| The Mohe tilapia "Guangfu No.1" was a hybrid between Oreochromis mossambicus(?)and Oreochromis hornorum(?),which has obvious heterosis compared with the parent in salinity and alkalinity tolerance.Based on the transcriptome sequencing,the expression profiles of miRNA and mRNA of the Mohe tilapia "Guangfu No.1" under alkali stress were analyzed in this study.The pairs of differentially expressed miRNA – mRNA were screened,and the expression characteristic in the Mohe tilapia and its parents were detected to understand the function of the differentially expressed miRNA-mRNA under alkaline stress.The results of this study have provided important reseach data for regulatory mechanism of significantly different expression miRNA and mRNA of tilapia under alkali stress and the molecular genetic mechanism of tilapia salt-alkalinity tolerance.1.Construction of miRNA expression profile and bioinformatics analysis of Mohe tilapia under alkaline stressThe gill tissues of Mohe tilapia were sampled for miRNA transcriptome sequence after 24 h of exposure to alkaline stress(concentration of 4 g/L)and to fresh water with alkalinity of 0%(the control group).The two groups with three replicates(each with 6fishes)were set up.The expression profile of Mohe tilapia miRNA under alkaline stress was constructed,and the bioinformatics analysis was performed on the results of transcriptome sequencing.The results showed that 180,185,186 known miRNAs and790,392,515 new miRNAs were found in A4,A5,and A6,respectively.83 significant differentially-expressed miRNAs were found under alkaline stress,41 of which were up-regulated and 42 down-regulated.GO and KEGG enrichment analysis were conducted to predicte target genes of differentially expressed miRNAs.57 pathways were enriched,including MAPK signaling pathway(ko04010),mineral absorption(ko04978),mTOR signaling pathway(ko04150),TCA cycle(ko00020)andbiosynthesis of fatty acids(ko00061)and so on.Eighteen miRNAs were randomly selected from significant differentially-expressed miRNAs for qPCR verification.The transcriptome sequencing results were consistent with the qPCR results and could be used for subsequent experiments.In this experiment,a large number of differentially expressed miRNAs,target genes and osmoregulation pathways were found in the miRNA expression profiles of the gill tissues of Mohe tilapia under alkaline stress,which provides basic reseach information for detect the molecular regulation mechanism of teleost fish under osmotic stress.2.mRNA expression profile and bioinformatics analysis of Mohe tilapia under alkaline stressThe gill tissue of the hybrid tilapia were sampled for mRNA transcriptome sequence after 24 h of exposure to alkaline stress(concentration of 4 g/L)and to fresh water with alkalinity of 0%(the control group).The two groups with three replicates(each with 6 fishes)were set up.The expression profiles of hybrid tilapia mRNA under alkaline stress was constructed,and the bioinformatics analysis was performed on the sequencing results of transcriptome.The results showed that 3412 differentially expressed genes were found under alkaline stress,of which 1472 were significantly up-regulated and 1940 were down-regulated.Based on the analyses of the differentially expressed genes,the results showed that there were 35 GO projects were enriched,and51 pathways were enriched,including proximal tubule bicarbonate reabsorption(ko04964),TNF signaling pathway(ko04668),p53 signaling pathway(ko04115),and steroid biosynthetic pathway(ko00100).Sixteen genes were randomly selected from the significantly different expression genes for qPCR verification.The transcriptome sequencing results were consistent with the qPCR results and could be used for subsequent experiments.In this experiment,a large number of differentially expressed genes and pathways were found in the mRNA expression profile of the gill tissue of Mohe tilapia under alkaline stress.Some of these genes are associated with osmoregulation.This research provided basic data for studying osmoregulation genes and pathways under osmotic stress.3.The expression analysis of differential expression miRNA and possible target gene of Mohe tilapia and its parents under alkali stressBy integrative analysis of the miRNA and mRNA transcriptome sequencing under alkaline stress,185 pairs of significantly different expression miRNA-mRNA werefound.Four pairs of miRNA-mRNA were selected and the relative expression levels were analyzed in the gill,intestine,liver,kidney and muscle of Mohe tilapia and its parents.The results showed that miRNA-1824-atic,miRNA-356-cant1,miRNA-766ppar? and miRNA-1352-sdpt1-a were expressed in gill,intestine,liver and kidney,muscle tissues of Mohe tilapia and their parents.The expression trends of miRNAs and the target genes were all showed opposite expression trends.By analyzing the function of the genes and comparing the expression of miRNAs and their target genes in Mohe tilapia and it parents,miRNA-1824-atic,miRNA-356-cant1,miRNA-766-ppar? and miRNA-1352-sdpt1-a may be involved in the osmoregulation of tilapia. |
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