Bitter taste perception detects bitter toxic sunstances and prevents organisms against bitter toxins which is a defense mechanism. The comnbination of bitter taste receptor coded by bitter taste receptor genes (T2Rs) with bitter ligands trigger the downstream signaling pathway and transduct external signal to cerebral cortex which cause aversion behavior to avoiding having toxic substances. Carnivores’ major food is animal tissues, which are lack of toxin, and their bitter taste perception is not so important for their living. Dog, as a first domesticated animal by human, there are less toxic bitter substances in their food and their dependence on bitter perception is decrease compared to its ancestor wolf. Thus, we hypothesize bitter taste receptor genes of dog are under relaxed selection constraints. We studied the microevolution of dog T2R family in the population level and the function of dog T2Rs.We chose 30 dogs from 15 dog breeds and got the 15 members of dog T2Rs, 5 dog T2R pseudogenes and 16 dog intergenic regions of each individual. We first compared the microevolution of T2Rs from dogs and other three placental mammals and then explored the function (binding sites) of dog T2Rs. After population genetic analysis, we first revealed that dog T2Rs has lower genetic diversity than that of intergenic regions. To further investigate the microevolution of different dog T2R genes, we groupes dog T2Rs into three kinds and found multiple to multiple ortholog genes have the highest polymorphism and one to one ortholog gene harbors the minimum polymorphism which indicates different T2R genes are under different selection constraints. This conclusion is consistent with that of other three placental mammals’ T2Rs. The genetic diversity of dog T2R14 is even higher than that of intergenic regions and there are 17 haplotypes among 30 dogs, which shows that it may be under relaxed purifying selection or diversifying selection.To characterize the function of dog T2Rs, we heteroexpressed dog T2Rs by in vitro cell heteroexpressed function assay system and challenged each dog Tas2R with 33 bitter compounds. We reported 5 ligands for 3 dog T2Rs for the first time, which are dTas2R1, dTas2R10 and dTas2R14. The 3 ligands for dTas2R1 are Colchicine, (-)-α-Thujone and 3-Methyl-1-pentyn-3-ol. Diethylene triamine pentaacetic acid (DTPA) is the ligand of dTas2R10 and the ligand of dTas2R14 is 6-Propyl-2-thiouracil (PROP). We challenged PROP structure related compounds with dTas2R14 and found the important chemical structure influenced the activation of dTas2R14. Because there are 17 alleles of dT2R14 and the ortholog gene of human is hT2R43-44 cluster which ligands are known, we conducted the function study of all the 17 dT2R14 alleles to reveal the functional differentiation of dog T2Rs in populations. By heteroexpressed 17 dTas2R14 haplotypes and function assays, we found amarogentin, the second natural ligand of dTas2R14. The response of amarogention is the same with that of PROP. Furthermore, we found the 17 dTas2R14 variations are divided into ‘functional’ and ‘nonfunctional’ types, 13 variations can be activated by the two ligands while the left 4 haplotypes can not. The amino acid site 91 of all the functional variations is serine (S) while that of all the nonfunctional haplotypes is asparagine (N). By the functional experiment of mutated dTas2R14 variations, we found amino acid site 91 is the critical site for the activation of dTas2R14. Additionally, we found another two amino acid sites, site 175 and site 185, also influence the function of dTas2R14. This research will provide further understanding of the relationship of T2R genes variations and the receptor function divergence.
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