Cloning,Expression And Functional Studies Of Carbonic Anhydrase(CAs)in Chlamydomonas Sp. ICE-L

A series of environmental problems,caused by the increasing atmospheric CO₂ concentration,become more and more serious,so an effective and environmental friendly method of reducing CO₂ concentration is in urgent need of improving the environment.Algae are characterized by their specific way of concentration and absorption of CO₂,which manifests the unique superiority in reducing atmospheric CO₂ concentration.CA is an important component of algae CCM,as a metalloenzyme capable of catalyzing CO₂ immobilization.Therefore,the research of CA in C.sp.ICE-L is very important and useful.In this paper,we mainly analyzed and discussed the expression and function of C.sp.ICE ?-CAs with the following aspects:1)The complete nucleotide sequences of ?-CAs were successfully obtained by molecular biological technique according to the four ?-CAs contig in the C.sp.ICE-L transcriptome.Bioinformatics analysis revealed that the four ?-CAs were consistent with the conserved sequence and His cluster of known ?-CAs.?-CA,?-CA2 and ?-CA4 were located in the chloroplast,while ?-CA3 in the cytoplasm.Phylogenetic tree further showed that ?-CA,?-CA2 and ?-CA4 aggregated with C.reinhardtii cyCA,and ?-CA3 with C.reinhardtii chCA.2)Transcription levels of ?-CA,under UVB,temperature and salinity conditions,were analyzed by qRT-PCR.It was found that the expression of ?-CA did not change significantly in the low stress condition?temperature 0 °C,salinity 64 ‰?,which revealed the adaptability and tolerance of C.sp.ICE-L on adversity.However,under high stress condition?UVB;temperature-20 °C,10 °C;salinity 96 ‰,128 ‰?,the expression was significantly changed,which indicated that algae may regulate their own metabolism by modulating the transcription levels of ?-CA to stress.3)We constructed the engineering bacteria expressing ?-CA Transetta?DE3?-pEASY?-Blunt E1-?-CA and Transetta?DE3?-pEASY?-Blunt E1 and the protein expressed by the two strain were analyzed by SDS-PAGE.Results showed that the former had a clear and thicker protein band at about 40 kDa,while the latter did not,and the molecular weight of the target protein was predicted to be 40.5 kDa.So it was confirmed that ?-CA was successfully expressed.4)In order to obtain pure ?-CA,the mixed protein sample was isolated and purified by nickel column affinity chromatography.The carbon dioxide hydration activity and the esterase activity of ?-CA were respectively measured.Results suggested that the former activity of ?-CA was 0.437 U/mg,and the other activity was higher than in the control as well.This is the first time to clone the four complete gene of ?-CAs in Antarctic ice algae C.sp.ICE-L.Subcellular localization of these four ?-CAs,predicted by bioinformatics,would help to further understand their respective functions and action mechanism.The transcriptional expression patterns of ?-CA under UVB,temperature and salinity stress indicated that the gene expression was affected by these stresses,which laid a foundation for self-regulated mechanism of stresses response.?-CA of C.sp.ICE-L was successfully expressed in the prokaryotic expression in vitro,and the protein with a high purity and enzymatic activity was obtained.