Function And Molecular Evolution Of Penguin Rhodopsin

The rhodopsin pigments is widely spread in vertebrate eyes,which plays a important role in scotopic vision.It means that we can use the gene to see the object included shape or dimension,however the colours of object we can not be discerned.The inhabitat of early vertebrates was quite similar,living in shallow waters.While the contemporary species is quite different,which will cause the body to adapt to the environment,resulting the functional changes in rhodopsin pigments.In this environment,rhodopsin in vertevrates can be distributed to 3 categories,which is surface(_?max500–507 nm),intermediate_{??max?490–495 nm}and deep-sea rhodopsin(_?max?480–485 nm).In most vertebrates,including birds,fish,amphibians,and mammals,the wavelength of maximum absorption peak of Rhodopsin is about500 nm,with relatively small changes and conservative functions.In aquatic environments,the diversity inhabitat is the driving force behind the evolution of opsin genes in aquatic species.In the deep sea,due to the low energy carried by long wavelengths that the lights can not be spread to deep water areas,resulting in extremely dark ambient light,which in turn causes blue-shift in the wavelength of maximum absorption peak of species.Existing bird species are divided into 40 orders,with various living habits,but only Penguin order species adapt to the deep-sea environment.To better adapt to the deep-sea environment,penguins have greatly improved their motion capabilities at the expense of loss flying ability.Such as,Aptenodytes forsteri,the emperor penguin,the representative species for penguin,has recorded the maximum depth of diving can reach to 444 m and the maximum duration is 21.3 min.When penguins quickly dive to the deep sea environment,and then from the deep sea environment to the surface quickly,the rhodopsin in the penguin's eyes may have special adaptability to light.Therefore,this paper focuses on the research of function in penguin rhodopsin in vito to explore the functional evolution of Rhodopsin and its molecular evolution mechanism during the origin and development of aquatic birds,and provide a certain reference for future research on the molecular basis of aquactic birds'visual inheritance and evolution.First,we expressed the rhodopsin pigment of three penguins?Aptenodytes forsteri,Pygoscelis adeliae and Spheniscus demersus?,and two control species?Nipponia nippon and Nannopterum harrisi?.When we verified the function by spectroscopy,we found that there is no significant difference in the wavelength of maximum absorption peak between penguin species and Nipponia nippon,Nannopterum harrisi.The wavelength range is just from 497 nm to 501 nm.There is no blue shift in the wavelength of maximum absorption peak of rhodopsin pigment in penguin species.Interestingly,we found that the retinal release rate of the pigments in penguin are faster than control species?Nipponia nippon and Nannopterum harrisi?when we mearsured by non-spectroscopy,and the retinal release rate of Aptenodytes forsteri and Pygoscelis adeliae were faster than Spheniscus demersus.Secondly,in order to study the molecular mechanism of the rhodopsin gene evolution in Penguin,we downloaded the complete coding sequence of rhodopsin gene for whole birds from the NCBI database to perform the molecular evolution analysis to calculate the ancestral sequence,and expressed the Rhodopsin pigments of ancestors of penguin and the common ancestor of the order of Sphenisciformes,Pelecaniformes and Suliformes.The results showed that the retinal release rate of penguins has changed during the ancestral state to better adapt to diving life,therefore the retinal release rate of penguin becomes faster than any other bird species.Due to some researches published a paper about genomic sequence of exsiting penguin species recently,we added the unused penguin species sequence to the original sequence,re-performed the molecular evolution analysis to calculate the ancestral sequence,and expressed the ancestral Rhodopsin pigment of the penguin to verify the function.The results show that the retinal release rate of ancestral protein is consistent with the previously estimated result.Finally,in order to clarify the molecular mechanism of the retinal release rate of Aptenodytes forsteri and Pygoscelis adeliae faster than the ancestral state,we constructed a site-directed mutagenesis on the amino acid at position 194 of Rhodopsin in Aptenodytes forsteri.The results showed that P194L was a key site,which led to a faster retinal release rate in Aptenodytes forsteri and Pygoscelis adeliae.This is the first time reported that the site can affect the retinal release rate.