Transcriptome Study And Immune-related Analysis Of Ctenopharyngodon Idellus Kidney Stimulated By Inactivated Aeromonas Hydrophila

Ctenopharyngodon idellus is an important freshwater fish cultured in China,and its yield and production value in recent years have been in the forefront of freshwater aquaculture fish.However,disease has become a bottleneck restricting in the development of grass carp cultivation,among which bacterial septicemia caused by Aeromonas hydrophila has always been an important disease in grass carp breeding.In this study,from both theoretical and practical considerations,C.idellus kidney(CIK)was selected as subject to conduct transcriptome studies aimed at discovering new immune-related genes in grass carp.It is hoped to provide information for the cloning of functional genes and study of immunology of grass carp,especially for the pathogenesis of bacterial septicemia in grass carp.In this study,CCK-8 and ELISA Kits were used to determine the cell activity and enzyme activity indexes of CIK stimulated by inactivated A.hydrophila,respectively.The optimal concentration and stimulus duration of inactivated A.hydrophila were determined by semi-quantitative RT-PCR,and transcriptome sequencing was performed on the cell samples under such stimulation conditions.Illumina sequencing technology was used to construct transcriptome library of CIK stimulated by A.hydrophila.Combined with bioinformatics analysis,the antibacterial immune response mechanism of CIK was explored.The main results are as follows:1.After stimulated by inactivated A.hydrophila,the number of CIK gradually decreased with the stimulus duration,and the viability index gradually weakened.After 12 hours of stimulation,a strong cytotoxic effect was observed,and the viability index decreased below 0.05.2.After stimulating CIK for 0,1,3,6,9,12,and 24 h with the pathogen concentration of MOI=100,the activity of T-AOC,SOD and ACP all showed a trend of significant increase at first(P<0.05),followed by a decrease,and then significantly increased(P<0.05).This showed that in the early stages of stimulus,CIK activated their defense mechanisms against pathogens caused by oxidative stress,and in the middle of the stimulation,T-AOC level significantly decreased.It was possible that CIK produced excessive reactive oxygen species and the antioxidant system of the cells could not resist the damage caused by oxidative stress,leading to the decrease of the total antioxidant capacity.3.The results of semi-quantitative RT-PCR showed that the expression levels of target genes IFN-?2 and NF-?B2 were different under different concentrations of inactivated pathogens and stimulation duration(P<0.05).The expression level of the genes were higher when the concentration of the inactivated pathogen was MOI=10and the stimulus duration was 1 hour,while the expression level were lower when the concentration of the inactivated pathogen was MOI=10 and the stimulus duration was6 hours.Finally,the cell samples of the control group and the treatment group with complex stimulation of MOI=10,1 h(group A)and 6 h(group B)were selected for subsequent transcriptome sequencing.4.A total of 30,430 genes were detected by transcriptome analysis,of which 23,241 were known genes and 7189 were new genes.A total of 1046 differentially expressed genes were identified in the group A,group B and control group.Compared with the control group,566 genes were up-regulated and 165 genes were downregulated in group A.In group B,the expression of 453 genes was up-regulated and the expression of 183 genes was down-regulated.321 genes were expressed in the two treatment groups.5.GO and KEGG were used to classify the functions of 1046 differentially expressed genes,and it was found that multiple genes were involved in the early immune responses such as receptor recognition,phagocytosis,coagulation and complement cascade.Among them,the GO items with high annotation rate of different genes of "ck VS A" and "ck VS B" were cellular process,single-organism process,response to biotic stimulus,cell and cell part,binding.The three pathways with the highest number of annotated genes were as follows: Signal transduction,Overview: cancer and the Immune system.It showed that differentially expressed genes play an important regulatory role in the immune processes such as pathogen recognition,cell surface receptor signaling and immune defense response.6.Screening of key immune function genes and qRT-PCR validation of some differential genes.Through the correlation analysis of the differentially expressed genes of "ck VS A" and "ck VS B" that were intensively annotated into the KEGG pathways of the immune system,12 key genes related to immune function were screened out.In order to verify the accuracy of the transcriptome sequencing results in this study,five differential genes in the "immune system" were analyzed by qRT-PCR.In this study,the transcription patterns of CIK stimulated by inactivated A.hydrophila were obtained by RNA-seq,and the immune genes and regulatory pathways associated with the antimicrobial response were systematically screened and identified.We believe that these important immune-related genes can provide important reference information for the bacterial septicemia in grass carp.It also provides genetic reference for the breeding of disease-resistant strains of grass carp.