| Rainbow trout(Oncorhynchus mykiss)is a typical cold-water fish,which has poor tolerance to higher water temperature.With the global warming,the impact of heat stress on rainbow trout is becoming more and more serious.Identifying the related genes of rainbow trout in response to high-temperature stimulation,and understanding the response strategy and regulation mechanism of heat stress of different genes expression level,can provide theoretical basis for the high-temperature genetic breeding and improvement of rainbow trout,and to explore various measures for rainbow trout to resist high temperature.MicroRNA is a recently discovered small molecule RNA that regulates the post-transcriptional regulation of genes.At present,the studies on miRNA in rainbow trout under heat stress have not been reported.Therefore,in this study,we selected rainbow trout from full-sibling inbreeding families as experimental materials.The liver and head kidney of three female fish from control group(18℃)and heat treatment group(24℃)were used to systematically analyze the miRNAs in the liver and head kidney of rainbow trout using llumina HiSeq 2500 sequencing platform and bioinformatics methods,and to reveal novel specific miRNAs in rainbow trout;Then,from the liver and head kidney RNA-seq and small RNA-seq results screen the stability express mRNAs(liver 5,head kidney 5)and miRNAs(liver 4,head kidney 5)respectively.Through geNorm NormFinder,BestKeeper and Comparative ΔCt four methods to evaluate these genes and four commonly used reference and a small non-coding RNA(U6)expression of stability,select under heat stress of liver and head kidney reference mRNA and reference miRNA,subsequently,small RNA-seq results were verified by RT-qPCR with the screened reference miRNA.Meanwhile,in order to study the regulation effect of miRNA on rainbow trout under heat stress,two miRMAs(ssa-miR-301a-3p and ssa-miR-7132b-5p)with differential expression was screened out from the results of small RNA-seq in this study.Bioinformatics software was used to predict their target genes,and dual-luciferase reporter gene detection system was used to verify their targeting relationship.The main results are as follows:1.Liver small RNA-seq results showed that 88,452,735 raw reads were obtained from six small RNA libraries.After removing low-quality reads,5’and 3’ joints,84,894,376 clean reads were obtained for subsequent analysis.The length of small RNA sequence in liver mainly distributes in the range of 21-23 nt,of which 22 nt sequence distributes most.A total of 363 conserved miRNAs and 636 novel miRNAs were detected.A total of 39 miRNAs were differentially expressed in response to heat stress,of which 20 were up-regulated and 19 were down-regulated.A total of 65 miRNAs-target gene negative correlation interaction sites were identified,including 18 up target gene-down-regulated miRNAs and 47 down target gene-up-regulated miRNAs interacting sites.GO and KEGG analysis showed that potential target genes were mainly involved in DNA replication and protein folding,mainly enriched protein processing in endoplasmic reticulum,NOD-like receptor signaling pathway,progesterone-mediated oocyte maturation and other pathways.2.Head kidney small RNA-seq results showed that 85,873,242 raw reads were obtained from six small RNA libraries.After removing low quality reads,5’joints and 3’ joints,82,295,067 clean reads were obtained for subsequent analysis.The length of small RNA sequence in head and kidney is mainly distributed in the range of 21-23 nt,of which 22 nt sequence is the most widely distributed.392 conserved miRNAs and 989 novel miRNAs were detected;79 miRNAs were differentially expressed in response to heat stress,of which 41 were up-regulated and 37 were down-regulated.;A total of 393 miRNAs-target gene negative correlation interaction sites were identified,of which 217 were up target gene-down-regulated and 176 were down target gene up-regulated.GO and KEGG analysis showed that potential target genes were mainly involved in protein folding,protein processing and metabolism,mainly concentrated protein processing in endoplasmic reticulum,NOD-like receptor signaling pathway and phagosome pathway.3.Through geNorm NormFinder,BestKeeper and Comparative ΔCt four methods to screen the reference mRNA and reference miRNA of rainbow trout liver and head kidney under heat stress,the results showed: β-actin and EF1-α are the most stable expression of reference mRNA in liver and head kidney respectively.Ssa-miR-26a-5p and ssa-miR-462b-5p are the most stable expression of reference miRNA in liver and head kidney respectively.Subsequently,RT-qPCR was performed to verify the differentially expressed miRNAs(8 in liver and 8 in head kidney)in the previous small RNA-seq results by screening the reference miRNA,and the results of RT-qPCR were found to be consistent with the results of small RNA-seq,confirming the reliability of the results of small RNA-seq.4.The target genes of ssa-miR-301a-3p and ssa-miR-7132b-5p(hsp90b2 and HERC4)were predicted by biological software.Hsp90b2 3′-UTR and HERC4 3′-UTR were cloned successfully,and recombinant vectors of wild type(hsp90b2 WT and HERC4 WT)and their mutants(hsp90b2 MUT and HERC4 MUT)were constructed for detection of miRNA targets.The dual-luciferase reporter gene detection system found that after 48 h of transfection with HEK 293 T cells,when the hsp90b2-WT was co-transfected,the fluorescence activity of the ssa-miR-301a-3p mimics group was significantly down-regulated compared with the mimics NC group(P<0.05);When co-transfected with hsp90b2-MUT,there was no significant difference in luciferase activity between mimics NC group and ssa-mi-301a-3p mimimics group.(P>0.05)。The results showed that ssa-miR-301a-3p could complement the 3′-UTR of hsp90b2,and there was a regulatory relationship between them.After transfection of HEK 293 T cells for 48 h,when the HERC4-WT or the HERC4-MUT were co-transfected,there was no significant difference in luciferase activity between the ssa-miR-7132b-5p mimics group and the mimics NC group(P>0.05),suggesting that ssa-miR-7132b-5p could not be targeted to the 3′-UTR region of the target gene HERC4,and there was no direct targeting relationship between them. |
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