Expression Studies Of TLR4 Signaling Pathway Related Molecules In Rana Dybowskii Under The Stress Of Aeromonas Hydrophila

In recent years, because of habitat destruction, water pollution, microbial invasion, epidemics and other factors, amphibian populations decreased annually. Studies have shown that Aeromonas hydrophila(Ah) as a ubiquitous in amphibians conditional pathogens, can cause frogs hemorrhagic septicemia (commonly known as "red legs disease "), which is an important factor in reducing its viability and population effects. After microbial invasion, Toll-like receptors (TLRs) can not only recognize pathogen-associated molecular patterns (PAMP) to start the innate immune response, but also activate signaling pathways to regulate the adaptive immune response.Primers of TLR4、MyD88、IRAK4、TRAF6 and NF-κB mRNA sequences were designed by high-throughput sequencing of R.dybowskii, we used quantitative real-time PCR to detect the expression levels of TLR4、MyD88、IRAK4. TRAF6 and NF-κB mRNA in R.dybowskii kidney, heart and liver under the stress of Ah and reveal the immune effects of TLR signaling pathway associated molecules, explore TLR signaling regulation mechanism in R.dybowskii after microbial invasion, and understand the immune response system in amphibians.The main results were as follows:1. When R.dybowskii was infected by Ah early, the TLR4 mRNA expression peaks appeared at 6h in its heart, liver and kidney. At this moment TLR4 mRNA in vivo was 4-7 times as the initial quantity.2. In R.dybowskii’s kidney, heart and liver, MyD88, IRAK4, TRAF6 and NF-κB mRNA reached the peak in the order was kidneys, liver, heart, which showed that TLR4 signaling pathway in kidney was first activated after Ah infection.3. The expressions of TLR4, MyD88 and IRAK4 mRNA peak in R.dybowskii’s kidney were significantly higher than that of heart and liver, which showed that kidney played a major role in R.dybowskii’s innate immunity under the stress of Ah.4. Compared with TLR4, MyD88, IRAK4 and TRAF6 mRNA, of the expression of NF-κB mRNA peak in heart, liver and kidney did the most obvious changes, especially in heart and kidney, NF-κB mRNA expression was much higher than that of other genes.It Indicated that the NF-κB was activated as a transcription factor in vivo to play a major role in the TLR4 signaling pathway linking innate immunity and adaptive immunity, and participated in the adaptive immune response. The heart and kidney.played a major role in the fight against late defense mechanisms.5. The results showed that Ah activated TLR4 signaling pathway in R.dybowskii’s kidney, heart and liver and induced the expressions of MyD88, IRAK4, TRAF6 and NF-κB mRNA, in the pathway, making it participate R.dybowskii body’s early immune response and play a role. TLR4 signaling pathway was activated when Ah invaded R.dybowskii. TLR4 was activated first and rapidly expressed, high expression of TLR4 mRNA activated the expression of MyD88 mRNA to make it increase, then the IRAK4 and TRAF6 gene were activated, it last played effect by the transcription factor NF-κB. It showed TLR4 signaling pathway played an important role in R.dybowskii’s antibacterial infection.We explained that TLR4 signaling pathway played a significant role during the invasion of microorganisms, which provide theoretical basis for understanding amphibians’ innate immune system and protecting the quantity and diversity of amphibian populations.