Study On Related Molecules And Physiological Adaptation Mechanism Of Leuciscus Waleckii Under High Alkali Environment

The Amur ide（Leuciscus waleckii）is a fish in the Cyprinidae family,is one of the"San Hua Wu Luo",a famous fish in Northeast China,and has important economic value.Compared with other Amur ide living in freshwater ecosystems,the Amur ide population in Lake Dali Nor of China is famous for its high tolerance to alkaline conditions of 54 m M（p H 9.6）.Yet,the ionoregulatory mechanism responsible for this remarkable alkaline adaptation remains unclear.This paper discusses the adaptation mechanism of the alkali form of Amur ide to the high alkali environment from the perspectives of fish physiology,molecular biology and omics.The high alkaline environment directly affects the osmotic pressure and acid-base balance of fish.Here,using a comparative approach,the alkali form of Amur ide and freshwater form of Amur ide were each exposed to 50 m M（p H 9.59±0.09）for 7 days at the same time,a level close to the alkalinity of Lake Dali Nor.This study highlighted differences in blood p H and serum ions(e.g.,Na⁺,K⁺,Cl^–and Ca²⁺),Na⁺/K⁺-ATPase（NKA）activity and its m RNA level of the two types of Amur ide after high alkalinity tolerance.At the same time,and m RNA expression of gill transporters（NHE2/3,NBC1,AE1,NCC,NKCC1,SLC26A5,and SLC26A6）and the expression abundance of some key proteins and their localization in gills were analyzed to find out their different adaptation to high alkali,and then understand how the alkali form of Amur ide regulates the osmotic pressure and acid-base balance to adapt to high alkali environment.The results showed that close relationships among the serum Na⁺and m RNA levels of NCC,NKCC1,and NHE,as well as NKA and NBC1,in addition to serum Cl^–and bicarbonate transporters（e.g.,SLC26A5 and SLC26A6）,characterized the alkali form of Amur ide.Combined with Western blot and immunofluorescence detection results of SLC26A6 and other proteins,we propose that this ecotype is able to ensure its transepithelial Cl^–and Na⁺uptake/base secretions are highly functional,by virtue of its basolateral NKA with NBC1 and apical ionic transporters,and especially NCC incorporated with other transporters（e.g.,SLC26）.This suggests an evolved strong ability to maintain an ion osmotic and acid–base balance for more effectively facilitating its adaptability to the high alkaline environment.The high alkaline environment will also hinder the excretion of ammonia nitrogen from the fish.In order to explore the ammonia excretion mechanism of alkali form of Amur ide in high alkaline environment,we compared the oxygen consumption rate,ammonia efflux rate,ammonia nitrogen and urea nitrogen content in blood and tissue of alkali form of Amur ide and freshwater form of Amur ide before and after treatment with 50 m M alkalinity for 7 days,and detected the m RNA expression levels of ammonia excretion-related genes Vacuolar H⁺-ATPase subunit ATP6V1A,RHBG and RHCG1 and the expression abundance of key proteins and their localization in gill tissues were also detected.The results showed that both alkali form of Amur ide and freshwater form of Amur ide could reduce the accumulation of toxic ammonia by increasing the content of non-toxic urea nitrogen in the body in the early stage of stress,so as to reduce the risk of ammonia poisoning,but alkali form of Amur ide could increase the expression levels of ammonia excretion related proteins RHBG and RHCG1 in gills in the late stage of stress,and at the same time restore the ammonia excretion rate to start the body’s ammonia excretion to reduce the ammonia content in blood and tissues,so as to adapt to the external high alkali environment.While freshwater form of Amur ide can not successfully initiate the ammonia excretion,resulting in increased ammonia content in blood and tissues,further increasing the risk of ammonia poisoning.Many key genes play an important regulatory role in the adaptation of alkali form of Amur ide to high alkali environment.Pre-transcriptional analysis identified a large number of differentially expressed genes that were related to salt/alkali adaptation.But it is speculated that that there is only limited scope for the regulation of adaptability to environments with high alkalinity at the level of the genome.Previous work established that epigenetic regulation can help organisms respond quickly to stressful external environments without modifying any DNA sequences.Therefore,it is suggested that the adaptation of Amur ide in the Dali Nor Lake to high alkali conditions may be achieved at the epigenetic level.We sequenced tissues from the gills and kidneys of Amur ide to identify lnc RNAs that were closely associated with the regulation of high alkaline adaptation.Fish were exposed to these conditions for 20 days and then screened for differentially expressed lnc RNAs.As a comparison,we also created additional conditions in which the concentrations of Na HCO₃ were 30 m M and 0 m M.A total of 159 million clean reads were obtained by high throughput sequencing and 41248 lnc RNA transcripts were identified.Of these,the mean number of exons and the mean length of the lnc RNA transcripts were 4.8 and 2079 bp,respectively.Based on the analysis of differential lnc RNA transcript expression,a total of 5244 and 6571 lnc RNA transcripts were found to be differentially expressed in the gills and kidneys,respectively.Results derived from GO and KEGG analysis of the coding genes were correlated with the lnc RNA expression profiles.GO analysis showed that many lnc RNAs were enriched in the following processes:"transporter activity","response to stimulus",and"binding".KEGG analysis further revealed that metabolic pathways were significantly enriched.A random selection of 16 lnc RNA transcripts were tested by q PCR;these results were consistent with our sequencing results.Further analysis of the sequencing results showed that the transcript LNC＿023391 co-localized with the encoding gene Rhcg1 was significantly differentially expressed at different alkalinities,suggesting that it may have an important regulatory effect on Rhcg1.Preliminary validation showed that LNC＿023391 is indeed a transcript sequence,not a genomic fragment,and is only expressed in gills.A total of 3674 bp transcripts were obtained by amplification,and the prediction result of coding potential was non-coding sequence.According to the prediction results of bioinformatics,it is speculated that LNC＿023391 may play an important role in regulating m RNA stability during Rhcg1 expression.We found that a large number of genes,with the same expression profiles as those with differentially expressed lnc RNAs,were associated with the regulation of acid-base balance,ion transport,and the excretion of ammonia and nitrogen.Collectively,our data indicate that lnc RNA-regulated gene expression plays an important role in the process of adaptation to high alkaline conditions in L.waleckii.This paper provides a regulatory model for the regulation of osmotic pressure,acid-base balance and ammonia efflux of alkali form of Amur ide in the process of adapting to extreme alkaline conditions,and predicts that lnc RNA may play an important role in the regulation of key genes in this process.This study will provide a solid foundation for the further analysis of its saline alkali tolerance mechanism,so as to cultivate new alkali tolerant fish species and make use of waste alkaline water.