Effects Of Alkalinities Stress On Phosphatase Gene Expressionin And Physiology Response In Leuciscus Waleckii

The saline-alkaline environment can lead to stress reactions in fish,and this extreme environment even threatens the lives of fish.However,some fish have evolved spontaneous regulation during long-term adaptation to balance the genetic mechanism of ion proportion in the body.Leuciscus waleckii belongs to Osteichthyes,Cypriniformes,Cyprinidae and Leuciscus,different geographic populations have different tolerances to salt and alkali.For example,the Dalinuoer Lake Leuciscus waleckii in Inner Mongolia can tolerate carbonate alkalinity of up to 53.57 mmol/L.Series of changes in the physiological indicators of fish under carbonate alkalinity stress,including phosphatase.Phosphatase can be divided into alkaline phosphatase(AKP)and acid phosphatase(ACP),which act on the growth and development of fish,energy metabolism,immune regulation and so on.This study focus on the differences between AW(Alkaline Waleckii)and FW(Freshwater Waleckii),and both were sampled from Dalinuoer Lake as alkaline water species and Songhua River as freshwater species.The water alkalinity was set to 0 mmol,30 mmol/L and 50 mmol/L,collect serum and tissues after twenty days.The serum alkaline phosphatase and acid phosphatase activities were analyzed,and the c DNA sequences of alkaline phosphatase and acid phosphatase from Leuciscus waleckii were cloned and the phosphatase gene expression in the organs was detected.The purpose of this study was to study the effects of phosphatase on the regulation mechanism of salt tolerance of Leuciscus waleckii under carbonate alkalinity stress and its role.The main results are as follows:(1)Using Automatic microplate reader and a serum phosphatase activity kit to determine AKP and ACP phosphatase activity in serum between AW and FW.The results showed that the alkaline phosphatase activity in the AW and FW showed different degrees of increase and decrease,and the changes of acid phosphatase and alkaline phosphatase were different.The serum AKP activity of FW at 0,30,and 50 mmol/L alkalinity was 9.71±0.17,13.18±1.38,and 12.4±0.52,and the serum AKP activity of AW was 10.56±0.45,8.72 ± 0.83 and 11.59±0.30,the serum ACP activity of FW was 99.36±6.01,97.40 ± 15.69 and 91.41±3.75,the serum ACP activity of AW was 99.14±1.32,117.39±10.22 and 107.15±4.24.Comparing the changes of the phosphatase activity of Leuciscus waleckii under different carbonate alkalinity stresses,high carbonate alkalinity increased the AKP activity of Leuciscus waleckii,and the AW at 30 mmol/L AKP activity is significantly higher than FW.Under high alkalinity,the ACP activity of AW increased significantly with FW Non-significant.It is speculated that the significant increase in AKP and ACP phosphatase activity at the carbonate alkalinity can reflect the tolerance of AW.Phosphatase activity can be used as an evaluation indicator of the alkali resistance mechanism of Leuciscus waleckii.(2)Referring to the AKP gene design primer of carp and transcriptome data of Leuciscus waleckii and design of primers,cloning and analysis of AKP and ACP genes of Leuciscus waleckii.The open reading frame(ORF)of AKP gene of Leuciscus waleckii was 876 bp in length and encoded 291 amino acids.The open reading frame(ORF)of ACP gene of Leuciscus waleckii was 528 bp in length and encoded 175 amino acids.The phosphatase gene of Leuciscus waleckii is highly homologous to Sinocyclocheilus rhinocerous,Cyprinus carpio,and Carassius auratus.(3)Referring to the AKP and ACP genes design real-time PCR primer of Leuciscus waleckii,and the expression of AKP m RNA in muscle,sputum,liver,brain and kidney of Leuciscus waleckii was determined by quantitative real-time PCR.The expression of AKP gene in different tissues of the two squid was different.In gills,AKP activity of FW was significantly higher than that of AW at 0 mmol/L,the expression of AKP genes of AW and FW down-regulated under high alkali environment.The ACP gene of AW showed a tendency to increase first and then decrease with alkalinity.FW showed that the expression of ACP gene was down-regulated at high alkalinity;In the liver,the AKP gene of AW is up-regulated with the increase of alkalinity while the expression of AKP with FW non-significantly.The ACP gene of AW is significantly increased at 30mmol/L alkalinity;In the kidney,the FW phosphatase gene is up-regulated in the kidney with increasing alkalinity,while the AW phosphatase gene is significantly upregulated less than FW.In the brain,the phosphatase gene is significantly up-regulated under high alkalinity of FW.The carbonate alkalinity of 50 mmol/L has a significant stimulation on FW.In muscles,the two types of Leuciscus waleckii showed a downregulated expression at high alkalinity,and the ACP expression level of AW was higher than that of FW.This paper believes that under carbonate stress,the phosphatase gene is differentially expressed in different tissues of Leuciscus waleckii,and phosphatase genes respond differently under carbonate stress.AW can cope with high carbonate alkalinity by regulating liver phosphatase gene expression,and FW by increasing kidney phosphatase gene expression.The phosphatase gene expression of the two groups of Leuciscus waleckii under alkali stress is tissue-specific.(4)Analyze the differentially expressed long-chain non-coding RNA associated with phosphatase under Leuciscus waleckii alkali stress,and use cufflinks software to calculate the expression level of lnc RNA transcript FPKM.A total of 299 differentially expressed lnc RNAs were screened.A total of 37 lnc RNAs related to acid phosphatase were detected between renal lnc RNA and coding genes.Lnc RNA was analyzed for GO enrichment,which included GO(phosphatase activity,GO: 0005488)items related to phosphatase activity.At carbonate alkalinity,Leuciscus waleckii between phosphataserelated long non-coding RNAs are differentially expressed,and there is lnc RNA predicted to function as lysosomal acid phosphatase.It is preliminarily inferred that the phosphatase activity of Leuciscus waleckii has changed in different carbonate alkali environments,AW raises phosphatase activity and up-regulates phosphatase gene expression in the liver under high-alkaline environment,inferred phosphatase can be used as an evaluation index for the salt-alkali tolerance of Leuciscus waleckii.AW can activate the related regulatory genes in gill,liver and kidney tissues in the salt alkali environment to ensure the ion homeostasis in the body.