Histological And Physiological Biochemical Response To Saline-alkali Stress And Transcriptome Analysis In Oreochromis Mossambicus

Oreochromis mossambicus has a high salinity tolerance and is often used as a parent to produce hybrid tilapia with more salinity tolerance,which is a reliable object to study the molecular regulation mechanism of osmotic pressure in fish.In this study,physiological and biochemical parameters of O.mossambicus were analyzed and measured to investigate the specific effects of saline-alkali stress on the tissue structure and antioxidant capacity of O.mossambicus.We also used transcriptome sequencing to sequence the gill tissues of O.mossambicus under saline-alkali stress to obtain the m RNA expression profiles of O.mossambicus in gill tissues under saline-alkali stress,and then analyzed the m RNA expression profiles.The differentially expressed genes related to osmolarity regulation were selected to understand the correlation between the expression changes and osmolarity regulation under saline-alkali stress by q RT-PCR.This study was conducted to understand the correlation between the expression changes and osmotic pressure regulation under saline-alkali stress,and to provide a basis for the study of osmotic pressure regulation mechanism in tilapia under saline-alkali stress.1 Effects of saline-alkali stress on the structure of gill,kidney,intestine and liver tissues and their antioxidant capacity in O.mossambicusO.mossambicus was subjected to saline-alkali stress（25 g/L Na Cl and 4 g/L Na HCO₃）and samples of gill,kidney,intestine and liver tissues（control group was untreated）were sequentially performed at 6 h,12 h,24 h,48 h,72 h and 96 h.Paraffin sections were made of the four tissues,and SOD,CAT,GSH-Px activities and MAD concentrations were also measured in the gill,intestine and liver tissues.The experimental results showed that saline-alkali stress causes pathological damage to the gill,kidney,intestine and liver tissue structures of O.mossambicus,with the most obvious changes in gill and intestine,but with the increase of saline-alkali stress time,the fish will adapt to the water environment and the level of damage of the tissue structures will be reduced.Meanwhile,the differential changes in SOD,CAT and GSH-Px enzyme activities and MDA concentrations in gill,intestine and liver of O.mossambicus were checked under saline-alkali stress,and the trend of SOD enzyme activities in gill,intestine and liver under saline-alkali stress were all significantly increased firstly and then gradually decreased（P<0.05）,but their peak time points were different,at 48 h,6 h and 12 h,respectively;CAT enzyme The trend of CAT enzyme activity in both gill and intestine was decreasing and then increasing,reaching the lowest value at 12 h and 6 h and the highest value at 24 h,respectively,but in the intestine the treated group was lower than the control group,and in the liver the trend was increasing and then decreasing,reaching the highest value at 6 h.The trend of GSH-Px enzyme activity in both gill and intestine was increasing and then decreasing with the time points at which it peaked were different,at 6 h and 24 h,respectively.The trend of GSH-Px enzyme activity in both gill and intestine was rising and then decreasing,but the time points of its peak were different at6 h and 24 h,respectively.The trend of MDA concentration in gill,intestine and liver was rising and then decreasing（P<0.05）,with the peak time points at 6 h in both gill and liver and at 24 h in intestine.2.Expression profile construction and bioinformatics analysis of O.mossambicus gill tissue under saline-alkali stressIn this study,transcriptome sequencing（RNA-Seq）was performed on gill tissues of Mozambique tilapia under saline-alkali stress for 24 h.Three replicates were set up for the saline-alkali treated（SA1,SA2,SA3）and control（C1,C2,C3）groups,and the gills of three fishes were taken from each replicate.High-throughput sequencing of the constructed gill tissue m RNA expression profile libraries showed that the number of raw data reads with an average length of 150 bp in C1,C2,C3 and SA1,SA2 and SA3 were43658340,46395938,44228314 and 39533176,44342584,44218796,respectively,and the number of bases was above 5.9 G.A total of 3801 significantly differentially expressed genes were screened,among which 1498 and 2303 were down-and up-regulated genes,respectively.GO enrichment analysis was performed on the screened significantly differentially expressed genes,and a total of 162 GO Terms were significantly enriched.Among them,98,46 and 18 GO items were enriched in biological process（BP）,cellular component（CC）and molecular function（MF）respectively.The most significantly enriched GO Term in molecular function was structural constituent of ribosome;the most significantly enriched GO Term in cellular component was ribosome;the most significantly enriched GO Term in biological process was translation.KEGG enrichment analysis of differentially expressed genes revealed that a total of 1132differentially expressed genes were involved in 151 pathways,of which 9 pathways were significantly differentially enriched（P-adj<0.05）,including Ribosome,Oxidative phosphorylation,and Cardiac muscle contraction.Cardiac muscle contraction,etc.Finally,14 genes were selected randomly among the significantly differentially expressed genes for real-time fluorescence quantitative PCR experiments for validation analysis.3.Effect of saline-alkali stress on the expression of Tau T2,AQP3,ATP1A1 and SLC25A6 genesThe expression characteristics of Tau T2,ATP1A1,AQP3,and SLC25A6 genes in nine tissues,including gill,kidney,intestine,liver,brain,heart,skin,spleen,and blood,and their m RNA expression levels in gill,kidney,intestine,and liver tissues at different time points during 96 h of saline-alkali stress were examined by fluorescence quantitative PCR（q RT-PCR）.The results showed that Tau T2,AQP3,ATP1A1 and SLC25A6 genes were expressed in the above nine tissues of fish with the different expression distribution characteristics.Under saline-alkali stress,the m RNA expression of Tau T2,ATP1A1 and SLC25A6 genes in gill,kidney,intestine and liver tissues was characterized by an increase and then a decrease,but the peak expression in each of the four tissues was at different time points.The expression of AQP3 m RNA in gill,kidney,intestine and liver decreased significantly to the lowest value at 24 h（P<0.05）,and then gradually increased.The changes of m RNA expression levels of the four genes showed regular correlation with saline-alkali stress,and the results can provide reliable data for the study of the mechanism of osmotic pressure regulation in fish.