| The effect of salinity stress on fish were described in this paper,respectively from the fish growth,survival,nutritional composition,antioxidant enzymes,Immunity,fish physiology and metabolism,which reveals the importance of the research on the effect of salinity stress on fish.The research progress of turbot on microsatellite genetic marker and fish salinity related genes and turbot transcriptome was reviewed,hopes to enrich the theoretical basis for studying the osmotic pressure of turbot by combining the results of previous studies with this experimental result.The expression of prolactin(PRL)gene and Na+-K+-ATPase?1 gene in the intestine and gill of juvenile turbot turbot under different salinity stress were detected by QPCR(Real-time Quantitative PCR Detecting System).The salinity 30 as control group,salinity 5,10,40,50 as experimental group,the result shows that two genes were expressed in both two tissues,and the expression of the gene was tissue and time specific.The expression of PRL and Na+-K+-ATPase ? 1 gene in intestinal tissues firstly increased and then decreased with the accumulation of stress time under salinity 50 and 5.The expression trend of PRL gene in the gill tissue was same as intestinal.However the Na+-K+-ATPase ? 1 gene expression did not change significantly in the 5 low salt conditions,the expression quantity changes with time accumulation showed firstly decreased and then increased in 50 high salt conditions.In intestinal tissue,the two genes have a very significant synergistic effect.With the increase of salinity,the expression of the two genes firstly increased and then decreased,and the correlation coefficients were closed to 1.In the gills,in the range of 10-40 salinity,there was a obvious antagonism on the expression of two genes.When the expression of PRL gene showed an increased trend(decreased),the expression of Na+-K+-ATPase ?1 gene showed a downward trend(increase),and the correlation coefficients of two genes were negative,It is confirmed that prolactin could inhibit the activity of Na+/K +-ATP enzymes,and provide a theoretical basis for the study of molecular regulation mechanism of salinity stress.The juvenile turbot salinity transcriptome database was constructed by the high-throughput sequencing technology.The kidney tissue of salinity 50 sea water stress and normal salinity was sequenced,aiming to comprehensive understand the expression of turbot gene after salinity stress,screening the candidate genes correlated with salinity,salinity related metabolic pathways were identified detected a variety of molecular markers and the quantitative expression analysis of some salinity genes was carried out.The results were as follows:1.After getting clean reads,182225 unigenes was spliced,which could be annotated to all 7 databases.The functions of all genes are clustered into 43 GO categories,and the most clustering genes are "cellular process","connection","metabolic process",and "single biological process".The KOG annotated genes were classified according to the KOG of group.The most genes were followed by "signal transduction mechanism","general function","translation modification","protein conversion",and "mate".According to the KEGG metabolic pathway,the most clustering genes are "signal transduction","endocrine system" and "immune system.2.The pathways associated with salinity of turbot was identified by the analysis metabolic pathways and gene function that other fishes,respectively bile secretion,mineral absorption,calcium signaling and collecting tube acid secretion.There are also 16 pathways that salinity genes significant(P<0.05)enrichment.3.DESeq was used for analysis and the threshold value was padj<0.05.262 up-regulated genes and 506 down-regulated genes were obtained.In contrast to other fish salinity transcriptome data,59 genes related to turbot salinity were identified;This is not only related to Cl-genes,such as NACC2,PAT1,PRLR,etc.,but also Na+ related genes,such as NHE-3,SGLT1,ASBT and so on.There are genes associated with H2 O,such as: AQP1,AQP4,AQP8,CA etc.;and energy related genes,such as ATP6V1E1,NKA,etc.;and metabolism related genes,such as CHST6,INO1,ANXA11,IDH2,ECH1,ECH8 and so on.HSP70 gene was both associated with antigen processing and stress,also PRLR gene was associated with growth and osmotic pressure regulation.4.According to the function of genes and the results of previous studies,10 genes were selected for quantitative analysis,showed that 9 gene in low salinity(salinity 5)have high expression of stress in the kidney,besides PAT1 gene;the expression of 10 genes in the intestine was less,especially the expression of PAT1 gene,APOC1 gene,APOM gene,CALM gene,FABP6 gene,HMDH gene and other 6 genes in the intestines of quantity(P < 0.05)was significantly lower than other groups.In this paper,182225 Unigenes sequences in the transcriptome of turbot were searched by MISA software.81314 SSR loci were detected,and the frequency of SSR was 31.17%.The length of the 78247 EST-SSR was 12-20 bp,in which dinucleotide repeats SSR most,totaling 34783.A total of 335 repetitive motifs were detected,in which the AC/GT of single nucleotide A/T and dinucleotide were the most,accounting for 31.17% and 31.05% of the total SSR respectively.In 30 primers,a total of 3 pairs of EST-SSR primers showed individual polymorphism,polymorphism rate of 10%,the number of allele SSR was 2,the average effective number of alleles was 1.8141,the average heterozygosity was 0.5514,the average expected heterozygosity was 0.4486,polymorphism information content was 0.6897 0.6474 and 0.5713,with an average of 0.6361.In this study,151 microsatellite markers with better polymorphism were selected from the early days of our laboratory to study the salinity characteristics of turbot.Combined with Bulked Segregation Analysis method,preliminary screening of 4 microsatellite loci in normal salinity group and low salt stress group difference bands,then four microsatellite loci were identified and analyzed by SPSS software.One significant(P<0.01)related locus and 2 significant(P<0.05)related loci were obtained. |
PDF Full Text Request