The Screening Of Carbonic Anhydrase Genes And The Effects Of Environmental Factors To CA Activity And Its Expression Of Chlorella Pyrenoidosa

Carbonic anhydrase (CA) is an important component of CO2concentratingmechanism (CCM), which plays an important role in carbon transport and carbonmetabolism of marine phytoplankton. Its basic function is to participate in hydration ofcarbon dioxide. It catalyzes inter-conversion between CO2and HCO3-, which will providesufficient CO2substrate for the carboxylation reaction of Rubisco. There are differenttypes and numbers of CA in algae. Although they are different in primary structure, theyhave similar catalytic mechanism, and their active site contains zinc ion or cadmium ion.In this study, Chlorella pyrenoidosa820was used as the experimental material. First,we obtained the sequences through the way of transcriptome sequencing (RNA-Seq), andthen analyzed the type and number of ca. Three kinds of ca genes of α-ca, β-ca and γ-cawere selected from Chlorella pyrenoidosa, and the physical and chemical properties,transmembrane domain, signal peptide, secondary and tertiary structures of ca isoformswere predicted through bioinformatics tools. Finally, in order to explore the responsepattern of carbonic anhydrase to environmental factors, and to improve the utilization ofinorganic carbon and algal biomass of Chlorella, the effects of CO2and bicarbonate,salinity on the growth, extercellular carbonic anhydrase activity and three subtypes of caexpression level of C. pyrenoidosa were also investigated in this study. This work wasexpected to provide reference for revealing CCM of Chlorella.We obtained the sequences by high-throughput RNA-seq technique in agenome-information deficient marine microalgae C. pyrenoidosa820. In the4.71Gsequences,36,826unigenes with1089nt mean length were assembled. A total of23,015unigenes were annotated in the NR, NT, SwissProt, KEGG, COG and GO databases withE-value lower than1e-5. In the annotated sequences,21unigenes were identified as cagenes.Three ca genes were selected from the21ca genes for bioinformatics analyses. The results showed that the molecular weight of α-CA, β-CA and γ-CA was28.9kDa,34.9kDa and24.8kDa, respectively. The three subtybes of CA genes were all acidic, slightlyhydrophilic and fat-soluble proteins. One signal peptide and a strong transmembranedomain were detected in α-CA, so α-CA was presumed an extracellular CA. There wereno credible transmembrane regions and signal peptides in the β-CA and γ-CA sequences.Combined with the subcellular localization results, β-CA might locate in the cytoplasm orchloroplast stroma, while γ-CA was in the mitochondria stroma. The results of secondarystructure prediction showed that the percentage of random coil in α-CA was the highest,which occupied64.66%. The proportions of alpha-helix and random coil were almost thesame in β-CA, which were44.76%and44.13%. And the proportions of alpha-helix andrandom coil in γ-CA protein accounted for39.48%and40.34%, respectively. The analysisof tertiary structure prediction showed that three dimensional structure of α-CA wereloose,but β-CA and γ-CA were relatively compact, and the γ-CA was found a repeatedfolding-structure.The effects of CO2and bicarbonate on the growth, extercellular carbonic anhydraseactivity and3subtybes of ca expression level of C. pyrenoidosa were investigated. Resultsshowed that the algal density cultured in double air CO2(high CO2level) could reach1.23-fold of that in air CO2(low CO2level). But the CA activity in high CO2level waslower, which were0.41-fold of that of low CO2level on the8thdays. The α-and γ-camRNA accumulations under high CO2level were slight higher, which were1.22times and1.47times of that of low CO2level. The growth of C. pyrenoidosa was promoted when thebicarbonate ranged from0to10mmol/L concentration. And the algal CA activity showedmaximum value under5mmol/L HCO3-group. The highest levels of α-, β-and γ-ca wereobserved under1mmol/L HCO3-group.The effects of salinity on the alggrowth, extercellular carbonic anhydrase activityand3subtybes of ca expression level were also investigated. Results showed that the algagrew faster under15and30salinity, and the algal density of45salinity was0.83-fold ofthat of15salinity. The carbonic anhydrase activity was inhibited with the increasingsalinity, which was especially obvious under45salinity and long time treatments.However, the expression level of three subtypes of ca genes increased from15to45salinity, which could reached from1.52-fold to2.16-fold of that of15salinity.It could be seen that there were three kinds of CA in Chlorella pyrenoidosa. Thephysical and chemical properties of the protein encoded by CA were different. It could beconcluded that the growth of C. pyrenoidosa was suitable under low and moderate salt, double air CO2concentration and higher bicarbonate concentration, and the CA activitywas induced by low salinity, air CO2and5mmol/L HCO3-concentration. However, the camRNA level was higher under high salinity, double air CO2level and1mmol/L HCO3-conditions.