| The metamorphosis of amphibian is a complex process regulated by thyroid hormones (THs), in which most of the organs and tissues undergo a wide range of remodeling. THs exert their functions by binding to thyroid hormone receptors (TRs). In order to explore the effects of THs in metamorphosis, we study the expressional change of TRs in Rana chensinensis during development. In this paper, we firstly designed specific primers according to the TRα and TRβsequences of Rana catesbeiana, then TRα and TRβ of R. chensinensis were cloned by PCR. Some biological softwares, DNAStar, SMART, ProParam and so on, were used to splice sequences, predict their protein molecular weight, protein secondary and tertiary structures. After that R. catesbeiana and Rana rugosa in GenBank were chose to conduct the comparison analysis of Amino acid structure domains. Next, R. catesbeiana, Pelophylax nigromaculatus, Eublepharis macularius, Gallus gallus and so on,10 species of vertebrate were selected for ClustalW analysis and then constructed evolutionary trees with MEGA5.1. Finally, the expression levels of TRa and TRβ mRNA were examined in liver, brain, skin and tail of R. chensinensis tadpoles during 33 to 46 stages with semi-quantitative PCR and quantitative Real time PCR (qRT-PCR). The analysis results are as follows:1.The cloned cDNAs using the liver of R. chensinensis adults are 1257 bp and 1122 bp encoding 418 and 373 amino acids, with a open reading frame respectively. They have been accessed by GenBank (Accession No. KJ579109.1, KJ579110.1), named rcTRα and rcTRβ respectively in this paper. Analysis results of softwares show that rcTRα and rcTRβ sequences both does not exist N-terminal signal peptide, meaning that they are non secretory proteins. (1) The start codon and termination codon of rcTRα is ATG and TAA, respectively. Molecular weight is 47.79 kDa and theoretical pI is 7.08. Secondary structure contains 48.09% α-helix,4.31% extended strand and 46.89% random coil. Three mainly functional domains in rcTRa were predicted including ZnF_C4 domain (58-131), coiled coil domain (137-165) and HOLI domain (228-386). In addition, rcTRa also contains VMC domain (10-64), IBR domain (44-99), HOX domain (149-199) and so on. (2) The start codon and termination codon of rcTRβ is ATG and TAG, respectively. Molecular weight is 42.28 kDa and theoretical pI is 6.76. Secondary structure contains 40.48% random coil,50.94% α-helix and 6.17% extended strand. ZnF_C4 domain (16-89) and HOLI domain (186-344) are found in rcTRp. In addition, rcTRp also contains MCM domain (1-372), RING domain (19-76), HOX domain (107-157) and so on.2. Alignment of predicted amino acid sequences reveals that rcTRα and rcTRβ include a A/B domain, a DBD, a D domain, and a LBD. Moreover, each sequence contains two putative cysteine-rich zinc fingers in DBD, which are characteristics of nuclear hormone receptors. The TRs identities of R. chensinensis, R. catesbeiana and R. rugosa are relatively high. So, we infer that the cloned TRs are the members of thyroid hormone nuclear receptors. There are some differences in amino acid alignment results, rcTRβ has a deletion of 42 amino acids in A/B domain compared with rcTRa,51 amino acids are different between rcTRa and rcTRβ in DBD, D domain and LBD.3. The phylogenetic tree shows that TRa and TRβ sequences are clearly separated and locate in two separated clades clearly. In both TRa and TRβ clades, R. chensinensis is grouped with R. catesbeiana, R. rugosa and P. nigromaculatus, which is consistent with these species being members of Rana Besides, rcTRα and rcTRβ have similar positions in each clade. The identities of nucleotide and amino acid sequences show that rcTRα and rcTRβ both have high identity with other species of anuran, relatively low with reptile, bird and mammal. Compared with TRα, TRβis higher in identity between R. chensinensis and other species investigated. Moreover, the identity of nucleotide sequences between rcTRα and rcTRβ was 72%, and that of amino acid sequences was 87%. Similar identity relationship in nucleotide and amino acid sequences between rcTRα and rcTRβ also exists in other species of vertebrates.4. The results of qRT-PCR are similar to that of semi-quantitative PCR. The results show that rcTRα and rcTRβ express in liver, brain, skin and tail but the expression patterns are different during the metamorphosis of R. chensinensis tadpoles. The expressional changes of rcTRa are moderate, while rcTRβ are dramatic. Additionally, the levels of rcTRβ mRNA are relatively low at pre-metamorphosis, and then peak around climax. The rcTRa expression of brain are most moderate among the four tissues and rcTRa expression maintains at a higher level during the whole metamorphosis. Tissue specific expressions of rcTRα and rcTRβsuggest that THs play various roles in different organs during metamorphosis of R. chensinensis. |
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