| Macrobrachium nipponense is one of the important aquaculture species in China,and it is widely distributed.Environmental stress has become one of the important factors hindering the healthy development of aquaculture.Ammonia and nitrite are the most common among many chemical pollution stress factors,and they are also the most significant stress on the physiological functions of the organism.In this study,the immune indicators of M.nipponense on ammonia stress(22.1 mg/L)and nitrite stress(10.0 mg/L)were studied,followed by stress experiments.In the nitrite stress experiment,six sequencing libraries were constructed,three libraries from treatment groups and other three from control groups.In the ammonia stress experiment,the hepatopancreas of M.nipponense as the research object.In total,two sequencing libraries from control groups and two sequencing libraries from treatment groups were constructed in the ammonia stress study.The library uses high-throughput sequencing platform to identify differentially expressed genes(DEGs)related to ammonia/nitrite stress in the hepatopancreas of M.nipponense.Analyzes the pathways and DEGs related to immunity under stress.Analysis and validation of DEGs associated with immune.Results are as follows:(1)The change of immune indicators was measured under the ammonia stress(22.1mg/L): SOD and T-AOC of hepatopancreas,hemocytes,muscle and gill have different trends under ammonia stress.The activity of SOD(hepatopancreas,hemocytes,muscle)?T-AOC(hepatopancreas,muscle)and PO is highest at the 48 h.The SOD and T-AOC activity of hepatopancreas is the highest of the four tissues.Therefore,hepatopancreas plays an important role in the body's resistance to ammonia stress.And 48 h is a key time point for M.nipponense against ammonia stress.(2)The change of immune indicators was measured under the nitrite stress(10.0mg/L): SOD and T-AOC of hepatopancreas,hemocytes,muscle and gill have different trendsunder nitrite stress.The activity of SOD in hepatopancreas,hemocytes and gill tissue,the activity of T-AOC(hepatopancreas,muscle)and PO is highest at the 48 h.Therefore,hepatopancreas plays an important role in the body's resistance to nitrite stress.And 48 h is a key time point for M.nipponense against nitrite stress.(3)Transcriptome sequencing under six libraries were constructed by Illumina HiSeq sequencing platform.A total of 137,690,587 clean reads were obtained.After GO analysis,unigenes mainly enriched in metabolic processes,catalytically active processes,binding and cellular components.KEGG pathway enriches 5529 unigenes.The most enriched path is ribosome pathway,RNA transport pathway and lysosomal pathway.There were a total of13,960 unigenes annotated into 26 KOG groups,and signal transduction mechanisms and general functional prediction were the most enriched groups.(4)Nitrite stress transcriptome sequencing identified 825 DEGs,including 762 significantly up-regulated genes and 63 significantly down-regulated genes.GO analysis of DEG is enriched in proteolytic metabolism,cell transformation and single organism processes.KEGG enrichment analysis shows that among the 60 pathways that are significantly enriched.The most abundant is the RNA transport pathway,lysozyme Body pathways and purine metabolic pathways.In the immune response related pathway,the major enriched pathways are the lysosomal pathway,glycerolipid metabolic pathway,the phagosome pathway and JAK-STAT signaling pathway.(5)21 immune response related DEGs were obtained,19 of which were significantly up-regulated and 2 were significantly down-regulated.21 DEGs are mainly enriched in the lysosomal pathway,alanine aspartic acid glutamate metabolic pathways,arginine and proline metabolic pathways and JAK-STAT signaling pathway.(6)Study on immune response related pathways and genes screened under nitrite stress.By studying the differentially expressed genes on the lysosomal pathway and JAK-STAT signaling pathway,we found that nitrite stress initiates the expression of immune response-related genes.It is proved that the lysosomal pathway,JAK-STAT signaling pathway and related genes have certain protective effects when the M.nipponense is stressed by nitrite.(7)Transcriptome sequencing under ammonia stress,Four sequencing libraries were constructed.The raw reads obtained from each transcriptome library were transcribed andspliced,and the clean reads were analyzed and screened.2858 unigenes were annotated by the five major databases.A GO annotation analysis of 63453 unigenes revealed that they were mainly enriched in metabolic processes,cellular processes,and a single organism process.(8)A total of 56663 expressed genes were obtained from the control group and the stress group by screening(fpkm>0.3).There were 27,466 genes co-expressed in the two groups.According to the screening conditions,the differentially expressed genes were screened and analyzed.GO analysis of differential genes revealed that 344 DEGs were enriched in a single organism process,up-regulated genes were mainly enriched in cellular processes(193),and down-regulated genes were mainly enriched in metabolic processes(188).KEGG analysis of DEGs revealed 20 pathways that were significantly enriched on complement and coagulation cascades,platelet activation pathways,and glycolysis/gluconeogenesis.(9)44 immune response related DEGs were obtained after screening,of which 23 were up-regulated and 21 were down-regulated.Cluster analysis of immune response related DEGs,significant aggregation into two categories.KEGG analysis of the gene revealed that it is mainly enriched in the immune system-associated complement and coagulation cascade pathways,platelet activation pathways,antigen processing and presentation pathways.(10)Under ammonia stress,studies on immune response related pathways and genes revealed that the complement and coagulation cascade pathways are the pathways that enrich the most DEGs.Studies on the genes C3 and CFI in the pathway revealed that nitrite stress initiates the expression of immune response related genes under ammonia stress.It is proved that the complement and coagulation cascade pathways and related genes have a certain protective effect on the organism when the M.nipponense is stressed by ammonia.In summary,the immune index of M.nipponense under stress(ammonia stress,22.1mg/L;nitrite stress,10.0 mg/L)was determined.The high-throughput sequencing was used to identify the differentially expressed genes associated with stress(ammonia/nitrite)in hepatopancreas of M.nipponense.The genes and pathways related to immunity and apoptosis were analyzed.A total of 825 DEG related to nitrite and 887 DEG related to ammonia.Immune response related pathways and key genes under nitrite stress were screened out.These molecular-level responses confirm that these stress-related genes and pathways play animportant role in the protection of M.nipponense against ammonia and nitrite stress.These genes and pathways can be used as molecular immunological indicators of the defense response of M.nipponense under stress.This study produced a large amount of data,which laid the foundation for further revealing the immune defense mechanism of shrimp.The data provide new ideas for the immune response of M.nipponans and laid the foundation for the cultivation of new varieties resistant to ammonia and nitrite stress. |
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