Cloning, Expression And Phylogenetic Analysis Of Tshr Gene From Amphioxus Branchiostoma Belcheri Tsingtauense

Thyroid stimulating hormone receptor(TSHR) which widely consisted in vertebrate especially in mammals,is a kind of Glycoprotein receptor,belongs to G-protein-coup receptor family.TSHR conjugated with Thyroid Stimulating Hormone(TSH) participated the signal transmission actions in animals.Amphioxus,a cephalochordate,has long been regarded as the extant invertebrate most closely related to the proximate ancestor of vertebrates.Studies on the gene structure,function and expression in amphioxus will contribute to the understanding of the origin and evolution of the vertebrates.In this paper, Tshr gene was cloned from the total RNA of the Branchiostoma belcheri tsingtaunese,and the structure,expression,evolution and expressions of proteins of Tshr gene have been studied.In this paper,the Tshr gene cloned by the methods of RT-PCR and RACE.The lull-length cDNA of Tshr gene is 1825 bp and it contains an open reading frame of 1377bp coding a poly peptide which molecular mass of approximately 50kDa and contains 459 amino acid residues.The 5'-untranslated region of Tshr gene contains 154 bp and 3'-untranslated region contains 295 bp.Amphioxus TSHR has 30～40%identity with other animals' TSHR.It is also between vertebrates and invertebrates in the phylogenetic tree, shows that Amphioxus TSHR is still in a primitive state.Section in situ hybridization shows that Tshr gene has the specific expression location at endostyle which has the evolution homologous to the thyroid of vertebrates.Embryos in situ hybridization and real-time PCR indicate the functions maybe have difference between embryos and adults.Amphioxus TSHR has been successfully expressed in Escherichia coli.The molecular weight was estimated 50 kDa.Western blot with TSHR antibodies showed that the antibodies also reacted with the protein we obtained.