Analysis Of MiRNA Expression And Identification Target Genes In Nile Tilapia (Oreochromis Niloticus) Under Alkali Stress

China has a large amount of saline-alkali water resources which are difficult to develop and utilize.The Nile tilapia(Oreochromis niloticus)is a euryhaline economic species with the characteristics of fast growth and strong resistance.It is beneficial to the development of saline-alkali water resources and the restoration of ecological environment to carry out the breeding of saline-alkali tolerant Nile tilapia.Study on the mechanism of adaptation to saline-alkali environment in fish can provide theoretical basis for breeding practice.This study performed high-throughput sequencing and bioinformatics analysis of miRNAs in gill tissue of Nile tilapia at different alkalinities;Real-time PCR was used to analyze the expression of 7 randomly selected representative miRNAs and target genes;The dual luciferase reporting experiment and injection experiment in vivo were used to verify the targeting relationship between miR-192 and SLC16A7.The main results are as follows:(1)Two alkalinity groups(AW4,4g/L Na HCO3;AW6,6g/L Na HCO3)and one fresh water group(FW)were prepared.After 72 hours of acute alkali stress,gill tissues were collected for high-throughput sequencing and bioinformatics analysis.A total of 276 known miRNA were found including 42 sustained up-regulated miRNAs and 21 down-regulated miRNAs with increasing of alkalinity(P < 0.05).In addition,there are miRNAs which differ in expression with increasing of alkalinity,but did not change continuously.4 sustained up/down-regulated miRNAs(miR-24 a,miR-30 d,miR-143,miR-155)and 3 non-sustained changed miRNAs(miR-122,miR-192 and miR-194a)were randomly selected for target gene prediction and KEGG enrichment analysis.It was found that calcium signaling pathway and MAPK signaling pathways,etc.were significantly enriched.(2)Acute alkali stress experiments(AW4,AW6)were performed.The expression of 7 miRNAs(miR-24 a,miR-30 d,miR-143,miR-155,miR-122,miR-192)and six predicted target genes(slc20a1a,nme3,SLC16A7,them4,SLC7A1,igfbp5a)were analyzed within 96 hours.The results showed that the expression of 7 miRNAs was consistent with the sequencing results,and 2 possible targeting relationships(miR-122 and slc20a1 a,miR-192 and SLC16A7)were found.(3)miR-192 and SLC16A7 was selected to further verify the targeting relationship.The double luciferase reporting experiment showed that miR-192 inhibited the expression of SLC16A7 by binding to the 3’UTR of SLC16A7.Injection of miR-192 antagomir reduced the expression of miR-192 and increased the expression of SLC16A7 in vivo.These results indicated that miR-192 and SLC16A7 were involved in the process of tilapia in response to alkali stress.