Orthologs are genes originated from a single ancestral gene by vertical descent, which is related to speciation. Paralogs are genes related by duplication. It is widely accepted that orthologs tend to have similar function and paralogs tend to have different functions. However, this idea is challenged with the rapid accumulation of large-scale genomic data and comparative genomic studies. In our research, we chose bat T2R16 to explore the evolutionary pattern and functional divergence between orthologs and paralogs, owing to T2R16 existing in bats in these two forms and the paralogs emerging in a short time. Through combining different methods of PCR (polymerase chain reaction) amplification, sequencing, real-time PCR etc, 26 sequences of T2R16 were obtained, including 14 orthologs and 12 paralogs which distributed in 4 species of myotis. And then, we conducted evolutionary analysis respectively. The selection pressure detection indicated that both T2R16 orthologs and paralogs underwent a similar overall purifying selection. Despite of this overall purifying selection, we indentified positively selected sites in both T2R16 orthologs and paralogs. Intriguingly, great differences were observed in terms of the site number and distribution, which may suggest that different evolutionary mechanisms acted on T2R16 orthologs and paralogs. At the mean time, we found out that, in T2R16 orthologs, the ancestor of Rhinolophoidea showed a positive selection and T2R16 gene tree conflict with bats species tree for the Pteropodidae clade. These two interesting finding indicated that functional divergence could happen in T2R16 orthologs. To test this prediction, we constructed T2R16 functional expression vectors and conducted the cell-based functional assays in vitro. The results implied that the fate of T2R16 orthologs and paralogs were similar. That is, both could encounter loss of function and sensitivity divergence for detecting a particular bitter compound. Additionally, two copies of T2R16 paralogs, MFI_1 and MFI_2, showed no response to salicin, while the other two copies, MFI_3 and MFI_4, maintained the ability of response to salicin, which could suggest that subfunctionalization had happened for T2R16 in species MFI.
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