The Adaptive Evolution And Protein Function Analysis Of Cetaceans MLN And MLNR

Cetaceas are a group of secondary aquatic mammals with important significance during evolution.Approximately 55-53 million years ago,the ancestors of cetaceas transformed from a fully terrestrial quadruped to an obligate aquatic habitat,along with the great change of morphology and fast adaptive,though gradually evolution became the dominant species of Marine mammals.In the process of entering into the sea,the ability to store energy and maintain normal body temperature is the most important challenge for Cetaceas.In order to cope with such extreme conditions,whale form a thick layer of blubber.This study focused on the adaptive evolution of the Motilin Receptor and Motilin,which coding an important endocrine regulator of gastro-intestinal motility.Motilin is secreted by duodenal mucosa of Mo cells,it plays an important role in the digestive of mammals including induces migrating motor complex during the interdigestive period.Genetic research showed MLNR was lost in rodents;however,besides Kangaroo Rats and Kangaroo Mice which retained an intact,and thus potentially functional,open reading frame for MLN,the MLN gene became a pseudogene in other rodents.Here,by polymerase chain reaction,we amplificated MLNR and MLM sequence of several represtative whales.Besides bowhead whale,the MLNR from 11 species of whales are existed as pseudogenes;MLN of 4 species of cetaceas are pseudogenes.After aligning the identified whale MLNR and MLN sequences with wild yak MLNR,we found multiple indels and premature stop codon,respectively.By mapping the first stop codon on secondary structure of MLNR,we found all these mutation lead to shorten of protein secondary structure,thus leading to the loss of MLNR and MLN.In cetaceas,bowhead whale is the only one that preserve the functional MLNR-MLN pair,while 11 species of cetaceas retain MLNR as pseudogene and complete MLN.Furthermore,our selective pressure analysis calculated by PAML and Datamonkey software revealed positive selection in MLNR and MLN of cetaceas.It suggested that adaptive evolution occurred in cetaceas.According to report in the literature and the analysis of the selection pressure,we suspected MLN stimulates proliferation and differentiation of preadipocyte and liqid storage,which benefits the thickness of blubber.We investigated functions of MLN protein on 3T3-L1 cells differentiate into mature adipocytes then measured intracellular liqid,expression of PPARy(marker of adipocyte differentiation),and fatty acid uptake.Results showed that MLN protein synthesized by chemical method based on killer whale promotes 3T3-L1 preadipocyte cells differentiate into mature adipocytes,and induces fatty acid uptake in mature adipocytes.Interestingly,the effects of MLN protein from killer whale is stronger than that from yak and human in the fatty acid uptake experiments.So MLN must have undergone adaptive evolution to provide a guarantee for its adaptation to an aquatic environment.Based on the result that Bowhead Whale is the only one that preserve the functional MLNR-MLN pair,we showed functional retainment in the MLNR-MLN pair of bowhead whale by calcium assay kit.Based on Evolutionary biology,we speculated that MLNR gained new function in bowhead whale.