| The Japanese flounder(Paralichthys olivaceus)is a typical model for studying the metamorphosis of fish.During metamorphosis,its right eye shifts to the left and its lifestyle transitions from planktonic to benthic.Therefore,the retinal photoreceptor system of flounder has also undergone adaptive changes.Thyroid hormone(TH)acts mainly through the thyroid hormone receptor(TR).Exogenous TH can promote the metamorphosis process and shorten the metamorphosis time in flounder,while exogenous thiourea(TU)slows down the metamorphosis process.Under the action of genes related to photoreceptor differentiation,photoreceptor precursor cells(PPC)can differentiate into rod cells and cone cells,which can synthesize rod opsin and cone opsin,respectively.The neural retina leucine zipper protein(nrl)and photoreceptor-specific nuclear receptor(nr2e3)promote PPC differentiation into rod cells.while growth differentiation factor 6a(gdf6a)and T-box transcription factor 2b(tbx2b)are key factors specifying the fate of blue and UV cone photoreceptors,respectively.Studies have found that TH can affect the expression of the opsin gene,and photoreceptor cells can synthesize opsin,so does exogenous TH affect the expression of the opsin gene by regulating photoreceptor cell differentiation? To test this hypothesis,we investigated the regulatory relationship between thyroid hormone-photoreceptor cell-opsin during the development of the photoreceptor system of Japanese flounder by molecular cell biology methods.The main results are as follows:1.Targeted regulation of opsin gene by thyroid hormone in flounderAn online website was used to predict the thyroid hormone response element(TRE)and TR transcription factor binding sites in the promoter regions of five opsin genes(rh1,sws2aβ,lws,rh2b-1 and sws1)of flounder.Subsequently,p3×Flag-TRαB over-expression plasmids and five opsin gene promoter region recombinant plasmids(p GL3-rh1/sws2aβ/lws/rh2b-1/sws1-pro)were constructed.The p3×Flag-TRαA,p3×Flag-TRαB and p3×Flag-TRβ over-expression plasmids were co-transfected with five recombinant plasmids of the opsin gene promoter region,respectively,in 293 T cells.In addition,T3 with a final concentration of 75 n M was set in the experiment.The dual luciferase reporter gene assay revealed that T3 and TRs positively regulate rh1,lws and sws2aβ genes,and that T3 may regulate sws2aβ gene expression through TRαA.The regulatory role of thyroid hormones on the opsin genes was confirmed.2.Regulation of gene expression related to photoreceptor differentiation in flounder during metamorphosisThe expression profiles of four genes related to photoreceptor cell differentiation(nrl,nr2e3,gdf6 a and tbx2b)were examined in flounder by q RT-PCR in the tissue expression profile of adult fish,the temporal expression in the metamorphic stage of the juvenile fish,the TH and TU treatment groups and the rescue group.The results showed that these four genes were the most highly expressed in the eye tissue of adult flounder and varied with the metamorphosis of flounder,indicating that these four genes are closely associated with the development of the photoreceptor system during the metamorphosis of flounder.The nrl and nr2e3 genes peaked at 36 dph,and the gdf6 a and tbx2 b genes peaked at 28 dph and 32 dph,respectively.After exogenous TH and TU treatment,nrl gene expression was positively regulated by TH and significantly reduced at 36 dph of TU treatment;nr2e3 gene was irregularly promoted and repressed by TH and TU;gdf6a gene was promoted by TH at the early stage of metamorphosis and repressed by TH at the late stage of metamorphosis,while TU had a significant negative regulation on gdf6 a gene;tbx2b gene was promoted by TH at the early and late stage of metamorphosis and irregularly promoted and repressed by TU.In addition,the expression of gdf6 a and tbx2 b genes were both upregulated in the rescue group,while the expression of nrl and nr2e3 genes were not significantly different.The above results indicated that nrl,nr2e3,gdf6 a and tbx2 b genes were affected by TH to varying degrees.3.Targeted regulation of photoreceptor differentiation related genes by thyroid hormoneWe preliminarily predicted the regulatory relationship between TH and four photoreceptor differentiation related genes(nrl,nr2e3,gdf6 a and tbx2b)by analyzing the TRE and TR transcription factor binding sites on the promoters of the four genes.Then,four photoreceptor differentiation related genes promoter region recombinant plasmids(p GL3-nrl/nr2e3/gdf6a/tbx2b-pro)were constructed and co-transfected into293 T cells with p3×Flag-TRαA,p3×Flag-TRαB and p3×Flag-TRβ over-expression plasmids,respectively,and T3 with a final concentration of 75 n M was added.The dual luciferase results showed that TH could regulate the expression of these four genes,and T3 might regulate nrl,nr2e3 and tbx2 b through TRαA,while T3 might regulate the expression of gdf6 a through TRβ.It was further confirmed that TH regulates genes related to photoreceptor cell differentiation.Moreover,we constructed p EGFP-nrl and p EGFP-gdf6 a green fluorescent protein plasmids.293 T cells were co-transfected with p EGFP-nrl and p GL3-nr2e3-pro,as well as p EGFP-gdf6 a and p GL3-tbx2b-pro.The results showed that the luciferase activities of nr2e3 and tbx2 b genes were significantly increased,which verified the regulation effect of Nrl on nr2e3 and Gdf6 a on tbx2 b.4.Targeting regulation of photoreceptor cell differentiation related genes to opsin genesIn order to study the effect of photoreceptor cell differentiation related genes on opsin genes of flounder,a green fluorescent plasmid p EGFP-nrl/nr2e3/gdf6a/tbx2 b was constructed.Then p EGFP-nrl and p EGFP-nr2e3 were co-transfected into 293 T cells with p GL3-rh1-pro rod opsin gene recombinant plasmid,respectively.p EGFP-gdf6 a and p EGFP-tbx2 b were co-transfected into 293 T cells with p GL3-sws2aβ/lws/rh2b-1/sws1-pro cone opsin gene recombinant plasmids,respectively,and the activity of opsin gene promoter was detected by dual luciferase.In addition,nrl,nr2e3,gdf6 a and tbx2 b genes were over-expressed in the brain cells of flounder,and the changes of opsin genes were detected by q RT-PCR.The luciferase activity test results showed that Nrl and Nr2e3,Gdf6 a and Tbx2 b could positively regulate the expression of rod opsin and cone opsin genes,respectively.However,after over-expression of these four genes in the brain cells of flounder,rod and cone opsin genes are affected irregularly,and the specific mechanism needs further study.In conclusion,this study mainly analyzed the expression profiles of photoreceptor cell differentiation related genes in adult tissues of flounder,larval metamorphosis stage,TH and TU treatment groups and rescue groups by q RT-PCR method,and initially screened out TH-regulated photoreceptor cell differentiation related genes.The regulation of TH on opsin genes and photoreceptor cell differentiation related genes in flounder and the effect of photoreceptor cell differentiation related genes on opsin genes in flounder were detected by molecular cell biology methods.By exploring the regulatory relationship between thyroid hormone,photoreceptor cell and opsin during the photoreceptor phylogeny of flounder,the influence of thyroid hormone on the photoreceptor phylogeny of flounder retina was initially revealed,which provided data reference for further research on the molecular mechanism of the photoreceptor development of flounder. |
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