Cloning Of Rana Dybowskii MyD88 And Expression Research Of Its Pathway Related Molecules Under The Stress Of Rana Grylio Virus (RGV)

In the past twenty years, the global amphibian population showed a sharp decrease. Studies showed that the emergences and spreads of infectious diseases were the main reason for the decline of amphibian populations. As a dominant amphibian species of Northeast China, the Rana dybowskii frog is also suffering the threats of pathogenic microorganisms. Ranavirus of the iridovirus family is one of the important factors. As an important component of the innate immune system, toll-like receptors can recognize pathogen-associated molecular patterns (PAMP) carried by microbial pathogens, thereby inducing the innate immune responses and regulating the adaptive immune responses. MyD88 is an important adaptor protein in the TLR signaling pathway and shared by all TLRs except TLR3.Firstly, rapid amplification of cDNA ends (RACE) technology was used to obtain the full-length cDNA sequence of Rana dybowskii MyD88. Conserved domain research, multiple sequence alignment, homology analysis, protein properties prediction, and phylogenetic tree construction were used to analyze Rana dybowskii MyD88. Secondly, Quantitative real-time PCR (qPCR) was used to examine the expression changes of Rana dybowskii MyD88 and several other key molecules IRAK4, TRAF6 and TRAF3 in heart, liver, kidney and skin during iridovirus stress.The full-length cDNA of Rana dybowskii MyD88 is 1472 bp, with an open reading frame of 855 bp, encoding a protein of 285 amino acid residues. The MyD88 amino acid sequence contains a death domain (DD) and a Toll/interleukin-1 receptor (TIR) domain. Rana dybowskii MyD88 was calculated as a hydrophilic protein with predicted molecular mass and pI of 32.79 kDa and 6.00, respectively. Multiple sequence alignment and homology analysis revealed that the most conserved part of MyD88 proteins are located in the DD and TIR domains. In addition to its homologue in amphibian, Rana dybowskii MyD88 shared highest indentities to its mammlian homologue. Phylogenetic tree analysis showed that, rather than to its fish counterparts, Rana dybowskii MyD88 has a closer evolutional kinship to its mammals, birds, and reptiles homologues. qPCR results showed that, after iridovirus infection, the expression of Rana dybowskii MyD88 transcripts in kidney, liver and heart was significantly increased, and the expression of IRAK4 transcripts in all four organs was also significantly increased. However, significant up-regulation of TRAF6 only appeared in the skin. TRAF3 expression exhibited significant up-regulation in three other organs except kidney. This result showed that MyD88 and IRAK4, two important molecules of the TLR signaling pathways have participated in the antiviral immune processes of Rana dybowskii, and IRAK3 plays a more important role than TRAF6 does in the anti-iridovirus immune responses.This research firstly characterized the full-length cDNA sequence of Rana dybowskii MyD88, one adaptor molecule of the TLR signaling pathways, and analyzed the expression patterns of the key molecules MyD88, IRAK4, TRAF6 and TRAF3. This study provides research basis for further understanding the innate immune system of the amphibians.