Molecular Cloning, Tissue-specific Expression Of Retinoid X Receptors And Retinoic Acid Receptors Of Lateolabrax Japonicus

Abstract of Thesis：Vitamin A（retinols）plays a fundamental role in diverse biological processesin fish. Retinoic acid (RA), the most active retinoid, is synthesized in two steps from retinol. The firststep, oxidation of retinol to retinaldehyde, The second step, oxidation of retinaldehyde to RA. Biologicaleffects of RA are primarily mediated through the action of two classes of specific receptors, RARS andRXRs localized in the nucleus of the target cells, which act as ligand-activated transcription factors. Inthese heterodimers, RARs are activated by all-trans RA (atRA) and9-cis RA (9cRA), whereas RXRsare activated only by9cRA.Three retinoid X receptor subtypes (RXRα, RXRβ and RXRγ) cDNAs were obtained fromLateolabrax japonicus by RT-PCR and RACE. The obtained cDNA sequence of RXRα was1686bp inlength, containing a5’-untranslated region (UTR) of364bp and an uncompleted CDS of1322bp,encoded a putative protein of440amino acids. The full length cDNA sequence of RXRβ was1741bp inlength, containing a5’ UTR of150bp,3’UTR of256bp and an ORF of1335bp which encoded aputative protein of444amino acids with an estimated molecular weight of49.4kD. The RXRγ cDNAwas1847bp in length with88bp of5’-UTR,397bp of3’-UTR and an ORF of1362bp which encoded aputative protein of453amino acids with an estimated molecular weight of49.9kD. As other RXRs, theDNA-binding domain (DBD) of Lateolabrax japonicus RXRα、RXRβ and RXRγ contain the P box andD box and the ligand binding domain (LBD) contains12α-helices and2β-sheets with aligand-dependent activation function AF-2in the COOH terminus of the H12. In the phylogenetic tree,Lateolabrax japonicus RXRα, RXRβ and RXRγ were clustered with corresponding subtypesrespectively.The tissue-specific expression of each RXR gene were explored using RT-PCR. The mRNAof RXRα were expressed in all tissues examined with low level. The mRNA of RXRβ expressedhomogeneously in all tested tissues excepting a low expression in heart. RXRγ mRNA were alsoexpressed in all tissues tested with high levels in muscle, intestine, kidney, fat and spleen.Full-length cDNA coding for RARα and RARγ were isolated from Lateolabrax japonicus liver inthis paper. RARα was shown to be2094bp, which included a124bp of5’-untranslated region （UTR）,a608bp3’-UTR and a1362bp open reading frame （ORF）. The predicted Lateolabrax japonicusRARα consisted of453amino acids with a molecular weight of56.20kD and the oretical isoelectricpoint of8.20. The whole length of RARγ cDNA was1671bp consisting of a36bp5’-UTR，a 129bp3’-UTR and a1506bp open reading frame coding501amino acids with a theoretical isoelectricpoint of4.96and molecular weight of56.20kD. RARα and RARγ shared63.1%identity in their aminoacid sequence. Lateolabrax japonicus RARα and RARγ share relatively high level of similarity（96.9%and95.2%, respectively） with those of Takifugu rubripes. BothRARα and RARγ have typicalnuclear receptor structure, unlike the RXRs, RARα and RARγ with AF-1in the A/B domain and with Fdomain. Lateolabrax japonicus RARα and RARγ were expressed in all ten tissues tested（liver, muscle,heart, eyes, intestine, kidney, fat, spleen, gill and brain）. The highest expression of RARα was in eyes,gill and intestine, the lowest in heart and brain. The highest expression of RARγ was in gill and spleen.The results lay the foundation for further study of the functions of RARs and RXRs.