Selection And Verification Of Target Genes Regulated By Thyroid Hormone Of Paralichthysolivaceus

The Japanese flounder(Paralichthysolivaceus)is an ideal model for studying the metamorphosis of fish.Thyroid hormone(TH)plays a decisive role in the development of flounder.The physiological effects of thyroid hormones are mainly exerted through Thyroid Hormone Receptors(TRs).There are two different types of TRs: TRα and TRβ.TR regulates the transcription of target genes by binding to the specific DNA sequence of thyroid hormone response element(TRE)-AGGT(C/A)A.In this experiment,P olivaceus was used as a research animal.This study aimed to use a new method to find the target genes directly regulated by TH.We expressed and purified the p3×Flag-TRαA and p3×Flag-TRβ fusion proteins to find target genes directly regulated downstream of TH by using a cyclic amplification and selection of target method(CAST).After comparing the genome of P olivaceus,target genes were identified by double luciferase report experiment and real-time quantitative PCR experiment.The main results are as follows:1.Construction of TRαA and TRβ eukaryotic expression vectors,expression and purification of Fusion ProteinsIn this section,the p3×Flag-TRαA and p3×Flag-TRβ recombinant eukaryotic expression vectors take were taken to transiently transfected HEK293 T cells after TA clones verified that the the CDS region of TRαA,TRβ genes were correct.The lysate containing the total protein were purified by G1 affinity chromatography column and BCA method was used to detect protein concentration.Western Blot test showed that p3×Flag-TRαA and p3×Flag-TRβ fusion proteins were successfully expressed in mammalian protein expression systems.2.Selection of target genes regulated by thyroid hormoneIn this section,two complementary single-stranded random sequences with specific amplification primers at both ends and random sequence in the middle were synthesized.The double-stranded random identify library synthesized by using the annealing synthesis system.Using the DNA-protein binding characteristics,the synthetic DNA double-strand random identify library was interacted with the p3×Flag-TRαA and p3×Flag-TRβ fusion proteins through the DNA-protein binding system.After six rounds of target gene cycle amplification and screening(CAST,Cyclic Amplification and Selection of Target),TA clonedthe final round of amplification products.We got 63 TRαA identify sequences and 59 TRβ identify sequences.3.Analysis and prediction of potential target genesThere are 12 specificidentify sequences containing TRE sequences in the TRαA identify library;13 specific identify sequences containing TRE sequences in the TRβ identify library;and four partially overlapping specific recognition sequences in the TRαA and TRβ recognition libraries.The identify sequence was compared with the promoter region of the P.olivaceus genome by using bioinformatics methods to select potential target genes whose promoter region can match the library identify sequence and contain the TRE sequence.Selection of the TRαA identify library found 12 potential target genes;selection of the TRβ identify library found 13 potential target genes.Compared the CDS regions of potential target genesthrough the NCBI(https://www.ncbi.nlm.nih.gov/)database,the TRαA recognition library selected 11 valid potential target genes;the TRβ recognition library screened 12 valid potential targets.Among them,TRαA and TRβ recognition libraries were screened to obtain four identical potential target genes: frizzled-3-like,TBC1 domain containing kinase(tbck),aminoacyl t RNA synthetase complex interacting multifunctional protein 1(aimp1),and MYC associated factor X(max).The remaining seven target genes screened from the TRαA recognition library are: mannose binding 2(lman2),RAB GTPase activating protein 1(rabgap1),adoption-enhancing nuclease-like,F-box and WD repeat domain containing 8(fbxw8),FAST kinase domain-containing protein 3 mitochondrial-like,tousled like kinase 2(tlk2),and family with sequence similarity 198 member A(fam198a).The remaining 8 target genes screened by the TRβ recognition library are: dual specificity protein phosphatase 7-like(dusp7),serine/threonine-protein kinase 26-like(stk26),syntaphilin-like,A disintegrin and metalloproteinase with thrombospondin motifs 10-like(admats-10),MAP kinase activating death domain(madd),teratocarcinoma-derived growth factor-like(tdgf),phosphordiesterase 4D(pde4d),and pleckstrin homology domain-containing family A member 5-like.4.Expression of potential target genesin different adult tissuesTRαA target genes,4 TRβ target genes,4 TRαA and TRβ target geneswereusedto detect geneexpression in different adult tissuesbyreal-time fluorescence quantitative PCR.The results showed that except for pde4 d and dusp7,which were not significantly expressed in brain tissues(P>0.05),other genes were significantly expressed in brain tissues(P<0.05).Heart and kidney tissues were significantly expressed(P<0.05)in TRαA potential target genes,TRβ potential target genes were also significantly expressed in heart and gill tissues(P<0.05),and TRαA and TRβ common potential target genes were only significantly expressed in brain tissues(P<0.05).5.Expression of potential target genesin different metamorphosis development stagesReal-time quantitative PCR was used to detect the expression of each gene at different metamorphosis development stages of P.olivaceus.The results show that all target genes can be roughly divided into two categories: the first type has a gradually increasing expression of 17-20 dph,the peak expression of 28 dph reaches a peak value,and the expression of 32-36 dph gradually decreases.It is presumed that the expression of such target genes is promoted by TH.Anothertype has the highest expression level in 17 dph,20-28 dph expression gradually decreases,28 dph expression reaches the lowest value,and 32-36 dph expression gradually increases.It is presumed that the expression of such target genes is inhibitedby TH.6.Expression of potential target genesin different metamorphosis development stagesof TH group and TU groupIn order to further infer whether the potential target genes are indeed regulated by TH,the exogenous TH and TU were used to treat the P.olivaceus,and the expression levels of each gene in different metamorphic developmental stages were detected byreal-time quantitative PCR.The results showed that the target genes promoted by TH:the expression level of 28 dph in TH group was basically no difference compared with NC group(P>0.05),and the expression level was the most significant in this group(P<0.05);compared with the NC group,the expression level of the group was significantly increased at 17 dph,20 dph,or 36 dph;the expression level of the TU group in each period was lower than that of the NC group and the TH group.The results showed that the target genes inhibited by TH: whether in the NC group,TH group,or TU group,the expression had the lowest levels at 28 dph(P<0.05);compared with TH group,the NC group wassignificantly increased at 17 dph,20 dph,or 24 dph(P<0.05);the expression of TU group was slightly higher than that of TH group in each period(P>0.05).7.Promoter transcriptional activity of potential target genesIn order to further verify whether the potential target genes selected are indeed regulated by TH,two TRαA potential target genes(fbxw8,apoptosis-enhancing nuclease-like),3 TRβ potential target genes(dusp7,pleckstrin homology domaincontaining family A member 5-likeand admats-10),and two TRαA and TRβ common potential target genes(frizzled-3-like,max)wereselected in this experiment to clone the promoter region containing the TRE site fragment,then construct a reporter gene recombinant plasmid to transfect HEK293 T,and use the dual luciferase reporter gene detection kit to verify each potentialtarget gene promoter transcriptional activity.The experimental results not only verify that the transcriptional activity of the selected target gene promoter is indeed regulated by TH,but also found that the more TRE sites in the promoter region,the more significant the TH regulation effect.In addition,it was found that binding TRαA andat the same time binding TRβ havemore significant regulation effectby TH compared with just binding TRαAor TRβ.Conclusion:THe target gene directly regulated by TH contains the characteristics of the TRE sequence-AGGT(C/A)A.In this experiment,11 potential target genes of TRαA and 12 potential targets of TRβ were selected by using the technical method of DNAprotein interaction.Among them,TRαA and TRβ have 4 identical potential target genes.They have been verified by real-time quantitative PCR and dual luciferase report experiments that these genes are indeed regulated by TH.These results complement the TH-regulated dentin allergy basic information on development networks.This method is an effective method for identifying proteins acting on DNA,providing an effective reference method for future protein-DNA interaction studies.