miR-192在尼罗罗非鱼应答碱度胁迫中的表达及靶基因

Expression of miR-192 in Nile tilapia in response to alkalinity stress and verification of target genes

采用实时荧光定量、生物信息学、双荧光素酶报告和体内miRNA抑制的方法,探究miR-192在尼罗罗非鱼(Oreochromis niloticus)应答碳酸盐碱度胁迫中的作用。结果如下:(1)实时荧光定量PCR实验表明,在急性碱度胁迫(6 g/L NaHCO3)尼罗罗非鱼6 h后,鳃组织中miR-192的表达显著下调,溶质转运蛋白基因(solute carriers16A7,SLC16A7)表达显著上调(P<0.05);(2)借助生物信息分析预测到SLC16A7可能是miR-192的靶基因;(3)利用双荧光素酶报告实验发现miR-192会与SLC16A7的3′UTR结合;(4)体内对miR-192抑制后,SLC16A7的表达显著上调(P<0.05)。证实了miR-192参与了尼罗罗非鱼应答碱度胁迫中的调控过程,SLC16A7是miR-192的直接靶基因,为探明miRNAs调控尼罗罗非鱼应答碱度胁迫的分子机制提供了一定的依据。

In order to explore the role of miR-192 in response to carbonate alkalinity stress in Nile tilapia,real-time PCR,bioinformatics,dual-luciferase reporter assay and miRNA inhibition in vivo were used.The results were as follows:(1)The real-time PCR experiments showed that the expression of miR-192 was significantly down-regulated,while solute carriers16 A7,SLC16 A7 was significantly up-regulated in gills of Nile tilapia after 6 hours acute alkalinity stress(6 g/L NaHCO3)(P<0.05);(2)Bioinformatics analysis revealed that SLC16 A7 might be the target gene of miR-192;(3)Dual-luciferase reporter assay showed that miR-192 directly regulated SLC16 A7 by targeting its 3′UTR;(4)Inhibition of miR-192 significantly increased the mRNA level of SLC16 A7 in vivo(P<0.05).In conclusion,miR-192 was involved in the adaptation of Nile tilapia to alkalinity stress,and SLC16 A7 was the direct target gene of miR-192.This study provides a basis for exploring the mechanism of miRNAs in the response to alkalinity stress in Nile tilapia.