| As an euryhaline marine teleost,the cobia,Rachycentron canadum,has the ability to tolerate the 22.5-44.5 ‰ of seawater salinity and grow healthily.Studies have shown that miRNAs are involved in the regulation of osmotic pressure in teleost,but there is no report on how cobia miRNAs participate in the regulation of osmotic pressure.In this study,differentially expressed miRNA and target genes were obtained by identifying the miRNA expression profile of cobia gill tissue under low salt adaptation,and at the same time,combined with the preliminary transcriptome data of the research group,the negatively regulated miRNA-m RNA was screened.It was verified by the dual-luciferase report experiment,and the salinity-related genes(NHE3,NKAα1a,PRLR1)were cloned and analyzed.Based on the whole-genome sequence information of cobia,the NHE and NKA gene families were identified and analyzed.The main findings are as follows:1.Identification and analysis of miRNA expression profile in cobia gill tissue under low salt adaptationCobia was adapted to 10‰ and 30‰ salinity for 4 weeks.The gill miRNA expression profile was detected by high-throughput sequencing,and 409 known miRNAs and 139 novel miRNAs were identified.According to the conditions(P-value <0.05,fold change> 2),56 differentially expressed miRNAs(DEM)were screened,of which 22 miRNAs were significantly up-regulated and 34 miRNAs were down-regulated.In addition,the functional annotations of DEMs target genes indicate that GO terms are mainly related to metabolism,immune response and ion transport,and the top 20 KEGG Pathway includes prolactin signaling pathway,tryptophan metabolism,fatty acid degradation,etc.Eight miRNAs(5 known miRNAs and 3novel miRNAs)were randomly selected for q PCR verification.The results of comparison analysis showed that eight miRNAs were basically consistent with the results of small RNA sequencing data.2.Dual-luciferase was used to verify the target relationship of miRNA-m RNAAccording to the existing transcriptome data of the research team,through conjoint analysis of miRNA expression profiles,the differentially expressed miRNA-m RNA target gene relationship pairs were screened,and two sets of relationship pairs(mi R-1335-3p and NHE3,mi R-1788-3p and NKAα1a)were selected.According to the target gene binding site,the target gene 3’-UTR was synthesized,construct the corresponding wild-type(NHE3-pmir GLO-WT and NKAα1a-pmir GLO-WT)and mutant(NHE3-pmir GLO-MUT and NKAα1a-pmir GLO-MUT)recombination plasmid,use dual-luciferase reporter experiment to test the functional relationship between the miRNA and target gene.The experimental results showed that after NHE3-pmir GLO-WT and NKAα1a-pmir GLO-WT were co-transfected with mi R-1335-3p and mi R-1788-3p,respectively,the relative luciferase activity was significantly reduced,while the mutant plasmid co-transfected results show that there is no significant difference in relative luciferase activity,indicating that there is indeed a negative regulatory relationship between mi R-1335-3p and NHE3,as well as mi R-1788-3p and NKAα1a.3.Cloning and expression analysis of NHE3,NKAα1a,PRLR1Based on the DEM target genes,the salinity-related genes NHE3,NKAα1a and PRLR1 were selected for full-length c DNA cloning,sequence comparison,phylogenetic analysis and q RT-PCR detection.The results showed that the full-length c DNAs of NHE3,NKAα1a and PRLR1 were 3579,3526,2629 bp,the ORF were2718,3075,1953 bp,and the amino acid lengths were 905,1024,and 650 aa,respectively.The results of q RT-PCR showed that NHE3,NKAα1a and PRLR1 genes were expressed in 9 tissues including gill,intestine and heart,among which the highest abundant gene expression of tissue was gill.With the decrease of salinity,NHE3 was significantly up-regulated in the gill,and the intestine were significantly up-regulated after low-and high-salinity adaption,while the kidney and the intestine changed in the opposite trend,which was a significant down-regulation.NKAα1a was significantly up-regulated in the gill and intestine after being stimulated by low-and high-salinity adaption,while it was significantly down-regulated in kidney after being stimulated by high-salinity adaption.The expression abundance of PRLR1 in the gill,intestine and kidney is inversely proportional to the salinity of the water body.4.Identification and analysis of NHE and NKA gene familiesCombining cobia whole-genome sequence information and biological information analysis software to identifiy the members of NHE and NKA gene family,and the members of cobia NHE and NKA gene family were successfully obtained.Using the MEME website and TBtools software to analyze the gene structure,the results showed that NHE and NKA contained 10 different motifs,and the motifs were arranged regularly in genes.q RT-PCR detects the expression patterns of NHE and NKA gene families.NHE and NKA are widely present in 9 tissues including gills,brain,and heart.The high-expressing tissues of NHE are gill,kidney and brain,while NKA is gill,brain,and heart.The results of salinity adaptation showed that NHE1 was significantly up-regulated in the intestine after being stimulated by high-and low-salinity adaption,and the kidney was directly proportional to the change in salinity.With the increase in salinity,NHE2 c showed a significant downward trend in the gill,intestine and kidney.After low-salinity adaptation,NKAα1b and NKAβ3a were significantly down-regulated in the gill,NKAα3a and NKAβ3a were significantly up-regulated and down-regulated in the intestine,while NKAα1b,NKAα3a and NKAβ3a were all significantly up-regulated in the body and kidney.After high-salinity adaptation,the changing trend of NKAα1b and NKAβ3a in the gill was consistent with that of low-salinity adaptation,while NKAα3a and NKAβ1b were significantly up-regulated. |
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