Screening And Expression Regulation Of Key Genes In The Immune Pathway Of Trionyx Sinensis

Trionyx sinensis is a high-value commercial freshwater species in China,with annual production of over 319,000 tons in 2018.However,in recent years,intensive cultivation and environmental deterioration had caused frequent diseases by Aeromonas hydrophila,which left great problems to T.sinensis breeding industry.In turtle diseases,the search for immune genes had become a research hotspot.In the present research,biochemical indices were measured on the blood of turtles injected intraperitoneally with A.hydrophila,and transcriptome analysis was performed on liver and spleen tissues to explore the immune response mechanism of A.hydrophila to turtle.The main research contents of this paper are as follows:1.Growth characteristics of A.hydrophila and its infect on the immune biochemistry of T.sinensisThe growth curve of A.hydrophila was produced by spectrophotometer and colony counting method.There were distinct stages for the growth of the bacterium,3-9h,9-15h,and17-24h were its lag phase,logarithmic growth phase,and stable phase,respectively.The turtle was injected intraperitoneally with a bacterial gradient of 2.59×10~8,3.70×10~8,1.85×10~9,3.70×10~9,5.55×10~9 CFU/m L,and the half-lethal concentration was 1.39×10~9CFU/m L.Infection with A.hydrophila reduced the ACP and ALP levels in the blood of turtles and increases ALT activity.The results of blood enzyme activity and anatomy showed that A.hydrophila could damage the liver and spleen of the turtle.2.Screening and identification of immune pathways and genes of T.sinensisTranscriptome sequencing was performed using the liver and spleen of infected turtles,and 4,092 and 5,793 differentially expressed genes(DEGs)were obtained in the liver and spleen,respectively.The transcriptome data were analyzed by the method of weighted gene co-expression network analysis(WGCNA),and 14 network modules were obtained.The ivory,brown,yellow-green,and blue-green modules were identified as tissue-specific modules by screening.Functional enrichment analysis of GO and KEGG in tissue-specific modules was carried out.GO enrichment showed that 53,20,34,and 337 DEGs were found in the ivory,brown,yellow-green and blue-green modules,respectively.KEGG enrichment results showed that most of the pathways in the cyan modules were related to immunity,these pathways were mainly the Cytokine-cytokine receptor interaction,Apoptosis,MAPK signal pathway,Toll-like receptors signaling pathway,Intestinal immune network for Ig A production,NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway,Toll-like receptors signaling pathway in most of the genes was activated,it may be the key pathways.The immune genes in the pathway were roughly divided into five categories:toll-like receptors,interleukins and receptors,interferon receptors,chemokines and receptors,and tumor necrosis factors and receptors.Thirty-nine immune genes from the cyan module were selected for the visualization of the co-expression network,and four essential genes,IKBKE,CSF2RB,IL15RA,and Pelodiscus＿sinensis＿new Gene＿68865 were found.Finally,to verify the reliability of transcriptome sequencing,nine immune-related genes were randomly selected for q RT-PCR verification,and the q RT-PCR result was consistent with transcriptome sequencing analysis results.This study successfully explored the critical pathways for the immune response of A.hydrophila infected by T.sinensis and found a large number of immune genes,which provided reference value for the study of its immune mechanism.