Research On Metabonomics Of Carassius Auratus Gill Under NaHCO₃ Habitat Stress

In this study,the Fangzheng silver crucian carp（Carassius auratus）was used as the research object.Under sodium bicarbonate（NaHCO₃）stress,the content of ammonia and urea nitrogen（BUN）in serum of the Fangzheng silver crucian carp and the antioxidant biochemical indexes in the gill tissue Changes.On this basis,by studying the pre-processing methods of target organ gill tissue metabolomics samples,and carrying out metabolomics analysis of crucian carp gills under different concentrations of NaHCO₃stress,89 differentially expressed metabolites were screened out（Differential Expressed Metabolites,DEMs）,and analyzed 12 metabolic pathways related to alkali stress.Finally,combined with the second-generation transcription sequencing technology,the differentially expressed genes（Differential Expressed Genes,DEGs）in the process of crucian gill tolerance to salt and alkali were screened out.From the molecular level,preliminary analysis of the salt-tolerant mechanism of Fangzheng silver crucian carp.1.Effects of sodium bicarbonate stress on biochemical indexes of Fangzheng silver crucian carpIn order to clarify the influence of NaHCO₃ alkali stress on blood ammonia,urea nitrogen（BUN）,and anti-oxidation biochemical indicators of gill tissue in Fangzheng silver crucian carp,this study set up 1 freshwater control group（Con）and 3 NaHCO₃treatment groups:20 mmol/L（CA20）,40 mmol/L（CA40）,60 mmol/L（CA60）.The experimental results showed that with the increase of NaHCO₃ concentration,the blood ammonia and BUN content in the serum of Fangzheng silver crucian carp showed an increasing trend.Compared with the Con group,the NaHCO₃ treatment group increased significantly（p<0.05）;compared with the Con group,the contents of malondialdehyde（MDA）,total superoxide dismutase（T-SOD）,catalase（CAT）,and glutathione peroxidase（GSH-PX）activities in gill tissue all showed Significant change（p<0.05）.In summary,the ammonia accumulation in Fangzheng silver crucian carp under carbonate-alkali stress can’t be excreted normally,which provides a direction for the research on the salt-alkali tolerance of the Fangzheng silver crucian carp.At the same time,Fangzheng silver crucian carp can adapt to the external saline-alkali environment through its own regulation.2.Effect of sodium bicarbonate stress on metabolism of Fangzheng silver crucian carpIn order to improve the extraction effect of metabolites,the pretreatment method of fish gill samples was optimized based on UPLC-Q-TOF/MS technology in the experimental process of studying fish gill target organs by non-targeted metabolomics.The effects of different organic reagents,extractant dosage and extraction temperature on the coverage rate and stability of extraction of metabolites from gill tissue were investigated.The results showed that:for 30 mg gill tissue required for metabolomics,480μL of 80%methanol aqueous solution（v/v）pre-cooled at 4℃was added,after being fully grinded by a tissue grinder,ultrasonicated in an ice water bath for 10 min,put it at-20℃and let it stand for 30 min,and finally centrifuge at 13000 rpm for 15 min at a temperature of 4℃during centrifugation.Take 300μL of the upper layer solution after centrifugation and filter it through a 0.22μm organic phase filter into the sample bottle,the metabolites collected by UPLC-Q-TOF/MS have the highest coverage rate and the best stability,and this method has the best extraction effect.At the same time,the quality control（QC）samples interspersed in the detection sequence were used for methodological investigation,and 6 representative endogenous metabolites with different polarity,including Glucose,L-Leucine and Citric acid were selected for evaluation.The instrument precision,method precision and sample stability of this sample pretreatment method are all good.The gill metabolomics sample treatment method established above was used to detect the metabolites in the gill target organs of crucian carp under NaHCO₃stress by UPLC-Q-TOF/MS technology,and the stable and reproducing original data of Con group and three NaHCO₃ treated groups were obtained respectively.After data preprocessing and statistical analysis of the original data,89 DEMs were identified,of which 50 were up-regulated and 39 were down-regulated.Pathway analysis of these DEMs revealed that 12 metabolic pathways were affected,among which glycerophospholipid metabolism（p value 0.0041）was significant.3.Comparative study on gill transcriptome of Fangzheng silver crucian carp after sodium bicarbonate stressIn this study,second-generation sequencing technology was used to analyze the differential expression of messenger ribonucleic acid（m RNA）in the gill tissues of the Con group and the CA60 group.18 categories were annotated by the Cluster of Orthologous Groups（COG）database.Modified proteins were the known pathways with the most clustered genes.Using Gene Ontology（GO）enrichment analysis,DMGs enriched 18 GO categories（p-adjust<0.5）such as ammonia transmembrane transport,ammonia transport,and one-carbon composite transport（p-adjust<0.5）;applied Kyoto Gene and Genome Encyclopedia Encyclopedia（Kyoto Encyclopedia of Genes and Genomes,KEGG）enrichment analysis,enriched the proximal tubule bicarbonate recovery,nitrogen metabolism,apoptosis and other 19 KEGG pathways（p-adjust<0.5）.On the one hand,this study enriched transcriptome data of Fangzheng silver crucian carp under NaHCO₃ stress,on the other hand,it also provided a new research idea for the response mechanism of this species under the stimulation of environmental factors,further clarifying the molecular mechanism of Fangzheng silver crucian carp under saline-alkali stress,and seeking the saline-alkali tolerance related gene set related signal pathway.To provide a new theoretical guidance for the pathogenic mechanism of Fangzheng silver crucian carp exposed to NaHCO₃.