Genome Wide Identification Of Carbinic Anhydrase Gene Family In Amur Ide(Leuciscus Waleckii) And Adaptive Evolution Analysis In Extremely Alkaline Environment

Carbonic anhydrase（Carbonic anhydrase,CA）is a set of zinc metalloenzyme,are widely distributed in the organizations,mainly involved in intracellular CO₂ hydrate process of reversible catalytic reaction(CO₂+H₂O?H⁺+HCO^3-),the ion transport,playing an important role in acid-base balance to maintain a stable internal environment.Different from land animals that rely on respiration for acid-base regulation,teleosts completely rely on ion transport and ion exchange for acid-base balance regulation.Therefore,the carbonic anhydrase regulation system providing H⁺and HCO^3-is a key regulatory system for teleosts to regulate acid-base balance.Carbonic anhydrase is an important gene family for teleosts to adapt the extreme alkaline environment.In this study,Amur Ide（Cypriniformes,Cyprinidae,Leuciscus waleckii）were studied.It is a kind of teleosts widely distributed in northeast Asia of China.It generally lives in the freshwater,but it also has a very high salinity tolerance.It can also survive in some high-salinity lakes,such as Dali Nur Lake,a typical saline Lake on the Inner Mongolia plateau in China.Amur Ide can survive in the Dali Nur Lake.It is a good material for studying the evolutionary analysis of extreme alkaline adaptability,which is helpful for us to further to explore the regulatory mechanism in the alkaline environment about how the carbonic anhydrase gene regulates the acid-base balance.In this study,18 carbonic anhydrase gene families were identified through genome-wide identification,and phylogenetic analysis and model analysis were performed.Secondly,m RNA expression differences of carbonic anhydrase were analyzed by transcriptome sequencing data in different habitats.Finally,through the analysis of genome-wide genetic polymorphism locus scanning and protein structure prediction,the purpose of this study is to deeply analyze the molecular regulation mechanism of carbonic anhydrase in different environments of fresh and alkaline water,which provides a research basis for the adaptive evolution analysis of fish in high alkaline water.The main conclusions of this study are as follows:1.Identification and evolution analysis of carbonic anhydrase gene family in Amur IdeCarbonic anhydrase of all 8 available species were downloaded from Ensembl and NCBI databases.Finally,carbonic anhydrase of zebrafish were used as queries to search against all available Amur Ide genomic resources by TBLASTN and BLASTP.18 carbonic anhydrase genes were identified in the Amur Ide of Dali Nur Lake.Subsequently,the position information,genome length,protein length and exon number of 18 carbonic anhydrase genes on the genome were counted.Phylogenetic analysis showed that the same genes in different species were clustered,indicating the conservation of carbonic anhydrase in the evolutionary process.The results of phylogenetic relationship were consistent with the results of subcellular localization,which was divided into three groups,namely,intracellular CA group,extracellular CA group and CA-RP group.Through the motif analysis of the carbonic anhydrase gene family,it was found that motif1,2,3 and 4 were shared by 18 carbonic anhydrase genes,and were located in theβ-fold region of the protein,participating in the catalytic CO2dehydration/hydration process,while other motif mainly played the role of receptor recognition.The genome re-sequencing data of liver,kidney and gill tissues of Amur Ide under two different environments were used to calculate the differential expression of carbonic anhydrase.In two different environments of fresh and alkaline water,CA2,CA6,CA15b and CA15c were highly expressed in gills,but relatively low in liver and kidney.CA2,CAhz and CA5a were all expressed in liver,kidney and gills.CA2,CA15b,CA15c and CA6 are only expressed in the gills but not in the liver and kidney,and there are tissue-specific expressions.CA5a was highly expressed in the liver in the fresh water and down-regulated in the alkaline water.CA2expression is up-regulated in the liver and down-regulated in the kidney and gills.CAhz is up-regulated in the liver.2.Statistics of carbonic anhydrase allele frequency and calculation of Fst in two habitatsIn order to detect the genetic polymorphism of the carbonic anhydrase,and the variation of the allele frequency of haplotype in the Amur Ide in the alkali environment population and the freshwater population,we analyzed the genetic polymorphism of the carbonic anhydrase gene by using the genome re-sequencing data of 10 River Ussuri freshwater populations and 18 Dali Nur Lake populations.The results showed that 801 SNPs were found in 18 carbonic anhydrase,and 108 SNPs were found in the exon region,among which CA15and CA4 had a large number of SNP sites.Then,the Fst value of carbonic anhydrase gene was calculated,and there were 4 SNP with the Fst value higher than 0.65,which were located on CA15b,CA15c,CA5a and CA6 genes respectively,showing significant allele frequency differences.The change of amino acids was caused by the change of glutamic acid（Glu）to aspartic acid（Asp）and serine（Ser）to arginine（Arg）.CA5a was mutated from methionine（Met）to valanine（Val）.However,CA6 has a synonym mutation at this site,which is still alanine Ala.3.Adaptive evolution analysis of carbonic anhydrase gene CA15b in alkaline environmentThe allele frequency of the CA15b had the largest Fst value of 0.962,indicating that the two populations had undergone great genetic differentiation under different environments of the fresh alkaline environment.To explore the effect of amino acid non-synonymous mutations caused by significantly different base site mutations on protein structure,CA15b was selected as an example for further analysis.The mutation site of binding amino acids showed that the amino acid site with non-synonymous mutation was located at amino acid 268 of CA15b.The CA15b amino acid sequences of stickleback,zebrafish,carp,crucians,pufferfish,tilapia,tetraodon,Amur Ide were compared and analyzed by multi-species amino acids.Three fish lives in fresh water environment,zebrafish,carp,crucians,stickleback,and tilapia,pufferfish,tetraodon in sea water environment of CA15b 268 amino acids are serine,and alkaline environment Amur Ide of CA15b 268 amino acid mutations to isoleucine.Therefore,it is inferred that this site is related to alkaline adaptation of fishes.Through the prediction of the three-dimensional structure of the protein,it was found that the three-dimensional structure of the CA15b gene protein was mainly composed ofα-spiral,β-fold and random curling,and 10β-fold around the zinc ion completed the main catalytic function of carbonic anhydrase.After the non-synonymous mutation of CA15b at the 268 amino acid site,the valence bond structure of this site changed,but the three-dimensional structure of the whole protein did not change.For the adaptation to extreme alkaline environment,the 268 amino acid mutation of CA15b only slightly represents the planar network of polarity residue binding,thus changing the activity of CA15b.The significant difference in the SNP site of 3′UTR resulted in non-synonymous mutations of amino acids,which affected the expression of CA15b gene.It may be an important reason for the up-regulated expression of CA15b in Amur Ide gills of the alkaline Dali Nur Lake populations.