Transcriptomic Analysis And Determination Of Immune-related Genes In Channa Argus Aftyr Challenge With Nocardia Serilae

Snakehead fish(Channa argus)is an important native fish species in China,its growth fast,has strong adaptability and high yield.Snakehead fish has high market demand in Southeast Asia countries and has higher economic value.In the natural environment,snakehead fish has strong vitality.However,with the expansion of farming scale and the increase of breeding density,the frequent occurrence of disease cause increases the scope of influence and loss.Nocardia disease mainly affects snakehead fish,Nocardia disease also called sarcoidosis,pathogenic is Nocardia seriolae.Nocardia seriolae was gram positive bacteria,and is a facultative intracellular pathogen.Infected fish initially does not have obvious symptoms on the body surface,only lack of appetite,floating at water surface.With the aggravation of the disease,part of the body surface becomes black,abdominal enlargement and fin congestion.Heart,spleen,kidney,liver and other internal organ appear white nodules with diameter of 1-5mm.some affected fish occurs exophthalmos,anal swelling and ascites.Currently,Illumina high-throughput sequencing technology used to study the structure of trancriptomic of gene function at overall level and can explain the molecular mechanism in the specific biological process when happen of disease.The target in this study was the infected snakehead fish by Nocardia seriolae and head kidney was used for trancriptomic analysis.Sequencing results were assembled using short sequence assembly software Trinity,transcriptome data were compare with NR,KEGG,KOG and Swiss-Prot database;GO function annotation,KEGG metabolic pathway,SSR repeat sequence annotation,predictive coding protein frame(CDS)and the difference of gene expression level between the experimental and control group was analyzed then screening out those genes related to Nocardia seriolae for functional research.Two classical signaling pathway of non-specific immune system was studied,which are Toll like receptor signaling pathway(TLR)and NOD like receptor signaling pathway(NLR).The results in this study are as follows:Clean reads of unigenes for control group and experimental group were 35779908 and 36574025 respectively.After assembly,133999 unignes were obtained.The longest and shortest unigenes were 16393bp and 224bp.Its N50 and N90 values are 1460 and 315.Blastx comparison of unigenes with protein database was done and 106319 unigenes(79.34%)have been annotated.5753(4.29%)unigenes were mapped into GO functional annotation.KEGG pathway analysis had 5885 unigenes(4.39%);NR has 54886 unigenes annotation(40.97%)while the Swiss-Prot has 399795 unigenes(29.69%).1509 unigenes were classified into molecular functions,biological processes and cellular composition.49070 was classified into 26 categories of the KOG database.5885 unigenes were annotated into 335 KEGG pathway.Immune response pathways(TLR and NLR)that focus in this study were all from biological systems pathway in KEGG signaling pathway.By blastx comparison,CDS forecast were 40342 unigenes.From the analysis of SSR software(microsatellite software,MISA),there are 6 types of base repeated.The six types of base repeats were monomers(13849),dimers(9203),trimmers(3222),quadmers(433),pentamers(55),and hexamers(15).Difference expression of gene from the transcriptomic analysis,3912 unigenes had significant differences in expression.Among them,1552(39.67%)unigenes showed up-regulated expression while 2360(60.33%)unigenes showed down-regulated expression.There were 19 unigenes appeared differences expression in the Toll like receptor immune pathway,12 of the unigenes were up-regulated and 7 were down-regulated.10 unigenes showed differences expression in the NOD like receptor immune signaling pathway,3 were up-regulated,7 were down-regulated.7 genes from toll-like receptor pathway and 9 genes from nod-like receptor pathway had been randomly selected.From toll-like receptor pathway,TLR7,MAP2K4,NFKBIA,TAK1,IL8,FOS,and IL12 had been selected while IKKA,NF-KB p105 TNFAIP3,IL8,IL6,IL1B,HSP90A,ASC,and MAP3KIP1 from nod-like receptor pathway had been selected to underwent determination of DEGs using qRT-PCR.Results showed that all the DEGs from toll-like and nod-like receptor signaling pathway are almost similar.Histopathology analysis had been carried out and observation using light microscope,head kidney underwent pathologicl changes where nodules were form in the tissue from the infected group.Steatosis of hepatocytes happened in the liver of snakehead fish infected by Nocardia seriolae.Some tissue of spleen had been broken down in the experimental group.Gill filament of the snakehead fish from experimental group showed tissue degeneration and broken down.The cardiac tissue underwent pathological changes where some of the cardiac tissue had been degenerated.