fish were pooled together to make one sample. For ontogenetic expression profiles, embryos and larvae during multiple developmental stages were collected following natural spawning of the brood stock. Every developmental stage was examined by microscope observation. 30 embryos at fertilized egg (0 hour post fertilization (hpf)), 2-cell (1 hpf), muti-cell (3 hpf), midgastrula (4 hpf), neural (11 hpf), muscle burl (21 hpf), prehatching (28 hpf), and post-hatching (30 hpf); 20 larvae before first feeding (96 hpf) and after first feeding (144 hpf) were immediately pooled and frozen in liquid nitrogen.

Quantification of the NHEs expression by quantitative real time-PCR (qRT-PCR)

cDNA was synthesized using the PrimeScript™ RT Reagent Kit (TaKaRa, Tokyo, Japan). Primer Premier 5 software was used to design the primers used for qRT-PCR (Supplementary material: Table S2). The reactions were carried out on the Bio-Rad CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). The reaction system (20.0 μL) included 10.0 μL of iTaq™ Universal SYBR® Green Supermix (Bio-Rad), 2.0 μL of cDNA, 1.0 μL of each primer (10 μmol/L), and 6.0 of PCR-grade DEPC water. Reactions were performed in triplicate per sample, and cycling parameter was set as following: 94 °C for 2 min, followed by 40 cycles of 15 s at 94 °C,30 s at 60 °C, and 45 s at 72 °C. The geometric means of β-actin, 18SrRNA, and GAPDH, housekeeper genes were used to normalize expression levels of NHE genes [58]. All samples were detected in triplicate and the relative expression levels of NHE genes were calculated using the 2-ΔΔCT method [59].

Statistical analysis

The data of C. nasus NHEs expression during salinity and ammonia stress were analyzed by two-way ANOVA. Kolmogorov-Smirnov and Shapiro-Wilk methods were used to test the normal distribution. Interactive effect was tested using conversed non-normally distributed data. When P < 0.05, two-way ANOVA was performed. Data analysis was performed on SPSS 20.0. All data were displayed as mean ± SE. P < 0.05 indicates a significant difference. Histograms were drawn via GraphPad 8.0.