Molecular Evolution And Functional Studies On Pancreatic Ribonuclease In Free-tailed Bats

Bats(Chiroptera)are a group of animals who have strong adaptability and can be survival almost all over the world.They have diverse diets,including nectarivory,frugivory,insectivory etc.Pancreatic ribonuclease(RNase1)belongs to a family of enzymes,which has been studied a lot.Researchers found that the enzyme has immune-related functions by degrading RNA viruses.RNASE1 has been found underwent adaptive functional evolution after gene duplication in ruminants and leaf-eating monkeys.Researchers found there could be a digestive function for these enzymes.In addition,gene duplication has also been found in some other groups,such as carnivora,which could also be related to digestion.Moreover,researchers found multiple RNASE1 gene duplications in a bat genus Tadarida.Some of the gene copies were under positive selections.However,no functional assays have been performed on these bat enzymes.In this study,we amplified and sequenced the RNASE1 genes from Tadarida brasiliensis,Desmodus rotundus and Miniopterus fuliginosus,and also obtained other bat RNASE1 sequences from databases.In total,we got the RNASE1 sequences from 35 bat speices.Interestingly,we found two new RNASE1 duplicates in Tadarida brasiliensis,which were named Tadarida brasiliensis-?4 and Tadarida brasiliensis-?5,respectively.There are also two duplicates found in Miniopterus fuliginosus and the genes were named Miniopterus fuliginosus-? and Miniopterus fuliginosus-?.However,only one gene copy was identified in Desmodus rotundus.By reconstructing a RNase1 amino acid tree,we found that the RNase1-? group and their ancestral branches are longer than that of RNase1-? group.In addition,our results further support that the genes from RNASE1-? group underwent significant positive selections.Here we inferred the ancestral RNASE1 gene sequenecs for Tadarida brasiliensis and then expressed the enzymes in vitro.Then enzymatic assays were carried out for these RNase1 using t RNA at different p H.The results of enzymatic activities showed that the RNase1-? group adapted to more acid environments while that of the RNase1-? group shifted to more basic environments,suggesting a functional divergence.We suggest that the RNase1 enzymes which adapted to acid environments may be related to a better digestive function for the t RNA from food.On the other hand,enzymes more adapted to basic environments may still play an immune role in organism.