Transcriptome And Metabolome Analysis Of Macrobrachium Nipponense In Response To Ammonia Nitrogen Exposure

The oriental river prawn,Macrobrachium nipponense is an important freshwater economy shrimp of China.With the development of the shrimp aquaculture industry,the problem of environmental stress has become increasingly prominent.Ammonia nitrogen is the most important environmental stress factor in shrimp culture which can seriously affect the growth and development of shrimp and physiological health.Therefore,studying the molecular mechanism of anti-ammonia nitrogen stress in shrimp can not only provide the theoretical basis for improving the disease resistance and immunity of shrimp,but also lay the foundation for protecting the germplasm resources and cultivating resistant varieties.In this study,we created a controlled of ammonia nitrogen stress environment in the laboratory,and the freshwater prawn were kept in the high ammonia concentration(22.1mg/L)of the environment.Randomly select lively individuals from the stress of 48 hours which were still alive.The hepatopancreas were extracted to analyze the transcriptome and metabolomics compared with the control group prawn under normal feeding conditions.Bioinformatics analysis of sequencing results found that immune-related genes were significantly enriched in the complement and coagulation cascade,Toll-like receptor signaling pathway,T cell receptor signaling pathway and B cell receptor signaling pathway.The metabolomics screened urea,uric acid and shrimp were closely related to ammonia nitrogen stress traits.The serine protease inhibitor(Mn-SPI)gene was selected from the complement and coagulation cascade pathway for cloning and tissue expression analysis.The results are as follows:(1)Using the Illumina sequencing platform HiSeq4000 for two-terminal sequencing,a total of 183,877,174 raw reads were obtained with an error rate of only 0.01%.After filtering this raw reads,get 176,228,782 clean reads.The number of sequencing sequences is multiplied by the sequencing length to obtain 26.43 G Clean bases.After obtaining a clean reading,use Trinity to splice the clean reads.75,742 transcripts and 63,453 unigenes wereobtained.Gene annotation for all Unigene,including NR,Swiss-Prot,KEGG,COG,KOG,GO and Pfam databases.All unigenes were annotated successfully.Through the analysis of differentially expressed genes,it was found that 887 differentially expressed genes were screened after ammonia nitrogen stress,481 genes were up-regulated and 406 genes were down-regulated,and they were mainly enriched in 16 immune-related pathways.Immune-related genes were significantly enriched in Complement and coagulation cascades,B cell receptor signaling pathway,T cell receptor signaling pathway and Toll-like receptor signaling pathway.According to its biological function,fifteen differentially expressed genes were selected and verified by qRT-PCR in the stress group and the normal group.The results showed that the qRT-PCR analysis results were consistent with the trend of the sequencing results.Complement and coagulation cascade pathway is the most important enrichment pathway.Therefore,the key gene serine protease inhibitor was selected for cloning and expression analysis.The results showed that the expression of Mn-SPI gene increased with the stress time,and the difference was significant(p<0.05),reaching the highest and then gradually decreasing.At the same time,the expression of hepatopancreas in immunohistochemistry was significantly higher than that in other tissues.It is suggested that serine protease inhibitor plays a major role in improving the ability of anti-ammonia nitrogen stress and enhancing its immunity.(2)The metabolites were annotated from the matrix obtained by the metabolic experiment,and 103 variables were finally obtained that could be annotated by the database.All the samples were analyzed by principal component analysis,and two main components were obtained during the automatic simulation,which can explain a total of 60.8% of the principal components,the first principal component(PC1)can explain 50.4% of the data model,the second principal component(PC2)can explain 10.4% data model.All the metabolites were statistically analyzed.The results found that 36 metabolites were down-regulated after ammonia-nitrogen stress including cadaverine,tetradecanoic acid,inosine,arachidonic acid hexadecanoic acid and so on.5 metabolites were up-regulated after ammonia-nitrogen stress including alanine,hydroxysuccinic acid,pyruvic acid,2-aminobutyric acid and fumaric acid.The results showed that the up-regulation of urea anduric acid was closely related to the anti-ammonia nitrogen stress of freshwater prawns,and provided new ideas for the screening of Macrobrachium nipponense with anti-ammonia nitrogen stress from the perspective of metabolomics.(3)Correlation Analysis was taken in DGE of transcriptome and metabolites obtained from metabolomics.Pyruvate,glycine and inosine were mainly concentrated in the glycolysis/ gluconeogenesis pathway,protein digestion and absorption pathway and purine metabolic pathway.Correspondingly,20,8,8 different genes were enriched in these pathways.The glycosylation / gluconeogenesis pathway plays a major role in the body's ammonia secretion.The body's digestion and absorption of the protein can improve the immunity of the body.The purine metabolic pathway is closely related to the detoxification mechanism of crustaceans under ammonia-nitrogen stress.In summary,the results provide the basic data for revealing the molecular mechanism of anti-ammonia nitrogen stress,which laid the foundation for further revealing the physiological metabolic mechanism of shrimp resistance to ammonia nitrogen stress and the breeding option of improving immunity.