Research On Physiological Response Mechanism Of Panulirus Ornatus To Ammonia Nitrogen Stress

Panulirus ornatus is one of the main breeding lobster species in China,which mainly distributed in the eastern tropical Indian Ocean,Southeast Asia,Australia and the Western Pacific.In China,it is mainly distributed in the South China Sea.At present,the artificial breeding of P.ornatus mainly relies on marine fishing for land-based factory farming.Under high-density culture conditions,ammonia nitrogen has become one of the most important environmental factors affecting the normal physiological metabolism of lobster.Studies have shown that high ammonia nitrogen content in aquaculture water environment will have significant effect on the physiological functions of crustaceans as well as their resistance to bacteria and viruses,and even lead to non-pathogenic infectious death of the organism.Therefore,an in-depth interpreting of the ammonia nitrogen detoxification metabolic pathways of P.ornatus can not only provide a theoretical basis for the study of lobster resistance mechanisms,but also lay a foundation for the protection of germplasm resources of lobsters and the cultivation of resistant varieties.Our study mainly studied the effects of ammonia nitrogen stress on the expression of enzyme genes such as GDH and related physiological and biochemical effects of P.ornatus,and conducted transcriptome sequencing analysis to explore the physiological response mechanism of P.ornatus to ammonia nitrogen stress preliminarily.This dissertation includes four parts,the main results are as follows:1.Transcriptome sequencing analysis of the hepatopancreatic tissue of P.ornatus under ammonia nitrogen stressUnder the ammonia nitrogen stress at a concentration of 20mg/L,the hepatopancreas of P.ornatus for 48 hours were taken as the experiment group(Geg group)and the control group(Gcg group)for transcriptome analysis.There were 252730696 clean reads in the 6 libraries,and the data volume was 37.9G,as well as Q20 was 97.76-98.27%,and Q30 was 93.47-94.73%.A total of 436 differential genes(DEGs)were screened between the Gcg group and the Geg group,and 12 DGEs were selected at random to be verified by real-time PCR to confirm the reliability of the transcriptome data.The enrichment analysis showed that the DEGs were mainly enriched in immune signaling pathways such as IMD signaling pathway,peroxisomes,nucleotide metabolism and fatty acid metabolism,and other related pathways.It showed that the ammonia nitrogen stress response of P.ornatus involved in immune function,oxidative stress,cytoskeleton remodeling and amino acid metabolism.2.Cloning and sequence analysis of GDH,GOT,ASL genes of P.ornatusThe RACE cloning technology was used to successfully obtain three genes related to the ammonia nitrogen metabolism enzyme genes of P.ornatus,which were named PoGDH,PoGOT,and Po ASL.PoGDH has a total length of 2431 bp,including 74 bp 5’NCR,1674 bp ORF and 683 bp3’NCR,encoding a total of 557 amino acids,with a predicted molecular weight of 61.599 k D,and p I of 6.46,and GRAVY is-0.280,which is a hydrophilic protein.PoGOT has a total length of 2270 bp,contains a 73 bp5’NCR,a 1284 bp ORF and a 913 bp 3’NCR.It encodes a total of 427 amino acids,with a predicted molecular weight of 47.105 k D,and p I of 8.84.GRAVY is-0.230,which is a hydrophilic protein;Po ASL has a total length of 2567 bp,including 101 bp of 5’NCR,1428 bp of ORF and 1038 bp of3’NCR.It encodes a total of 475 amino acids and has a predicted molecular weight of 53.697 k D,p I of 5.27,GRAVY is-0.115,it is a hydrophilic protein.The results showed that the amino acids of PoGDH and PoGOT genes had the highest homology with Litopenaeus vannamei,91.92% and 86.08%respectively.Po ASL is closely related to Procambarus clarkii.3.The expression of PoGDH,PoGOT and Po ASL genes in various tissues of P.ornatus and their expression analysis under ammonia nitrogen stressRT-qPCR was used to detect the PoGDH,PoGOT and Po ASL genes expression distribution in various tissues of P.ornatus and their expression under the ammonia nitrogen stress.The results showed that PoGDH,PoGOT,and Po ASL genes were expressed in all tissues,and PoGDH and Po ASL were both expressed in muscle with the highest level,but lower in the eyestalk,intestine,and stomach,and the difference was not significant(P>0.05).PoGOT expression was the highest in hepatopancreas,followed by gills,muscle and heart,which had significant differences compared with other tissues(P<0.05).Under the stress of ammonia nitrogen,PoGDH and Po ASL showed an overall upregulated trend and then a downward trend.The relative expression of PoGDH in the gills,hepatopancreas and muscle tissues of each concentration group showed obvious timeliness.Except for some individual time points,the relative expression levels of PoGDH and PoGOT were significantly higher than control(P<0.05).The maximum relative expression of PoGOT mostly appeared at 12 h under the stress of ammonia nitrogen,which was significantly different from the control group(P<0.05).In the initial stage of stress,the relative expression of Po ASL in each tissue was significantly higher than control(P<0.05),and showed a significant dose effect,and then gradually decreased.At 48 h,the relative expression of Po ASL was still significantly higher than control(P<0.05).According to the above results,PoGDH,PoGOT and Po ASL genes might play important regulatory roles in the process of responsing ammonia nitrogen stress.4.The effect of ammonia nitrogen stress on the activities of metabolic enzymes and antioxidant enzymes of P.ornatusThe 48-hours acute ammonia nitrogen stress experiment was performed on P.ornatus,and the activities of anti-oxidant enzymes and ammonia nitrogen metabolism-related enzymes in the tissues were determined.The concentrations of each group were 1 mg/L,10 mg/L,and 20 mg/L.The results showed that the body responded quickly to ammonia nitrogen stress,and the antio-xidant enzyme activity in the tissues increased.The activities of T-AOC and SOD at 6h～12h under ammonia nitrogen stress were significantly higher than control(P<0.05),and gradually decreased after reaching its peak value.At 48 h,The activities of T-AOC and SOD in 20mg/L group were inhibited,and the other two groups were still significantly higher than control(P<0.05).The content of LPO in the tissues gradually increased under ammonia nitrogen stress.At 48 h,each experiment group was significantly higher than control(P<0.05).The activities of ammonia metabolism-related enzymes such as GS and GDH showed an overall trend of increasing first and then decreasing.Compared with control,they all increased more or less.At 48 h,the activities of some nitrogen metabolism enzymes in the 20mg/L group were inhibited.Therefore,we speculated that the high concentration of ammonia nitrogen would cause damage to the body structure,and then affect the related physiological functions.The aboving results indicated that the P.ornatus synthesized non-toxic glutamine mainly through the interaction of GS,GDH as well as other metabolic related enzymes to avoid excessive accumulation of ammonia in the body,thereby achieving the metabolic transport of ammonia.