Role Of TLR Pathway Response Molecules Of Chinese Rare Minnow (Gobiocypris Rarus) In The Detection Of Four Pollutants' Immunotoxicity

With the increasing severity of environmental pollution,a lot of pollutants and pathogenic bacteria in the aquatic environment,which have potential harm to aquatic organisms.The immune system is the first line of organism to defense,and the toll-like receptor?Toll-like receptor,TLR?signaling pathway is the key pathway to activate the immune response,which plays an important role in protecting the organism from exogenous pathogenic bacteria.Rare minnow?Gobiocypris rarus?as a native species plays an increasingly important role in aquatic ecotoxicology,whereas the scarcely research related to immunotoxicity about the rare minnow was found.In order to further develop and excavation the important genes which take participate in the immune response of rare minnow and the role of immunotoxicity monitoring in the immune response.In the present experiment,we first clone,sequencing and analysis the 5 immune response factors in the TLR signaling pathway of rare minnow.However,in order to explore the immune toxicity monitoring of these genes role in the immune response of rare minnow,typical pesticides?Chlorpyrifos?CPF?and Avermectin?AVM??and heavy metal(Cu²⁺and Cd²⁺)contaminants in water were selected to exposed rare minnow under the environment related concentrations.The expression of these 5 genes and other genes in the TLR pathway were detected by quantitative PCR and the changes of physiological and biochemical parameters of rare minnow were detected by ELISA to evaluate the immune toxicity effects of water pollutants on rare minnow;In addition,the further effects of the existence of environmental contaminants on the body's ability to resist exogenous pathogens was also explored,an intraperitoneal injection of exogenous pathogens was carried out after the pollutants exposure rare minnow for 7 d,after 48 h infection the mRNA transcription levels of these genes and the levels of physiological and biochemical parameters were detected to assess the effects of pollutants on rare minnows ability of host resistance.Main results are as follows:1.TLR pathway immune response factors gene Myeloid differentiation primary response88?myd88?,Interleukin-1-receptor associated kinase-1?irak-1?,Interferon regulatory factor 5?irf5?,Interferon regulatory factor 7?irf7?and Interleukin-8?il-8?of rare minnow were cloned;the phylogenetic analysis,analysis of amino-acid domain,and the basal expression of these 5genes in difference tissue of rare minnow were implemented.The results showed that these 5 genes share a highly homology with other cyprinoid species and share 47.5%,79%,89.8%,81.2%and 91.2%similarly with zebrafish respectively,and contents similar domains with other fish which plays immune function,confirming their potential role in the immune response.The basal expression presents a high level of expression in the immune organs spleen and kidney implies the presence of tissue-specific function of these genes.2.CPF and AVM exposure of rare minnow and subsequent bacterial challenge experiments showed that CPF and AVM can affect the expression of genes in immune response pathway mediated by tlr3 and tlr5 and altered the physiological and biochemical parameters of Alkaline phosphatase?ALP?,C-reactive protein?CRP?,Complement 3?C3?,Lysozyme?Lys?and Immunoglobulin M?IgM?,Which immune function were influenced and might be induce an immunotoxic effects on rare minnow by CPF and AVM exposure.Subsequent bacterial infection experiments show that compared to AVM,the toxic effects of CPF exposure was stronger,which can significantly reduce the resistance of the body to exogenous pathogens.3.The results of Cu²⁺and Cd²⁺exposures indicate that environmental-related concentrations of Cu²⁺and Cd²⁺exposure can eliciting the immune response signaling pathway gene mediated by the tlr3 and tlr5,and changing the biochemical parameters of ALP,CRP,C3,Lys and IgM in rare minnow immune response;subsequent bacterial infection of rare minnow shown that heavily metal can produce toxic effects to inhibit the certain expression of immune response genes and reduce the content of physiological and biochemical parameters for immune responses by affect the function of these immune factors in immune response.