Data on molecular characterisation and expression analysis of the interferon-related developmental regulator 2 (IFRD2) gene from red sea bream, Pagrus major

The interferon-related developmental regulator 1 (IFRD1) protein is expected to play a role in the regulation of inflammatory responses in adult mice, since it is known to repress transcription of NF-κB in myoblasts that regenerate skeletal muscle after traumatic injury Micheli et al., 2011. The IFRD2 gene is expressed in many tissues including skeletal muscle, kidney, heart, brain, lung, placenta and liver in adult humans and is highly expressed in adult human skeletal muscle and heart. In mice, interferon-related developmental regulator 2 (IFRD2) may be associated with early haematopoiesis after gastrulation and in the hepatic primordium Buanne et al., 1998. In this study, we analysed the molecular characteristics of the IFRD2 gene identified from Pagrus major (PmIFRD2) and performed multiple alignments and phylogenetic analyses of the protein sequence. In addition, we examined the expression pattern of IFRD2 in healthy red sea bream tissues and the temporal expression pattern after challenging with various pathogens [Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV)]. This study characterises the non-specific immune response of the red sea bream after viral and microbial infections.


a b s t r a c t
The interferon-related developmental regulator 1 (IFRD1) protein is expected to play a role in the regulation of inflammatory responses in adult mice, since it is known to repress transcription of NF-kB in myoblasts that regenerate skeletal muscle after traumatic injury Micheli et al., 2011. The IFRD2 gene is expressed in many tissues including skeletal muscle, kidney, heart, brain, lung, placenta and liver in adult humans and is highly expressed in adult human skeletal muscle and heart. In mice, interferon-related developmental regulator 2 (IFRD2) may be associated with early haematopoiesis after gastrulation and in the hepatic primordium Buanne et al., 1998. In this study, we analysed the molecular characteristics of the IFRD2 gene identified from Pagrus major (PmIFRD2) and performed multiple alignments and phylogenetic analyses of the protein sequence. In addition, we examined the expression pattern of IFRD2 in healthy red sea bream tissues and the temporal expression pattern after challenging with various pathogens [Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV)]. This study characterises the non-specific immune response of the red sea bream after viral and microbial infections.
© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Data
The interferon-related developmental regulator 1 (IFRD1) protein has been reported to play a role in the regulation of inflammatory responses [1]. Also, interferon-related developmental regulator 2 (IFRD2) in mice may be associated with early haematopoiesis after gastrulation and in the hepatic primordium [2]. The open reading frame (ORF) of the PmIFRD2 cDNA was identified from red sea bream injected with S. iniae and consisted of 1308 bp, that encoded 435 amino acids (aa). The predicted domains of IFRD2 included the IFRD2 domain (27e335 aa) and the IFRD2 C-terminal domain (380e432 aa) (Fig. 1). The isoelectric point and molecular weight of the PmIFRD2 protein were predicted to be 6.0 and 48.2 kDa, respectively. Multiple alignment analyses of the IFRD2 amino acid sequences between red sea bream, and other teleosts and mammals revealed that IFRD2 from the large yellow croaker was the most homologous to PmIFRD2 at 92.64%. Among the teleosts, IFRD2 from the zebrafish was the Specifications Table   Subject area Immunology and Microbiology More specific subject area Gene expression analysis Type of data Figure  How

Data format
Analysed and Real-time PCR

Experimental factors
Open reading frame (ORF) of PmIFRD2 cDNA was obtained from next generation sequencing (NGS) analysis from liver of rea sea bream challenged with S. iniae. PmIFRD2 gene expression level profiles were compared between healthy fish and fish challenged with various pathogens.

Experimental features
This experiment could be provided as a basis for analysing the functional characteristics of the PmIFRD2 gene in the non-specific immune system of red sea bream.

Data source location
Gyeongsang National University, Tongyeong, Republic of Korea Data accessibility The data are available for this article Value of the data These data provide a basis for predicting the function of IFRD2 through phylogenetic analysis of IFRD2 in Pagrus major and other species. These data provide a basis for understanding the role of PmIFRD2 in the immune system of red sea bream infected with various pathogens. PmIFRD2 mRNA expression analysis results can also be used in comparative analyses of IFRD2 gene expression in other fish species.
least homologous to PmIFRD2 (78.49%), and IFRD2 from house mouse (53.85%) was the least homologous to PmIFRD2 among mammals (Fig. 2). To confirm the phylogenetic location of PmIFRD2, the phylogenetic tree was divided into teleost and mammalian clusters, and PmIFRD2 was most closely related to the Japanese flounder and large yellow croaker in the teleost cluster (Fig. 3). Quantitative real-time PCR (RT-qPCR) was used to confirm the expression levels of PmIFRD2 mRNA in healthy and infected red sea breams. The expression analysis of PmIFRD2 mRNA in healthy red sea bream, showed 82.42-fold more expression in the head kidney than in the trunk kidney (Fig. 4). The expression patterns of PmIFRD2 mRNA in gills, liver, kidney and spleen were confirmed after challenging red sea breams with E. piscicida, S. iniae or RSIV (Fig. 5). After challenging with E. piscicida, the expression of PmIFRD2 mRNA was slightly elevated in the liver at 1 hour post-infection (hpi) and increased to 1.81fold at 12 hpi. In the spleen, the expression was 1.57-fold higher at 1 day post-infection (dpi), but there was no significant difference (Fig. 5-A). After infection with S. iniae, the expression of PmIFRD2 was significantly upregulated in the gills at 12 hpi, in the kidney at 1 dpi, and in the liver at 12 hpi and 7 dpi (Fig. 5-B). After challenging with RSIV, the expression of PmIFRD2 mRNA was significantly upregulated in the gills at 3 dpi ( Fig. 5-C).

Sequence and phylogenetic analysis of PmIFRD2
PmIFRD2 was acquired from the liver of a red sea bream and the ORF identified by NGS analysis. Sanger sequencing was performed to verify the cDNA sequence of PmIFRD2. The amino acid sequence of PmIFRD2 was predicted using the GENETYX ver. 7.0 program (SDC Software Development, Japan) and the NCBI BLAST program. The molecular weight and isoelectric point of PmIFRD2 were predicted using the ProtParam tool of the ExPASy Proteomics Server, and the location of the specific domains of PmIFRD2 was predicted using SMART web software. Multiple sequence alignments were analysed by ClustalW between the predicted amino acid sequence of PmIFRD2 and the IFRD2 amino acid sequences of other species registered in the NCBI peptide sequence database. In addition, the phylogenetic analysis of PmIFRD2 was performed using the neighbour-joining (NJ) method of the Mega 4 program. Support for each node was derived from 2000 bootstrap replicates.

Fish
Experimental healthy red sea bream (weight: 68.5 ± 10 g, body length: 14.3 ± 1 cm) were supplied by Gyeongsangnam-do Fisheries Resources Research Institute (Tongyeong, Republic of Korea), kept in a seawater tank (water temperature: 20e23 C) for 2 weeks and fed daily on a commercial diet during the acclimatisation period. Three healthy red sea bream were anaesthetised with benzocaine (Sigma, USA) before tissue collection. For the bacterial and viral challenge experiment, S. iniae (1.5 Â 10 5 CFU/

Various tissue samples of healthy fish for PmIFRD2 mRNA expression analysis
Three healthy red sea bream were anaesthetised, and 12 tissues were sampled including the head kidney, heart, muscle, spleen, skin, gills, intestine, liver, stomach, brain, eye and trunk kidney, which were aseptically isolated to profile tissue mRNA expression. All samples were stored immediately frozen in liquid nitrogen at À80 C until they were used for total RNA extraction.

Total RNA extraction and cDNA synthesis
Total RNA was extracted from various red sea bream tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Briefly, 500 mL of TRIzol was added to each sample and then homogenised. A total of 100 mL of chloroform (Invitrogen) was added, and the samples were vortexed and centrifuged at 14,000 rpm for 10 min. The supernatant was transferred to a new 1.5-mL tube, equilibrated with PCI (phenol:chloroform:isoamyl alcohol) and centrifuged at 14,000 rpm for 10 min. The supernatant was transferred to a new 1.5-mL tube and then mixed with 500 mL of isopropanol (Sigma), 5 mL of Dr. Gen (TaKaRa, Japan), and 30 mL of 3 M sodium acetate (TaKaRa) and then centrifuged at 14,000 rpm for 10 min. After removing the supernatant, 600 mL of 75% DEPC ethyl alcohol was added and centrifuged at 14,000 rpm for 5 min. Finally, the supernatant was removed, the RNA was allowed to dry naturally at room temperature for 10e15 min, and then it was resuspended in 30e40 mL of DEPC DDW. After extraction of total RNA, samples were treated with RNase-free DNase (Promega, USA) according to the manufacturer's instructions. cDNA synthesis was carried out using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer's instructions.

RT-qPCR analysis
The tissue expression profile of PmIFRD2 mRNA was assayed by RT-qPCR with a DICE Real-Time System Thermal Cycler (TaKaRa). The specific primer sets were designed by Primer3 ver. 3 (http:// bioinfo.ut.ee/primer3-0.4.0/) based on the cDNA sequence of PmIFRD2 (forward: 5 0 - . The levels of PmIFRD2 transcripts were normalised to EF-1a levels. The data are presented as the mean ± SD from three independent cDNA samples with three replicates for each sample. The asterisks represent significant differences compared to the control (PBS) group by ANOVA (*P value < 0.05 and **P value < 0.01).
CATCCTCACCGCCATTCT-3 0 , reverse: 5 0 -AGTCCTCTCCATCTTCAGCA-3 0 ). For RT-qPCR, 1 mL of cDNA template, 1 mL of forward and reverse primers, 9.5 mL of DDW and 12.5 mL of TB Green were mixed in a total volume of 25 mL using TB Green premix Ex Taq™ (TaKaRa). The cDNA mixture was used in the following reaction, conditions: incubation for 4 min at 50 C, an initial denaturation step for 10 min at 95 C, and then 45 cycles of 20 s at 95 C and 30 s at 60 C, followed by a final dissociation stage for 15 s at 95 C, 30 s at 60 C and 15 s at 95 C. The degree of PmIFRD2 mRNA expression was compared with the expression level of elongation factor 1 alpha (EF-1a) (forward: 5 0 -CCTTCAAGTACGCCTGGGTG-3 0 , reverse: 5 0 -CTGTGTCCAGGGGCATCAAT-3 0 ) mRNA and three repetitions were performed for each gene for the accuracy of the experiment. The relative mRNA expression levels were calculated using the comparative Ct (2 ÀDDCT ) method and normalised to EF-1a.

Expression of PmIFRD2 after challenge with pathogens
Healthy red sea bream were randomly divided into three groups and then challenged by intraperitoneal injection with 100 mL of S. iniae (1.5 Â 10 5 CFU/fish), E. piscicida (1.5 Â 10 5 CFU/fish) or RSIV (1 Â 10 5 copies/fish) suspension, respectively. The control group was injected with the same volume of phosphate buffered saline (PBS). The gills, liver, kidney and spleen from the three fish at 1 and 12 hpi, and 1, 3, 5 and 7 dpi from each group were isolated and frozen in liquid nitrogen. All samples were obtained and analysed in triplicate, and total RNA extraction, cDNA synthesis and RT-qPCR were performed as described above.