| Paralichthys olivaceus is a typical metamorphic model organism of fishes.It needs to undergo a dramatic metamorphosis process during its growth,and its right eye moves to the left and its lifestyle changes from a planktonic type to a benthic type,resulting in the visual photosensitive system of the Paralichthys olivaceus has changed.Thyroid Hormone(TH)can induce and accelerate the abnormal process of Paralichthys olivaceus larvae by binding to the Thyroid Hormone Receptor(TR).Among them,PDE6 s is specifically expressed in the eye and plays an important role in the light transduction cascade.The inhibitory subunit and catalytic subunit of cone cells were encoded by pde6 h and pde6 c gene,respectively,and the inhibitory subunit of rod cells was encoded by pde6 g gene.In addition,the upstream gene gnat2 of PDE6 s is the first step to mediate light transduction,and the downstream gene cngb3 of PDE6s is the last step that mediates light transmission.In addition,MicroRNA(miRNA)is important regulatory factors of the metamorphosis process of flounder to act on the 3’UTR region of the target gene for post-transcriptional regulation,and are regulated by thyroid hormones.Therefore,5 target genes of visual transduction pathway as research objects of this study,TH and miRNA as regulatory factors,and used RT-qPCR technology and dual luciferase experiment to initially reveal the interaction of TH,visual conduction cascade,and miRNA in the visual transduction cascade in the metamorphosis of Paralichthys olivaceus.It lays a foundation for further study of fish visual transduction system The research content is as follows:1.The expression of visual transmission related genes in Paralichthys olivaceus tissues and metamorphosisRT-qPCR technology was used to detect the expression levels of visual conduction-related genes in adult Paralichthys olivaceus tissues,larval metamorphosis stages,TH and TU treatment groups and rescue groups.The results of the tissue expression profile showed that the five genes(pde6h,pde6 c,pde6g,gnat2 and cngb3)were all significantly and highly expressed in the eyes,which indicated that these genes played an important role in vision.The results of the metamorphosis period showed that the expression levels of pde6 h,pde6c,pde6 g and gnat2 genes were significantly up-regulated in the late metamorphosis stage of Paralichthys olivaceus,while the expression levels of cngb3 were significantly down-regulated during the metamorphosis process.The results of TH and TU treatment group showed that the five genes were irregularlyup-regulated or down-regulated under the action of TH and TU.However,TH had significant positive regulation on pde6 h,pde6c,pde6 g during the whole process of metamorphosis,as well as gnat2 and cngb3 in the middle and late stages of metamorphosis.However,TU has significant negative regulation effects on pde6 h,pde6c,pde6 g in the whole metamorphosis stage and on gnat2 in the late metamorphosis stage.The experimental results of the rescue group showed that the Paralichthys olivaceus larvae in the TU+TH and TU+NC groups can be rescued to continue the metamorphosis process;In addition,the expression levels of these 5genes in the TU+TH and TU+NC groups are all significantly up-regulated,indicating that TH has a regulatory effect on these 5 genes.2.Verification of the regulatory relationship between TRαA and TRβ receptors,and gnat2 on PDE6sAnalysis of the promoter regions of pde6 h,pde6c,and pde6 g,according to the TRE site sequence,showed that the promoter region of the pde6 h gene contains 1DR4 type TRE site and 5 half-TRE sites;The promoter region of the pde6 c gene contains two T3 R transcription factor binding sites and one half-TRE site;the promoter region of pde6 g gene contains four half-TRE sites.The promoter sequences containing the TRE sites of the three genes were constructed on the p GL3-basic vector,and transferred to the 293 T cells with the internal reference plasmid and p3 x Flag-TRαA or p3 x Flag-TRβ.Exogenous T3 was added,and the results of the dual luciferase report showed that: T3 can positively regulate pde6 h and pde6 c to initiate transcription activity through TRαA,and can negatively regulate pde6 g to initiate transcription activity through TRβ.When T3 acted on TRαa and TRβ receptors,the activation transcriptional activity of pde6 c and pde6 g was higher than that of TRαA receptor,and the activation transcriptional activity of pde6 c was significantly lower than that of TRβ receptor.These results indicated that pde6 h,pde6c and pde6 g were all target genes of T3.In addition,when the gnat2 gene was constructed on pc DNA3.1and co-transformed with the promoters of the three genes,the results showed that gnat2 had a positive regulation effect on pde6 h and pde6 c,but do not regulation on pde6 g.3.Verification of the targeting relationship between visual transmission-related genes and miRNAUsing the target gene online prediction software to do prediction of target binding sites to the 3’UTR regions of pde6 h,pde6c,pde6 g,gnat2,and cngb3 genes and miR-21/miR-124/miR-193/miR-199/let-7e/let-7i according to the prediction principle,the results showed: pde6 h and pde6 c each have binding sites with 6miRNAs;pde6g has binding sites with miR-21,miR-193,let-7e and let-7.5;gnat2have binding sites with miR-199,let-7e and let-7i;cngb3 has binding sites with miR-193,let-7e and let-7i respectively.Then,according to the predicted results,the3’UTR region containing the binding site was constructed on the pmir-GLO and p EGFP-C2 vector co-transfected into 293 T cells with miRNA,respectively.The results of the dual luciferase report showed: pde6 h has a targeting relationship with miR-124,miR-193,miR-199 and let-7e,respectively.pde6 c has a targeting relationship with miR-124,let-7e and let-7i,respectively.gnat2 only has a targeting relationship with miR-199,pde6 g and cngb3 did not target miRNAs with predicted binding sites.Further according to the results of the expression of green fluorescent protein: mRNAs with relatively weak negative regulatory effects were further verified through the mutation binding sites,and the results showed that they had a real targeting relationship,consistent with the results of green fluorescent screening.In addition,RT-qPCR results showed that miRNA-124 and miRNA-193 were significantly expressed in the eye and brain tissues,and miRNA-199 was significantly expressed in the kidney,stomach and eyes,compared with other tissues.These results indicate that these three miRNAs play an important role in vision.This study mainly used the RT-qPCR method to analyze the expression levels of5 genes related to visual transduction in Paralichthys olivaceus tissues,developmental stages,TH groups,TU groups and rescue groups,and the dual luciferase experiment verified that T3 regulates PDE6 s through TRαA and TRβ receptors before transcription,and also verified the regulation of PDE6 s by gnat2,then miRNAs post-transcriptionally regulate target genes.The dual luciferase report experiment was finally used to explore targeting relationship between 6 miRNAs related to vision and5 genes related to visual transduction.The interaction among TH,visual transduction genes and miRNA in the visual conduction cascade reaction of Paralichthys olivaceus during metamorphosis was preliminarily explored,and deeply understand the mechanism of change of the visual photosensitive system of Paralichthys olivaceus during metamorphosis. |
PDF Full Text Request