Identification and Functional Analysis of Ras-Related Associated with Diabetes Gene (rrad) in Edwardsiella piscicida-Resistant Individuals of Japanese Flounder (Paralichthys olivaceus)

Abstract

Ras-related associated with diabetes (RRAD) is a member of the Ras GTPase superfamily that plays a role in several cellular functions, such as cell proliferation and differentiation. In particular, the superfamily acts as an NF-κB signaling pathway inhibitor and calcium regulator to participate in the immune response pathway. A recent transcriptome study revealed that rrad was expressed in the spleen of disease-resistant Japanese flounder (Paralichthys olivaceus) individuals compared with disease-susceptible individuals, and the results were also verified by qPCR. Thus, the present study aimed to explore how rrad regulates antimicrobial immunity via the NF-κB pathway. First, the coding sequence of P. olivaceus rrad was identified. The sequence was 1092 bp in length, encoding 364 amino acids. Based on phylogenetic and structural relationship analyses, P. olivaceus rrad appeared to be more closely related to teleosts. Next, rrad expression differences between disease-resistant and disease-susceptible individuals in immune-related tissues were evaluated, and the results revealed that rrad was expressed preferentially in the spleen of disease-resistant individuals. In response to Edwardsiella piscicida infection, rrad expression in the spleen changed. In vitro, co-culture was carried out to assess the hypo-methylated levels of the rrad promoter in the disease-resistant spleen, which was consistent with the high mRNA expression. The siRNA-mediated knockdown of rrad performed with the gill cell line of P. olivaceus affected many rrad-network-related genes, i.e., dcp1b, amagt, rus1, rapgef1, ralbp1, plce1, rasal1, nckipsd, prkab2, cytbc-1, sh3, and others, as well as some inflammation-related genes, such as bal2 and Il-1β. In addition, flow cytometry analysis showed that rrad overexpression was more likely to induce cell apoptosis, with establishing a link between rrad's function and its potential roles in regulating the NF-κB pathway. Thus,. the current study provided some clarity in terms of understanding the immune response about rrad gene differences between disease-resistant and disease-susceptible P. olivaceus individuals. This study provides a molecular basis for fish rrad gene functional analysis and may serve as a reference for in-depth of bacterial disease resistance of teleost.

Keywords: Japanese flounder; NF-κB; RNAi; disease-resistant and disease-susceptible individuals; methylation; rrad.

Figure 1

cDNA and predicted amino acid sequences of Po-rrad. The small letters show nucleotides, and capital letters denote predicted amino acid sequences. The letters in the red box indicate the start codon (ATG). The double underline marks the poly-A sequence. The black star represents the stop codon (TGA). The conserved domains are shown based on prediction. The green, blue, red, and black line represent G1, G2, G3, and G4 box, respectively.

Figure 2

Structural domains and rrad amino acid sequences of P. olivaceus and other vertebrates. All sequences were aligned using DNAMAN. The red box represent RGK domain.

Figure 3

A phylogenetic tree was constructed with the neighbor-joining algorithm in MEGA 4.0. The relative genetic distances are indicated by the scale bar and the branch lengths. The protein sequences of the different species used to build the tree were as follows: Oreochromis niloticus rrad (XP_003445756.1), Labrus bergylta, rrad (XP_020508528.1), Larimichthys crocea rrad (XP_019116206.1), Amphiprion ocellaris, rrad (XP_003445756.1), Acanthochromis polyacanthus, rrad (XP_022057232.1), Seriola dumerili rrad (XP_022623546.1), Paralichthys olivaceus rrad (XP_019935401.1), Cynoglossus semilaevis rrad (XP_008308414.1), Melopsittacus undulatus, RRAD (XP_005152231.1), Gallus gallus RRAD (NP_001264535.3), Canis lupus familiaris RRAD (XP_038520230.1), Trichechus manatus latirostris, RRAD (XP_004371570.1), Homo sapiens RRAD (AAB17064.1), and Pan troglodytes RRAD (XP_001143391.3). The black triangle represents the target species.

Figure 4

rrad expression level in P. olivaceus evaluated using qPCR. (A) Relative rrad mRNA expression in the various tissues of normal fish. (B) Relative rrad mRNA expression in the immune-related tissues of disease-resistant and disease-susceptible individuals. The mean ± SEM values from three separate individuals (n = 3) are shown. The different letters “a” and “b” indicate significant differences (p < 0.05).

Figure 5

qPCR analysis of rrad expression profile in different immune-related tissues (spleen, liver, kidney, and intestines) after E. piscicida infection. The results were determined at different time points (0, 6, 12, 24, and 48 h), and PBS was used as the control. The transcription levels were normalized using β-actin levels. Data were analyzed using IBM SPSS Statistics 19 with the independent samples t-test. A asterisk stand for a significant difference in comparison with the 0 h group (p < 0.05), two asterisks represented significantly different comparison with the 0 h group (p < 0.01).

Figure 6

In vitro stimulation of rrad in response to LPS, PGN, and poly I:C in the gill cell line of P. olivaceus. The data were measured using quantitative RT-PCR and normalized using β-actin gene as an internal control. The data are presented as the mean ± standard deviation of three biological replicates. The expression levels with different letters were significantly different, including a asterisk represented significantly different comprared to control (p < 0.05), two asterisks represented significantly different comprared to control (p < 0.01).

Figure 7

DNA methylation of rrad in the promoter and gene body. Methylation differences in the rrad promoter and gene body in the spleen tissue of disease-resistant (DR_Po_Sp) and disease-susceptible (DS_Po_Sp) individuals. Red and blue vertical lines illustrate the methylation level of cytosines, whereas solid rims denote methylation and unmethylation positions, respectively, in disease-resistant individuals, and blue indicates disease-susceptible individuals. The red box is the difference in promoter region methylation between disease-resistant and disease-susceptible individuals.

Figure 8

Methylation analysis. Luciferase assays for methylated and unmethylated recombinant plasmids. The x-axis shows different recombinant plasmids, and the y-axis shows the relative luciferase activity. The letters “a” and “b” indicate significant differences (p < 0.05).

Figure 9

Effect of rrad on the transcriptional activity of NF-κB. (A) Effect of overexpression and siRNA treatment of rrad on the transcriptional activity of NF-κB luciferase reporter gene for 48 h, after which the luciferase activity was measured. (B) After co-transfection of Po-rrad-pEGFP-N3, Po-rrad-siRNA, and LPS with the NF-κB luciferase reporter gene, cells were stimulated for 24 h. The letters “a” “b” and “c” indicate significant differences (p < 0.05).

Figure 10

Analysis of PPI interaction and siRNA effects after rrad RNAi in P. olivaceus gill cells. (A) The expression of rrad, dcp1b, amagt, rsu1, rapgef1, ralbp1, plce1, rasal1, nckipsd, prkab2, cytbc-1, sh3, bcl2, and Il-1β was analyzed in cultured gill cells after RNAi. (B) Three-dimensional protein prediction and PPI analysis of rrad. The correlation of proteins was predicted using the STRING 11.0 online database. (C) Compared with the control, the mRNA levels of the interaction predictor of rrad and other immune response genes, i.e., dcp1b, amagt, rsu1, rapgef1, ralbp1, plce1, rasal1, nckipsd, prkab2, cytbc-1, sh3, bcl2, and Il-1β, were detected after RNAi. The stars represented a significant differentce copared to the control group (p < 0.05).