The Expression Of TRβ Gene In Paralichthys Olivaceus Metamorphosis And Identification Of Target MiRNA

Paralichthys olivaceus is mainly distributed in the coastal areas of China and Japan and is a very important marine cultured fish in China.In the process of P.olivaceus metamorphosis,the morphology and physiology have undergone drastic changes at the same time,covering many biological changes.Thyroid hormone（TH）plays an important role in regulating the metamorphosis of P.olivaceus by binding to its receptors（TRs）.Proteins with TRs that bind to chromatin in the nucleus have α and β types and have high affinity for DNA recognition sites.TRs can be divided into different subtypes including TRαA,TRαB,and TRβ.Studies have shown that the expression of these three genes is elevated during the metamorphosis of P.olivaceus,and return to a lower level after metamorphosis,and TRβ is more abundant in the metamorphic peak than TRαA,TRαB.expression.Thus,thyroid hormone receptors,especially TRβ,play a crucial role in the metamorphosis of the P.olivaceus.At present,there are few reports about the mechanism of action of TRβ on the metamorphosis of P.olivaceus.MicroRNAs（miRNAs）are a class of endogenous non-coding singlestranded RNA molecules that are approximately 20-25 nt in length.In animals,they are generally 3’-untranslated regions of their target genes binding.Thus,it can effectively inhibit the synthesis of its transcripts,or lead to the gene silencing of degradation of target miRNAs.Then,during the metamorphosis development of P.olivaceus,is there any relationship between miRNAs and TRβ 3′-UTR? Currently unknown.To investigate the relationship between miRNAs and P.olivaceus TRβ 3′-UTR,we designed 3′-RACE-outer and 3′-RACE-inner primers according to the sequence of NCBI TRβ,and then used the 3′-RACE kit.Explained that,the amplified RACE PCR product was gel-recovered,ligated,transformed,cloned and sequenced,and the 3′-UTR cDNA sequence of the P.olivaceus TRβ gene was obtained.The full-length TRβ gene is 1740 bp,with a 5’-UTR of 115 bp,a 3’-UTR of 437 bp,and a CDS of 1188 bp,encoding 395 amino acids with a poly（A）tail at the 3’-end.The comparison of protein homologous sequences showed that the amino acid sequence of P.olivaceus TRβ is between 85% and 99% homologous to other species and has a ligand binding region LBD and a highly conserved DNA binding region DBD.Phylogenetic analysis of the TRβ gene revealed Epinephelus coioides（ABP62962.1）,labrus bergylta（XP₀20494287.1）and Acanthochromis polyacanthus（XP₀22047585.1）.Maylandia zebra（XP₀04561076.1）is closer to the evolutionary relationship.The above species are all located on the branch of fish and are therefore consistent with evolutionary theory.The highly conserved domain of the TRβ gene and its high homology with its species suggest that it is functionally conservative.Real-time PCR was used to detect the expression of the P.olivaceus TRβ gene.The results showed that the P.olivaceus TRβ gene was expressed in the adult tissues（brain,muscle,heart,kidney,gill,stomach,liver,intestine,and gonad）and metamorphosis-17 days posthatching up to 36 dph.TRβ is expressed in higher levels in the kidney and heart and is relatively low in muscle and gonads.The expression level gradually increased at 17dph-32 dph in the larval stage and peaked at 32 dph.The above conclusion is consistent with the theory that TRβ is involved in the regulation of it metamorphosis.Treatment of metamorphosis larvae with exogenous TH and TU was performed to detect the expression of TRβ in the P.olivaceus of metamorphosis larvae by Real-time PCR.The results showed that the TH group was in contrast to the control group.The expression level was higher than that in each period;in the TU group,the expression of TRβ was almost unchanged,and all of them were lower than the NC group.The results showed that TH can promote the expression of TRβ gene in the metamorphosis stage of P.olivaceus,while TU inhibits the expression of TRβ gene.The 3’-UTR sequence of TRβ gene was cloned from the total RNA by 3’-RACE method;then the PCR primers for the desired restriction sites SacI and XbaI required for the recombinant plasmid containing dualluciferase reporter gene were designed and synthesized according to the cloned 3′-UTR sequence of TRβ gene.After PCR amplification,the target 3’-UTR cDNA sequence of TRβ gene containing specific restriction enzyme site,was obtained.The obtained target gene fragment and pmiRGLO vector were digested by double enzymes.The ligation reaction was performed with T4 DNA Ligase and transformed into DH5α competent cells.The recombinant plasmid was extracted,and then double enzyme digestion,agarose gel electrophoresis and sequencing validation were performed.The results showed that the vector was successfully constructed and named as pmiRGLO-TRβ-3’-UTR.In this experiment,we also used the RNAhybrid bioinformatics website to predict the miRNAs that might interact with the target genes and select the most likely five miRNAs: pol-miR-125a;pol-miR-214;pol-miR-460-5p;pol;-miR-138;pol-miR-125a*.The recombinant plasmid and microRNA mimics were transfected into 293 T cells for culture and dual luciferase activity assay.The results showed that pol-miR-125 a,pol-miR-214,pol-miR-460-5p and pol-miR-138 all target miRNAs of P.olivaceus TRβ,while pol-miR-125a* is not a target miRNA of TRβ.In this study,stem-loop real time PCR was used to respectivel detect pol-miR-125 a,pol-miR-214,pol-miR-460-5p,and pol-miR-138 in normal,TH,and TU treatments of metamorphic larvae（20 dph and 28 dph）.The results showed that pol-miR-125 a expression was decreased in NC control group and TH treatment group,but no significant change was observed in TU group;pol-miR-214 expression was decreased in NC control group and TH treatment group.However,in the TU group,there was no significant change in the expression level;the expression level of pol-miR-138 was same to pol-miR-125 a and pol-miR-214;the expression level of pol-miR-460 was slightly decreased in the NC control group,but increased in the TH and TU treatment groups.In conclusion,the 3’-RACE method was used to clone the cDNA sequence of TRβ-3’-UTR and analyze its amino acid sequence.In addition,the expression of TRβ gene in the tissues and early developmental stages of P.olivaceus was quantitatively detected.And the effect of exogenous TH and TU on the TRβ gene;secondly,construct a dual luciferase reporter gene recombinant plasmid containing the target fragment;and the miRNAs that may interact with TRβ-3′-UTR were predicted using the RNAhybrid website;Dual luciferase reporter gene recombinant plasmids and miRNA mimics were transfected into cells,respectively,and analyzed by dual luciferase reporter instrument.Results showed that pol-miR-125 a,polmiR-214,pol-miR-460-5p and pol-miR-138 is a target miRNAs for P.olivaceus TRβ,while pol-miR-125a* is not a true target miRNA for TRβ.These results provide basic data for the further study of the function and mechanism of miRNAs and TRβ in the metamorphosis of P.olivaceus.