| Rainbow trout（Oncorhynchus mykiss）is a typical cold-water fish,which has low tolerance and high sensitivity to high temperature.With the intensive and large-scale development of rainbow trout farming industry,the survival challenges faced by rainbow trout in summer are becoming increasingly severe and it is easy to die or reduction of production in heat stress.In our group’s previous study on high-throughput sequencing of mi RNAs that were differentially expressed before and after heat stress of rainbow trout（18°C and 24°C）,it was found that there were two novel miRNAs in the head kidney tissues of rainbow trout,before and after the heat stress.The differential expression was extremely significant(**P<0.01),tentatively named novel-434 and novel-242,the target genes were predicted and analyzed.It was found that novel-434 and novel-242 negatively regulate important target genes such as HSPA4L and CRT by miRanda,respectively.HSPA4L is a target gene for novel-434 to be validated.CRT serves as a target gene for novel-242 to be validated.Its dual-luciferase reporter system is used to verify its target relationship,and Gene Ontology（GO）annotation and Kyoto Encyclopedia of Genes and Genomes are used.（KEGG）analysis analyzed its regulation mechanism.The specific test results and conclusions as follows:1.Successfully cloned pmirGLO-HSPA4L 3’-UTR and pmirGLO-CRT 3’-UTR and their mutation vectors pmirGLO-mut-HSPA4L 3’-UTR and pmirGLO-mut-CRT 3’-UTR,and determined a better transition the Transfection system provides the basis for further cell experiments.After transfection of 48 h,compared with the mimics NC group in transfection with the recombinant plasmid pmirGLO-HSPA4L 3’-UTR,the luciferase activity in the novel-434 mimics group was significantly lower than that in the mimics NC group(5.69 fold,**P<0.01);The transfection of the recombinant vector plasmid pmirGLO-mut-HSPA4L3’-UTR was not significantly different from the mimics NC group and the novel-434 mimics group（P>0.05）;in the transfection of the recombinant vector plasmid pmirGLO-novel-434experiments,compared with mimics NC group,the luciferase activity in novel-434 mimics group was down-regulated and the difference was extremely significant(**P<0.01).The above experimental results showed that miRNA:novel-434 has a targeting relationship with HSPA4L gene,and novell-434 can be targeted and controlled by way of partial sequence complementation with the 3’-UTR region of target gene HSPA4L.Kinase-associated miRNAs are involved in the regulation of the development of rainbow trout-induced heat stress through post-transcriptional inhibition of the heat shock protein gene HSPA4L.2.After transfection of 48 h,the activity of luciferase in the mimics NC group and novel-242 mimics group was not significantly different（P>0.05）in transfection of the recombinant vector plasmid pmirGLO-CRT 3’-UTR;In the experiment of plasmid pmirGLO-mut-CRT 3’-UTR,the difference between mimics NC group and novel-242-mimics group was not significant（P>0.05）;in the experiment of transfecting the recombinant vector plasmid pmirGLO-novel-242,with mimics compared with the NC group,the pmirGLO-novel-242 luciferase activity decreased significantly(**P<0.01),indicating that novel-242 could not be targeted to the 3’-UTR region of the target gene CRT predicted by bioinformatics.3.novel-434 is involved in the regulation of the heat stress process of rainbow trout by targeting the HSPA4L.GO analysis show that HSPA4L is involved in 11 biological processes such as cell composition and synthesis,development and cell metabolism;KEGG enrichment analysis shows that HSPA4L is enriched in the endoplasmic reticulum associated degradation pathway of the endoplasmic reticulum protein processing pathway,thereby promoting the degradation of damaged proteins and participating in the regulation of adaptive protection under heat stress in the body. |
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