Genome-Wide Identification, Phylogenetic Analysis And Expression Pattern Profiling Of The Aquaporin Family Genes In Leuciscus Waleckii

Amur ide（Leuciscus walleckii）is a cold water cyprinid fish widely distributed in northern China,including both alkaline and freshwater（such as L.walleckii in the Wusuli River）species.Among them,the L.walleckii located in Dali Lake（DL）,Inner Mongolia(carbonate alkalinity=53.57 mmolol·L^-1,p H=9.6)has extremely strong alkali resistance,which makes L.walleckii an important experimental material for studying the molecular mechanism of salt and alkali tolerance.Aquaporin（Aqp）is a transmembrane-specific channel for small molecules that help in regulating homeostasis in fishes when adapting to changing environments.Studies have shown that Aqp is involved in the response of fish to alkalinity stress,but its role in this process in L.walleckii remains to be revealed.The aim of this paper is to investigate the response of Aqp gene family of L.walleckii to alkaline stress and the screening of genes related to high alkaline environment,which can provide theoretical support for the subsequent further uncovering of the mechanism of alkaline adaptation in L.walleckii.Firstly,the members of the Aqp family were identified from the whole genome,and then the phylogeny,motif and gene structure of the Aqp family were analyzed;Secondly,RNA-seq was used to analyze the expression of Aqp gene under different conditions and the gene expression profiles of aqp7,-8aa,-9a were analyzed by qRT-PCR;Then,the SNP analysis between the populations of Dali Lake and Wusuli River was carried out by using whole-genome resequencing;Finally,the amino acid sequence of the selected important genes was analyzed:1.Identification and sequence analysis of the Aqp gene family of L.walleckii.In this study,a total of 18 Aqp genes have been identified from L.walleckii genome,and the location and length,etc.of these 18 Aqp genes have been listed.It is worth noting that compared with zebrafish,there are three copies of Aqp3a gene in L.walleckii,of which aqp3ap1 and aqp3ap2 are pseudogenes.We have constructed phylogenetic trees of several species.According to the clustering relationship and topological structure,the Aqp gene can be divided into four grades:classic aquaporins,aquaglyceroporins,ammoniaporins and unorthodox aquaporins.Analysis of the gene structure revealed aquaporins of the same rank with the same or similar number of exons.The result of motif analysis shows that the closer the homologous relationship is on the evolutionary tree,the more similar its motifs are,and most of the proteins contain motif 3 and 52.Calculation of differential expression of three different conditions.First of all,the data of the differential expression of the two geographical populations of Ardisia wallichii in Dali Lake（alkaline water）and Wusuli River（fresh water）showed that aqp3a,-3ap1,-3ap2,-7 were highly expressed in gill,but low in kidney and intestine;In gill,except for aqp1aa,the expression of all Aqp genes in WSL L.walleckii was higher than that in DL L.walleckii.We then focus on the expression of Aqp gene in the DL L.walleckii under different conditions.The tissue expression profile of DL L.walleckii after seven days of exposure to alkaline water(carbonate alkalinity50 mmol·L^-1)showed that aqp3a,-3ap1,-3ap2 were mainly expressed in gills and skin;aqp10b,-8ab are mainly expressed in the intestine;aqp8aa was highly expressed in intestine,muscle and skin;aqp9a is highly expressed in brain,gill and skin.The results of the experiments on the different days under alkalinity stress(carbonate alkalinity 50 mmol·L^-1)and its recovery of the DL L.walleckii showed that the expression of aqp0a,-3a,-3ap1,-7,-8aa,-9a in gills generally showed a trend of up-regulation in alkaline water environment and down-regulation in fresh water;On the contrary,aqp1aa,-1ab,-3ap2,-11 generally showed a downward trend in alkaline water environment,then upward trend,and finally upward trend in fresh water.Three genes that were upregulated in alkaline water and downregulated in freshwater were selected for qRT-PCR validation,and the results are as follows:After 3d of stress,the expression of aqp7 decreased to 0.56-fold.After 3 d of stress,the expression of aqp7 decreased to 0.56-fold（p<0.01）.After 3 d and 5 d of stress,the expression of aqp8aa increased to 1.86-fold（p<0.01）and 1.63-fold（p<0.05）,respectively.After 5 d and 7 d of stress,the expression of aqp8aa increased to 1.95-fold（p<0.05）and 3.29-fold（p<0.01）,respectively.3.SNP analysis and protein analysis of important sites in the DL and WSL L.walleckii.We identified 1460 single nucleotide polymorphism（SNP）markers in the Aqp gene between DL and WSL L.walleckii.There are 114 SNP markers in CDS area,and the average Fst value is 0.177±0.256.Five SNPs were ultimately obtained by selecting the SNP markers with the top 5%of the Fst value in the CDS region.The five sites were Aqp3a.1,-3a.2,-3a.3,-3a.4,and-11b,and their Fst values were 0.816,0.910,0.906,0.822,and 0.816,respectively.Among them,there are two non-synonymous mutations.The Aqp3a.1 is a nonsynonymous SNP that caused the substitution of glutamicacid for asparticacid;a nonsynonymous substitution occurred at Aqp3a.4,replacing alanine with serine.Multiple species alignment of the amino acid sequences of Aqp3a.4 with larger FST values in the two loci showed that DL L.walleckii was serine,while except zebrafish,WSL L.walleckii and other freshwater species was alanine.The Aqp3a was mainly composed of six transmembraneα-helices and two short helices.Its secondary structure mainly includesα-helical and random coils.The 66th amino acid of Aqp3a.4（Ser66）was in the center of H2 and near the Ar/R residue phenylalanine（Phe63）.The mutation of the amino acid at position 66 did not cause many changes in the three-dimensional structure of Aqp3a,but may change the penetration of solutes across the membrane.In summary,the above research results show that Aqp genes are involved in the response of L.waleckii to alkaline stress,and Aqp3a is one of the key genes involved in regulating L.waleckii’s adaptation to alkaline environments.From this,we had access to all members of the Aqp family of L.walleckii and their classification;Understanding its gene expression under different conditions,and this facilitates the initial exploration of Aqp gene function;The analysis of SNPs and Aqp3a is beneficial to understand the adaptive evolution of L.walleckii.Through the above studies,we can explore the role of Aqp genes in alkalinity stress and provide a theoretical basis for further research,which is beneficial to the uncovering of the mechanism of saline alkaline tolerance adaptation in L.walleckii.This will be beneficial for the cultivation of saline–alkaline tolerant species further enhancing the utilization of saline–alkaline waters and promoting the development of aquaculture in China.