The Functional Study Of Thyroid Hormone And Critical Genes Of Its Singnaling Pathway In The Pacific Oyster Crassostrea Gigas

The Pacific oyster(Crassostrea gigas),living in the intertidal area,is a world-wide aquaculture species.Due to its remarkable ecological and economic value,the Pacific oyster has become one of the most well-known species in lophotrochozoa.In this study,we evolutionary studied the Thyroid hormone system and nuclear receptor family members in the Pacific oyster.The following were the main results and conclusions: 1 The functional study of thyroid hormone and critical genes of its singnaling pathway in the Pacific oyster Crassostrea gigasThyroid hormones(THs)play important roles in development,metamorphosis,and metabolism in vertebrates.During the past century,TH functions were regarded as a synapomorphy of vertebrates.More recently,accumulating evidences have gradually convinced us that TH functions also occur in invertebrate chordates.To date,however,the TH-related study in non-chordate invertebrates is limited.In this study,THs were qualitatively detected by two reliable methods(HPLC and LC/MS)and quantitative measured by a electrochemical immuno-methods in the development of C.gigas.Molecular biology methods were used to study the critical genes in the THs signaling pathway,including four thyroid hormone peroxidases(PERT),two iodothyronine deiodinase and one throid hormone receptor(TR).The gene function of them were characterized by various molecular biology methods,including qRT-PCR,western blot,subcellular localization,prokaryotic recombinant protein expression,yeast two-hybrid assay,electrophoretic mobility shift assay(EMSA)and dual-luciferase reporter assay.The results suggested that there is an active TH system in oysters.THs may involve in embryogenesis,growth and metamorphosis of C.gigas.Oyster embryos may synthesize THs autonomously via CgPERT1.The deiodinase activity of transformation from T4 to T3 was detected in the Pacific oyster C.gigas in vivo and the identified oyster deiodinases(CgDx and CgDy)were speculated to be responsible for this deiodinase activity.The promoters of CgDx and CgDy were activated by CgTR in a dual-luciferase reporter system.Furthermore,the atypical TH responsive elements(TREs)in the promoters could be bound by recombinant CgTR.Thus,CgDx and CgDy are also TH-responsive genes and THs may transcriptional regulate the expression of oyster deiodinases via CgTR binding to TREs in their promoters,suggesting a conserved TH feedback regulation mechanism in mollusks for the first time.As a first step in elucidating the TH signaling cascade,an ortholog of vertebrate TH receptor(TR),the most critical gene mediating TH effects,was cloned in C.gigas.The sequence of CgTR has conserved DNA-binding and ligand-binding domains that normally characterize these receptors.Experimental results demonstrated that CgTR can regulate gene expression through binding to promoters of target genes and can interact with oyster retinoid X receptor(CgRXR).Moreover,CgTR mRNA expression was regulated by TH and CgTR proteins,and an atypical thyroid hormone response element(CgDR5)was found in the promoter of CgTR and verified by EMSA assay.These results indicated that some of the CgTR function is conserved,and suggest that these TR functions had developed in the common ancestor of protostome and deuterostome TR.However,differences were also found in the DNA binding specificity and transcriptional activation activity of CgTR when compared with vertebrate TRs.the functions of CgTR provide significant evidence in understanding evolution of the TH system.This is the first systematic study on physiological effects and molecular mechanism of THs in non-chordate invertebrates.On the basis of our findings in this study and the sporadic evidence from other lophotrochozoa,it is reasonable to conclude that the origin of THs could date back to the lophotrochozoa.2 Functional characterization of CgRXRIn this study,we also cloned another NR member,CgRXR.qRT-PCR results indicated that CgRXR may be involved in embryogenesis in C.gigas.A recombinant full-length protein of CgRXR localized to the nuclei of HeLa cells.Dual-luciferase reporter assay results indicated that 9-cis RA or DHA could activate the transcriptional activation activity of CgRXR.This means that the ligand binding activity of CgRXR is conserved in C.gigas.Furthermore,TBT or TPT could activate the transcriptional activation activity of CgRXR,suggesting that the toxicity of organotin compound to oysters were madiated by CgRXR.CgRXR could binding to DR0-DR5,except for the weakness binding of DR4,the binding intensity of the other probes were comparable.Thus,the DNA binding property of CgRXR is different from vertebrates and gastropods.3 Evolution of a novel nuclear receptor subfamily with emphasis on the member from the Pacific oyster Crassostrea gigasNuclear receptors(NRs)belong to the transcription factors superfamily that regulates development,homeostasis,differentiation,and reproduction in metazoans via control of gene expression.Recently,rapid advances in genome projects for various metazoans provided new opportunities to study the evolution and function of NRs.Typically structured NRs are divided into six subfamilies.Here,the gene for a typically structured NR(CgNR8A1)was cloned from the Pacific oyster Crassostrea gigas.However,this novel receptor could not be assigned to one of the known NR subfamilies.Through data mining,nine other CgNR8A1 gene homologs were identified in metazoans,including cnidarians,mollusks,annelids,echinoderm,hemichordate,and cephalochordate.Phylogenetic analysis showed that these receptors were part of a novel NR subfamily,herein designated as NR8.Evolutionary analysis revealed that the NR8 subfamily was phylogenetically the third-oldest NR subfamily,originating from a common ancestor of Eumetazoa,and several gene loss events occurred independently in anscestors of vetebrates,ecdysozoans and platyhelminthes,which do not have NR8 member.Furthermore,the function of CgNR8A1 was investigated to give insight into the function of this novel NR subfamily.A nuclear localization signal peptide GKHRNKKPRLD was identified in CgNR8A1 and a recombinant full-length protein of CgNR8A1 localized to the nuclei of HeLa cells.The mRNA expression profile of CgNR8A1 suggested that it may be involved in embryogenesis in C.gigas.EMSA experiments showed that CgNR8A1 bound strongly to conserved DNA core motifs DR0,DR2,and DR4 and weakly to DR1,DR3,DR5,Half,and Pal0.In summary,the novel NR8 subfamily identified here extends the evolutionary understanding of NRs,and the functional analysis of CgNR8A1 provided further insights into the function of the NR8A1s.