Mechanism Of The Role Of CPS Ⅰ In The Detoxification Of Ammonia In Pelteobagrus Fulvidraco

With the rapid development of intensive aquaculture,feed residues and animal excreta make ammonia ammonia nitrogen accumulate in the aquaculture system,resulting in excessive ammonia nitrogen in the water column,which has become a regular environmental problem.The vast majority of fish are very sensitive to non-ionic ammonia（NH₃）,and a large accumulation of NH₃ in high concentrations in fish can cause hyperventilation,hyperexcitation,convulsions and even death.Under normal conditions,most fish can excrete ammonia directly through the gills,but when the concentration of ammonia reaches high levels in the environment,ammonia detoxification must be completed by the urea cycle,and carbamyl phosphate synthase I（CPSⅠ）is a key gene of the urea cycle,which plays a key role in the detoxification process of ammonia in fish.In this thesis,the full-length c DNA sequence of hepatic CPSⅠgene was obtained by RACE technique using yellow catfish（Pelteobagrus fulvidraco）;the role of CPSⅠgene in the detoxification process of ammonia nitrogen was analyzed by suppression of CPSⅠgene by acetaminophen（APAP）;the effect of increasing CPSⅠgene on endogenous ammonia nitrogen was analyzed by N carbamoyl-L-glutamate（NCG）activator to analyze the effect of raising CPSⅠin the case of elevated endogenous ammonia nitrogen.1.Cloning of CPSⅠgene and analysis of gene functionAccording to the sequence of the CPSⅠgene of yellow catfish in NCBI（registration number:XM＿027155265）,the yellow catfish liver tissue was provided by laboratory and the gene was cloned and sequenced by RACE technology.The sequence analysis showed that the full length of the CPSⅠgene is 5034 bp,with an open reading frame of 4461 bp,a5’non-coding region of 502 bp and a 3’untranslated region of 71 bp.The gene is presumed to encode 1486 amino acids,with a predicted molecular weight of 163.40 k Da and a theoretical isoelectric point of 4.49.Homology and phylogenetic analysis revealed that yellow catfish had a maximum of 92.87%homology with Pangasianodon hypophthalmus and a relatively low 72.66%homology with Homo sapiens.The phylogenetic tree showed that the CPSⅠgene was most closely related to the Pangasianodon hypophthalmus and the Italurus punetaus and was highly conserved after multiple sequence alignment.The results of tissue fluorescence quantitative RT-PCR showed that the CPSⅠgene was expressed in liver,brain,kidney,gill,muscle and skin,with the highest relative expression in kidney followed by liver.9μmol/g CH₃COONH₄ solution(96 h LC₅₀)was used in a 96 h ammonia stress experiment on yellow catfish,and the serum ammonia concentration increased significantly at 6-12 h and returned to normal levels after 24 h.However,the CPSⅠgene was significantly up-regulated in the liver at 24 h,indicating that it may be involved in the detoxification process of ammonia nitrogen,However,the urea cycle involved in CPS I needs to be further investigated.2.Immune responses and urea cycle-related enzymes in primary cells of yellow catfish treated with acetaminophen（APAP）under ammonia nitrogen stressPrimary culture of Pelteobagrus fulvidraco liver cells was performed,and the experiments were set up as control group（Control）,ammonia nitrogen group（NH₃,1 g/L）,inhibitor group（APAP,8 m M）,and ammonia nitrogen+inhibitor group（NH3+APAP）.After 6 h of stress,the urea cycle enzymes,antioxidant enzymes,immunity and apoptosis indexes were investigated in each group;the experimental results showed that the total antioxidant capacity（T-AOC）,superoxide dismutase（SOD）,and the total antioxidant capacity（T-AOC）of cells in NH₃and NH₃+APAP groups under ammonia nitrogen stress.dismutase（SOD）,catalase（CAT）and malondialdehyde（MDA）levels were significantly increased in the NH₃ and NH₃+APAP groups,and the cells promoted tumor necrosis factor（TNFα）,interleukin 1（IL-1）,and interleukin 8（IL-8）were significantly increased,and the protein content of apoptosis genes P53,Bax,Caspase 3,Caspase 9,and Cytochrome C m RNA expression was up-regulated,but Bcl2 m RNA expression was down-regulated.CPSⅠprotein content was inhibited to different degrees in the APAP and NH₃+APAP groups,with the best inhibition in the NH₃+APAP group.CPS I,argininosuccinate synthetase（ASS）,argininosuccinatelyase（ASL）,arginase（ARG）,ornithine transcarbamylase（OTC）,and glutamine synthetase（GS）were significantly increased in the NH₃ group.NH₃ group were significantly up-regulated.The protein contents of carbamyl phosphate synthaseⅢ（CPSⅢ）,ASS,ASL,ARG,OTC and GS were significantly up-regulated in the NH₃+APAP group.It showed that acute ammonia and nitrogen stress increased cellular antioxidant enzyme activity,induced inflammatory response and apoptotic pathway,and alleviated ammonia toxicity by synthesizing glutamine and promoting urea cycle response.In the study,it is necessary to apply the results of acute experiments to practical production.Therefore,it is significant to explore the role of urea cycle in culture experiments.3.High protein feed supplementation with N-carbamoyl-L-glutamic acid（NCG）promotes the effect of CPS I on urea cycle-related enzymes in yellow catfishThe experiment was divided into four groups,protein level 40%（P40）,protein level60%（P60）,protein level 40%feed supplemented with 0.02%NCG（P40+NCG）and protein level 60%feed supplemented with 0.02%NCG（P60+NCG）.Juvenile yellow catfish（1.5±0.31）g were reared and domesticated on commercial diets for 14 d.The fish were randomly placed in 12 tanks of 30 fish each at 300 L.Three replicates were set up and cultured for 8 weeks.The feed was fed twice daily on a satiated basis and strictly recorded.Experiments were conducted to investigate the growth performance and urea cycle enzymes with ammonia excretion effect of the optimum protein versus high protein after the addition of NCG.The analysis yielded that for growth;the P40+NCG group had significantly higher weight gain rates,specific growth rates and feed coefficients.The survival rate and weight gain rate of the P40+NCG group were significantly higher than the control group when the feed level was increased to 60%.For urea cycle enzymes;the addition of NCG promoted the gene expression and protein content of CPSⅠ,OTC and ARG in both P40+NCG and P60+NCG groups.And the GS protein content of P60+NCG group was significantly higher than that of the control group.The results showed that the addition of 0.02%NCG promoted the growth of yellow catfish and promoted the expression of urea cycle-related enzymes.