Molecular Network Study Of TH Regulating Photosensitive System Of Japanese Flounder Larval

The Japanese flounder(Paralichthys olivaceus)is an important economic marine fish in China.Its typical post-embryonic metamorphosis is an ideal model for studying the mechanism of metamorphosis.During metamorphosis,the right eye migrates to the left side of the fish and the habits of life change from planktonic to benthic life,causing changes in its photoreceptor system to adapt to ecological changes.Thyroid hormone(TH)plays a key role in the regulation of larvae metamorphosis,and accelerates the metamorphosis of larvae through thyroid hormone receptor(TR).In addition,microRNAs(miRNAs)are also important regulators in the metamorphosis and are regulated by thyroid hormones.miR-124/96/182/183 is a key regulator in the development of vertebrate retina.The opsin gene(Opsin)mainly includes Rhodopsin,Red opsin,Green opsin,Blue opsin and UV opsin.It is a class of transmembrane protein with 350 amino acid.It mainly forms visual pigment with chromophore.The adaptation of fish to the external light environment creates an important way for organisms to receive external information.In order to preliminarily explore how TH,miRNAs and opsin regulate the development of their photoreceptor system during larvae metamorphosis.In this study,the expression level of opsin genes during the early development of larvae was detected by RT-PCR,and feed opsin genes regulated by TH.Then the bioinformatics method was used to predict the action elements of the opsin promoter regulatory region and TRE binding site,and the reporter gene vector and TR?A expression vector were co-transfected into 293 T cells to verify whether they were regulated by TR?A.Finally,the miRNAs target gene verification experiment was used to verify the post-transcriptional regulation level of miRNAs on the opsin gene.The interactions of TH,opsin and miRNAs in the development of larvae photoreceptor system were revealed,which provided basic data for further study on the regulation of larvae metamorphosis.1.Analysis of the expression difference of opsin genes in the process of larvae metamorphosisIn this experiment,larvae was used as experimental material,and the expression of larvae optic protein gene in adult tissues,metamorphosis and development,and the effects of thyroid hormone and thiourea on its expression level were detected by RT-PCR.The results showed that the opsin genes was expressed in different levels in adult tissues,but all were highly expressed in eye tissues.The five opsin genes showed different expression patterns during metamorphosis.The expression levels of Rhodopsin,Red opsin and UV opsin increased gradually from 17 dph and peaked at 28 dph,and gradually decreased at the late metamorphosis.The expression of Green opsin was abnormal.At the early 24 dph,the expression level decreased with the metamorphosis.The expression of Blue opsin gradually increased from 17 dph until the 32 dph reached the highest value in the late metamorphosis,and then gradually decreased and the expression level of 41 dph metamorphosis was still significantly higher than the initial stage of metamorphosis.The results of TH and TU treatment of metamorphosis larvae showed that under the action of TH and TU,Rhodopsin,Green opsin and UV opsin were up-regulated or inhibited in different degrees,but the regulation of exogenous TH on Red opsin and Blue ospin Significantly,it has significant positive regulation,while TU has obvious inhibitory effect on Red and Blue opsin.The rescue effect of TH on Red opsin and Blue ospin was further verified by rescue experiments.The results showed that TU group larvae completed metamorphosis after Red opsin The expression level decreased and the expression of Blue opsin increased,indicating that TH can regulate the expression of Red opsin and Blue opsin.2.Verification of the relationship between TR?A receptor and red opsin and blue opsin geneIn this experiment,bioinformatics methods were used to predict the elements of the promoter regions of Red opsin and Blue opsin,and the binding site of transcription factor TR,the thyroid hormone response element(TRE),to clone the opsin gene by PCR amplification.The sub-regulatory region sequences were ligated into the promoter-free pGL3-basic vector to construct pGL3-basic-Red opsin and pGL3-basic-Blue opsin recombinant plasmids,and co-transformed with the internal reference plasmid pRL-TK and p3×flag-TR?A expression vector.293 T cells were stained and their luciferase activity was measured.The results showed that theluciferase activity of only the transfected recombinant plasmid and the internal reference plasmid was higher than that of the empty vector transfection group,indicating that they all had promoter activity.The luciferase activities of the pGL3-basic-Red opsin,pGL3-basic-Blue opsin and overexpression vector p3×flag-TR?A co-transfected groups were 1.25-fold and 1.95-fold,respectively,of the control group.To further verify whether TR?A can promote the transcription of Red opsin and Blue opsin under T3 stimulation,T3 solution with a final concentration of75 nM was exogenously added during the transfection of 239 T and the experimental control group was set up.The results showed that under the stimulation of T3,the luciferase activities of pGL3-basic-Red opsin and pGL3-basic-Blue opsin and the overexpression vector p3×flag-TR?A were significantly higher;Compared with the T3 group,the activity increased by 0.4 times and 2 times,respectively.It indicated that Red opsin and Blue opsin are regulated by TR?A,but the regulation of Blue opsin is stronger,which may be through the thyroid hormone regulatory pathway of T3-TR?A-Blue opsin.3.Target gene prediction and verification of miR-124/96/183/182In this experiment,the regulation of miRNAs on the post-transcriptional level of photoreceptor-related genes was verified by the method of target gene verification.Firstly,the target gene on-line prediction website was used to predict the target genes of miR-124/96/183/182,and the genes related to photoreceptor development,such as Red opsin,Blue opsin and Otx2,were cloned,and the 3'UTR region sequences of these three genes were cloned.The recombinant plasmid was constructed by ligating the pmir-GLO dual luciferase reporter gene vector and transfected into 293 T cells to detect luciferase activity.The results showed that pol-miR-124 can target the negative regulation of the expression of Blue opsin and Otx2 genes.pol-miR-182 targets the negative regulation of the Blue opsin gene.This indicates that pol-miR-124/182 can regulate post-transcriptional regulation of Blue opsin and Otx2.In conclusion,this study analyzed the expression profiles of five opsins in various tissues and developmental stages of larvae,and the effects of exogenous thyroid hormones on optic protein in the period of larvae metamorphosis;further verified by promoter reporter gene experiments in T3 under the action,Red opsin and Blue opsin were regulated by TR?A.Finally,the target gene Blue opsin,Otx2 and pol-miR-182 target gene Blue opsin of pol-miR-124 were tested by target gene verification,and THand opsin were preliminarily explored.And the interaction of miRNAs in the development of larvae photoreceptor system,providing new basic data for the development mechanism of larvae metamorphosis.