Study Of Subcellular Localization Of Dinoflagellate Rhodopsin By Transformation And Expression In Mammalian Cells

The energy that supports the life activities in the ocean ultimately comes from sunlight,via photosynthesis as the main energy conversion mechanism.Rhodopsin is a newly discovered protein in microbial organism,which can convert solar energy to ATP,the main energy currency of cells,without the help of photosystems.Rhodopsins are now found in all domains of life including thousands of organisms across archaea,bacteria,fungi,and algae,which are light-activated,seven-a-helical,retinylidene trans-membrane proteins.Some rhodopsin function as out-ward proton pumps,some as inward chloride pumps whereas others function as light sensors or ion channels.Although they share a high degree of sequence identity and thus similarity in structure,many unique functions have been discovered and characterized among them.Based on amino acid sequences,rhodopsins,similar to that encoding proton-pumping rhodopsin in bacteria,have been found in dinoflagellates.Some studies indicated that rhodopsin of Prorocentrum donghaisense transcript abundance exhibited a strong diel rhythm.Its expression level was high in the light period and low in the dark.In the heterotrophic dinoflagellate Oxyrrhis marina,when starved,expressed a higher level of the proton pump rhodopsin,coincident with higher population growth,than under dark condition,suggesting a role of this protein as an energy supplementing mechanism.However,the function of dinoflagellate rhodopsin is still poorly understood.No evidence has been shown that the rhodopsins of dinoflagellates are functional as proton-pump and that their subcellular localization is on the plasma membrane.Utilizing the sequence of rhodopsin from P.donghaiense and Alexandrium catenella,the present study established an expression system of dinoflagellates rhodopsin in mammalian cells.In this mammalian cell expression system,subcellular localization of dinoflagellates rhodopsin was studied.Major results are as follows:(1)Rhodopsin from P.donghaiense and A.catenella was recombined into an expressed plasmid PB513B-1.The reconstructed reporter plasmid and the transposase expression vector PB200-PA were successfully co-transfected into HEK293T cell line to create mammalian cells expression system through Piggy Bac system.(2)Based on the successful creation of mammalian cells expression system,subcellular localization of dinoflagellates rhodopsin was studied with the aid of immunofluorescence technology,which indicated that rhodopsin from P.donghaiense and A.catenella was expressed and the proteins were localized at the plasma membrane in the mammalian cells expression system.