Transcriptome Analysis Of The Kuruma Shrimp(Marsupenaeus Japonicus) Under High Ammonia Nitrogen And Low PH Stress And Functional Study Of MjChi4 Gene

The kuruma shrimp(Marsupenaeus japonicus)is one of the most valuable shrimp species in the world.High ammonia and low pH are important environmental factors affecting the effect of its culture.Due to the accumulation of food and excreta under the factory farming model,human activities and climate warming,the water environment is deteriorating,and the impact of high ammonia and low pH stress on the culture industry of M.japonicus is becoming more and more serious.Therefore,it is necessary to enhance the tolerance to high ammonia and low pH of M.japonicus,and to breed excellent varieties of M.japonicu with enhanced tolerance to ammonia and pH stress.However,traditional breeding methods have high cost and long cycle,and the effect of improving low heritability traits is not obvious.Therefore,in this study,the transcriptome sequencing was used to analyses the molecular mechanism of M.japonicus in response to high ammonia and low pH stress,and identified the related stress response genes.The stress response gene chitinase 4(MjChi4)full length c DNA was cloned,qRT-PCR and RNA interference analysis were used to further verify that it may be related to high ammonia and low pH tolerance of M.japonicus.This study is of great significance for accelerating the breeding of new varieties of M.japonicus tolerance to high ammonia and low pH.The main results are as follows:(1)Transcriptome analysis of M.japonicus in response to high ammonia stressTranscriptome analysis of the hepatopancreas of M.japonicus under high ammonia stress used RNA-seq method,obtained 44,735,330–56,680,154 clean reads from 10 libraries,and found that 80.20%–82.38% of reads could be mapped to reference transcriptome.Comparative transcriptome analysis identified 2294 differentially expressed genes,many of which were related to immune function,oxidative stress,apoptosis,molting,and membranes.And totals of 36 gene ontology(GO)terms and 177 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were enriched.In addition,the expression levels of MjChi4 was significantly altered,and the genesencoding heat shock protein 90(HSP90),HSP10,and antioxidant enzymes(catalase and glutathione S-transferase)were significantly upregulated by high ammonia stress.Finally,amino acid metabolism,lysosome,peroxisome,m TOR,PI3 K Akt and AMPK signaling pathways were enriched.(2)Transcriptome analysis of M.japonicus in response to low pH stressTranscriptome analysis of the hepatopancreas of M.japonicus under low pH stress used RNA-seq method,obtained 46,250,194–68,423,114 clean reads from 12 libraries.Comparative transcriptome analysis identified 2702 differentially expressed genes,which were enriched to 56 GO terms and 222 KEGG pathways.And the expression levels of genes encoding MjChi4,digestive enzymes,antioxidant enzymes(cytosolic manganese superoxide dismutase,glutathione peroxidase,and glutathione S-transferase)and HSPs were significantly altered by low pH stress.Moreover,linoleic acid and arachidonic acid metabolism,lysosomes,ovarian steroidogenesis,and the peroxisome proliferator-activated receptor and AMP-activated protein kinase signaling pathways were significantly enriched.(3)Functional analysis of stress response gene MjChi4The stress response gene MjChi4 was successfully obtained by transcriptome analysis.The full-length c DNA of MjChi4 gene,2124 bp,encoding 613 amino acids was obtained by RACE cloning technology.Bioinformatics analysis revealed that the amino acid sequence of MjChi4 has a Sec signal peptide(Sec/SPI),a Glyco?18 and two Cht BD2 functional domains,and has no transmembrane region exists.The qRT-PCR analysis showed that MjChi4 was the highest expressed in the hepatopancreas;the expression pattern of MjChi4 increased first,then decreased,and then increased under ammonia stress;under pH stress,it showed a cyclical change of first decreased and then recovered during the first 24 h,and an expression pattern of first increased and then recovered after 24 h.Finally,after RNA interference with MjChi4,M.japonicus survival rate decreased and death time accelerated significantly under high ammonia and low pH stress;the expression of chitinase(MjChi1,MjChi2,MjChi3),antiviral protein,C-type lectin,C-type lysozyme and prophenoloxidase b were significantly decreased,and the expression of cytosolic Mn SOD and Imd signaling pathway transcription factors relish were significantly upregulated.