Identification and Functional Analysis of Ras-Related Associated with Diabetes Gene () in -Resistant Individuals of Japanese Flounder ().

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 was expressed in the spleen of disease-resistant Japanese flounder () individuals compared with disease-susceptible individuals, and the results were also verified by qPCR. Thus, the present study aimed to explore how regulates antimicrobial immunity via the NF-κB pathway. First, the coding sequence of was identified. The sequence was 1092 bp in length, encoding 364 amino acids. Based on and structural relationship analyses, appeared to be more closely related to teleosts. Next, expression differences between disease-resistant and disease-susceptible individuals in immune-related tissues were evaluated, and the results revealed that was expressed preferentially in the spleen of disease-resistant individuals. In response to infection, expression in the spleen changed. In vitro, co-culture was carried out to assess the hypo-methylated levels of the promoter in the disease-resistant spleen, which was consistent with the high mRNA expression. The siRNA-mediated knockdown of performed with the gill cell line of affected many -network-related genes, i.e., , , , , , , , , , , , and others, as well as some inflammation-related genes, such as and . In addition, flow cytometry analysis showed that overexpression was more likely to induce cell apoptosis, with establishing a link between '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 individuals. This study provides a molecular basis for fish gene functional analysis and may serve as a reference for in-depth of bacterial disease resistance of teleost.