Tissue Distribution And Temporal Expression Of Immune Related Genes And Proteomic Analysis In The Gill Of Carassius Auratus Infected By Aeromonas Hydrophila

Crucian carp（Carassius auratus）is one of the main fish in freshwater culture in China.Hemorrhagic septicaemia in crucian carp caused by Aeromonas hydrophila has long been one of the important disease prevention and control problems in aquaculture in China.In this study,crucian carp infected by A.hydrophila was used as an experimental animal,with its gills as the main research object.Histopathological observation showed that a large number of inflammatory cells infiltrate at the basal part of the gill lamellae,necrotic and exfoliated in respiratory epithelial cells.The differential proteomics of the control group and infected group（6h+12h）of its gill tissues was studied by isotope labeling relative and absolute quantification（i TRAQ）.The expression of differentially expressed proteins was verified by real-time quantitative PCR（q RT-PCR）.The results showed that 430 differentially expressed proteins were identified in the gill of crucian carp,among which 177 were up-regulated and 253 were down-regulated.The results of KEGG enrichment analysis indicated that the gills immune-related pathways were mainly involved in the signaling pathways such as mucus-secreted,complement and coagulation cascade reaction,antigen presentation and processing.Twenty-one immune-related proteins of gill were screened,10 up-regulated expression protein including membrane bound complement regulatory proteins（g Tecrem-2）,lysozyme C（Lys C）,ATP-dependent RNA helicase DDX5（ddx5）,heat shock protein（hsp90）,alpha-2-macroglobulin（α-2M）,complement（C9,C3,C7）,complement control protein factor I-A（Cf I-A）and solute carrier（slc4a1a）,eleven down-regulated proteins including calmodulin（Ca M）,nuclear transcription factor Y,alpha（nfyal）,apoptotic chromatin condensation inducer（acin1b）,proteasome activator subunit 2（psme2）,splicing factor 3B（sf3b5）,RNA binding protein（rbm8a,rbm25）,pre-m RNA splicing factor 18（prpf18）,ras GTPase activation binding protein 2（g3bp2）and small nuclear ribonucleoprotein（snrpf,snrpd3l）.The m RNA levels of21 protein were basically consistent with the quantitative analysis results of i TRAQ protein by q RT-PCR.The results indicated that the immune response in the early stages of infection may resist the invasion and infection of A.hydrophila,which is helpful to elucidate the molecular mechanism of local mucosa immune response in the gill of crucian carp.To further study the tissue distribution and sequence expression of differentially expressed genes（MHCⅡ、ddx5、Ca M、caln、C3、C9、hsp90、sf3b5）in crucian carp infected by A.hydrophila with q RT-PCR.The results of showed that the above 8 target genes had constitutive expression distribution in different tissues of crucian carp,among which the expression levels were relatively high in skin,intestinal,liver,spleen,muscle and other tissues,and low in gill,heart,brain and kidney.At the same time,MHCⅡand ddx5 gene showed an obvious up-regulated expression pattern in mucosal tissues such as gill,skin and intestinal at 6h-48h;Ca M and caln gene were down-regulated and then up-regulated in gill and skin tissues,and were mainly up-regulated at 48h,while were down-regulated in the intestinal,caln was mainly up-regulated at 24-36h.The expression of C3,C9,hsp90 and sf3b5 gene was up-regulated in the skin and intestinal,while down-regulated in the gills,among them,C3 and C9 was only significantly upregulated in the gill at 36h.At the same time,C3,C9,MHCⅡ,hsp90,sf3b5 and ddx5 showed obvious up-regulated expression patterns in liver,spleen and kidney after 6h-48h infection,while caln showed a trend of down-regulation,up-regulation and down-regulation.Ca M was only up-regulated in the brain.It is worth mentioning that the time-series expression of these 8 genes in infection-induced heart,brain and muscle tissues all showed significant differences（p<0.01）.The above results indicated that immune-related genes in the gill of crucian carp not only participate in local mucosal immune response,but also play an important role in the immune response of fish body.The full-length sequence and expression characteristics of Lys C and ITLN c DNA were analyzed by bioinformatics method.The results showed that Lys C gene,which codes for 145aa,contains one signaling peptide,one lysozyme domain（LYZ1）,and two conserved catalytic sites(Glu⁵³,Asp⁶⁹).The results of tissue distribution showed that Lys C gene was relatively high in intestine,liver,spleen,kidney,heart and muscle.Lys C was up-regulated in infected tissues,and significantly overexpression was induced in the gill,skin,intestine,spleen,muscle,brain after infected 36h（p<0.05）.ITLN gene encodes 314aa and contains one signaling peptide,C-terminal fibrinogen domains（FRe D）and two conserved Cys(Cys⁵⁵、Cys⁸⁴).ITLN was constitutively expressed in all tissues of crucian carp,among which it was relatively distributed in skin,intestine,liver,spleen and muscle tissues.ITLN was mainly up-regulated in infected tissues（p<0.01）.These results provide a scientific reference for further study of the functions and the exact mechanisms of mucosal immune response of Lys C and ITLN genes in fish.