Effects Of Ammonia Nitrogen Stress On The Expression Of Immune Genes Semaphorin-4A And IL-8 In Micropterus Salmoides

Ammonia nitrogen is an important environmental stress factor in aquaculture.In this experiment,the 96-hour static water withdrawal method was used to carry out the acute toxicity test of ammonia nitrogen on Micropterus salmoides.Based on the LC50 of total ammonia nitrogen of M.salmoides for 96 h,the control(group A,0 mg/L)and the ammonia nitrogen stress group(group B,30 mg/L)were designed.After 24 hours of ammonia-nitrogen stress,dissected the tissues of liver,kidney,gills,brain,muscle and intestines of each individual.Three fish from each group were randomly selected and mixed to transcriptome sequencing.The sequencing obtained 335539 Transcripts,154485 unigenes,and 5680 differential genes,of which 2801 were up-regulated and 2879 were down-regulated.The pathways with the highest degree of up-regulated differential gene enrichment are all related to amino acid metabolism,while those with the highest degree of down-regulated differential gene enrichment are signal transduction pathways.Among them,the differential gene enrichment degree on the Ras signaling pathway is the highest.TCR plays an important role in the Ras signaling pathway.By analyzing the expression of TCR in M.salmoides under ammonia nitrogen stress,it was found that the expression of TCR was significantly decreased.Ammonia nitrogen stress causes a large amount of exogenous NH3 to influx through the gills and skin and accumulate in the tissues,which damages the immune system of fish.TCR is inhibited and thus affects the activation of T cells.The reduction of TCR inhibits the synthesis of ZAP70,LAT,PLCy,etc.Eventually the RAS pathway is inhibited and the immune pathway is destroyed.Partial sequences of IL-8 and Semaphorin-4A genes were obtained from the M.salmoides transcriptome data and cloned to obtain the full length of IL-8 and Semaphorin-4A genes.IL-8,also known as CXC L-8,is a member of the CXC chemokine subfamily,which plays a vital role in inflammation and immune response,and is activated in response to various diseases.The cloned IL-8 gene has a full-length sequence of 879 bp,encoding 526 amino acids,with a molecular mass of 58.04 KDa and an isoelectric point of6.83.Semaphore proteins are a group of proteins.Sema4 A is a member of the fourth type of semaphore family and has multiple roles in the immune system,nervous system,and metabolic system.The gene helps to form cells,body fluids and innate immune response to promote T cell activation and inflammation.The cloned Semaphorin-4A gene has a full length of 2,267 bp encoding 99 amino acids with a molecular mass of 10.99 KDa,and an isoelectric point of 9.24.Using M.salmoides as the experimental object,two ammonia nitrogen stress groups were designed as the control group(0 mg/L)and the ammonia nitrogen stress group(30 mg/L)based on the semi-lethal concentration of ammonia nitrogen.The head,kidney,brain,liver,gill,and spleen tissues were selected at 48,72,and96 h.The q RT-PCR method was used to analyze the effects of acute ammonia nitrogen stress on the expression of IL-8 and Semaphorin-4A genes in different tissues in M.salmoides.The results showed that IL-8 and Semaphorin-4A genes were expressed in all tissues.The expression levels of IL-8 and Semaphorin-4A genes in the intestine,kidney,brain,liver,gills,and spleen changed significantly under ammonia nitrogen stress.This study provides theoretical data for the future research on the mechanism of effect of ammonia nitrogen stress in M.salmoides.