Cloning And Expression Regulation Of Aquaporin Gene CiMIP1 Of Chlamydomonas Sp. ICE-L From Antarctica

As one of the most basic activities of cells,rapid transmembrane transport of water molecules is an important factor for organisms to adapt to the changes in the osmotic pressure of the cell and outer environment quickly.Aquaporins localize on biological membranes,and are pore-forming membrane proteins which belong to the superfamily of the main intrinsic protein.They can facilitate the passive transport of numerous small polar substances,such as water,and other small solutes,particularly glycerol but also urea ammonia,metalloids or carbon dioxide,across cell membranes in all living creatures.Antarctic ice microalgae are the phytoplankton or microalgae that thrive in the pelagic,ice and meltwater environments of the Antarctic,which are the main sources of primary production in an otherwise barren region.Antarctic microalgae possess unique adaptations that allow them to proliferate in extreme conditions characterized by low or freezing temperature,seasonal light,strong ultraviolet radiation and high salinity fluctuations.Thus,understanding the underlying mechanisms of their acclimation has gained strong interests in the past years.Antarctic ice algae Chlamydomonas sp.ICE-L is a eukaryotic and single cell algae and it's also one of the most common and representative algae among Antarctic area.The aquaporins contained in it play an important role in mediating water transport,sustaining cell osmotic pressure balance and coping with various adversity stress.Therefore,it is necessary to study Antarctic ice algae aquaporins(CiMIP1).In this paper,we mainly analyzed and discussed the cloning and expression regulation of aquaporin gene CiMIP1 of Chlamydomonas sp.ICE-L with the following aspects:1)According to the Chlamydomonas sp.ICE-L sequencing results of transcriptional group,the aquaporin gene CiMIP1 was successfully obtained through molecular biology technology,the complete open reading frame of CiMIP1(GenBank ID:KY316061)was 927 bp and encoded a polypeptide of 308 amino acids with molecular weights of 32.43 kDa.Bioinformatics analysis revealed that CiMIP1contained six putative transmembrane domains.Besides,we were surprised to find that a third NPA motif existed at the carboxy terminus of the target protein except for two highly conserved ones.Subcellular localization predicted that Ci MIP1 protein was located in the cytoplasm.Phylogenetic tree analysis demonstrated that CiMIP1protein belonged to MIPD subfamily and was most closely related to CrMIPD1;1from Chlamydomonas reinhardtii and VcMIPD1;1 from Volvox carteri.2)Transcription levels of CiMIP1,under temperature and salinity conditions,were analyzed by quantitative real-time PCR.Results showed that the expressions of CiMIP1 was apparently up-regulated in a short time at-20?and 0?,while at 10?,the CiMIP1 gene needed a relatively long time to achieve the up-regulated expression.In addition,under high salinity conditions of 64‰and 128‰,the Ci MIP1 gene expression increased continuously during the first 48 h,when the salinity decreased to16‰,it was inhibited.Taken together,these results showed that the transcriptional level of Ci MIP1 gene was regulated differentially under temperature and salinity stress in order to adapt to these environmental changes.3)The recombinant of prokaryotic expression plasmid pEASY~?-Blunt E1-CiMIP1 was constructed and transformed to Transetta(DE3)Chemically Competent Cell to express CiMIP1 protein with the condition of 0.5 mmol/L concentration of IPTG,at 16?for 12 hours.SDS-PAGE revealed that the molecular weight of the target protein was similar to theoretic molecular weight and CiMIP1protein was expressed as inclusion body.4)The E.coli cell-free expression system was prepared and the corresponding plasmid was successfully constructed.Then add the plasmid into the system to express CiMIP1 protein after the extraction and purification of plasmids.After the reaction completed,inject the protein solution into dialysis cassettes,after dialysis,the CiMIP1 was isolated and purified by nickel column affinity chromatography,and the pure CiMIP1 was obtained.To the best of our knowledge,this is the first time to clone the aquaporin gene in Chlamydomonas sp.ICE-L,the bioinformatics analysis of the gene and its encoded protein will help to further understand its function and mechanism.The study of expression pattern of Chlamydomonas sp.ICE-L aquaporin gene under different temperature and salt stress may show that temperature and salinity can affect gene expression,which lays a foundation for clarifying the molecular mechanism of its temperature and salinity adaptation.Through the prokaryotic expression and cell-free expression,it also provides a certain material basis for the functional characterization of aquaporin.