Abstract
In this study, the transcriptome of the marine microalga, Chlorella pyrenoidosa 820, was sequenced by high-throughput RNA-seq using Illumina HiSeq 2000. Because the genome sequence of this species is unknown, the resulting transcriptome was assembled de novo and annotated. This resulted in 4.71G clean nucleotides with a 56.91% GC content and yielded a total of 36,826 unigenes with a mean length of 1089 nt. Among these, 23,015 unigenes were annotated in the NCBI-NR, NCBI-NT, Swiss-Prot, KEGG, COG, and GO databases with a cutoff E-value of 10-5. In the annotated sequences, 21 unigenes were identified as carbonic anhydrase (CA), which is an important enzyme in the CO2-concentrating mechanism (CCM), and were homologous to nine α-, eight β-, and four γ-CAs. Interestingly, the expression of three CA subtypes, including one α-, one β-, and one γ-CA, analyzed by real-time quantitative PCR, showed induction by high salinity. These results will enrich the CA gene information in the database and will help understand CCM in the genus Chlorella.
Acknowledgments
This work was funded by the National Natural Science Foundation of China (30700610), Natural Science Foundation of Zhejiang Province (LY13D060007), and the Project of Science and Technology Innovation Team of Zhejiang Province (2010R50025-25). This research was also sponsored by the K.C. Wong Magna Fund from the Ningbo University.
References
Asamizu, E., K. Miura, K. Kucho, Y. Inoue, H. Fukuzawa, K. Ohyama, Y. Nakamura and S. Tabata. 2000. Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii. DNA Res. 7: 305–307.10.1093/dnares/7.5.305Search in Google Scholar PubMed
Badger, M.R., A. Kaplan and J.A. Berry. 1980. Internal inorganic carbon pool of Chlamydomonas reinhardtii. Evidence for a carbon dioxide concentrating mechanism. Plant Physiol. 66: 407–413.10.1104/pp.66.3.407Search in Google Scholar PubMed PubMed Central
Blanc, G., G. Duncan, I. Agarkova, M. Borodovsky and J. Gurnon. 2010. The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis, coevolution with viruses, and cryptic sex. Plant Cell 22: 2943–2955.10.1105/tpc.110.076406Search in Google Scholar PubMed PubMed Central
Bozzo, G.G., B. Colman and Y. Matsuda. 2000. Active transport of CO2 and bicarbonate is induced in response to external CO2 concentration in the green alga Chlorella kessleri. J. Exp. Bot. 51: 1341–1348.10.1093/jexbot/51.349.1341Search in Google Scholar
Conesa, A., S. Götz and J.M. García-Gómez. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674–3676.Search in Google Scholar
Deng, Y., X. Sun, N. Xu and R. Yang. 2012. The cloning and expression analysis of Rubisco activase gene in the unicellular green alga Chlorella pyrenoidosa. Oceanologia et Limnologia Sinica43: 41–46 [in Chinese].Search in Google Scholar
Duncan, D.B. 1955. Multiple range and multiple F tests. Biometrics 11: 1–42.10.2307/3001478Search in Google Scholar
Field, C.B., M.J. Behrenfeld, J.T. Randerson and P. Falkowski. 1998. Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281: 237–240.10.1126/science.281.5374.237Search in Google Scholar PubMed
Fisher, M., I. Gokhman, U. Pick and A. Zamir. 1996. A salt-resistant plasma membrane carbonic anhydrase is induced by salt in Dunaliella salina. J. Biol. Chem. 271: 17718–17723.10.1074/jbc.271.30.17718Search in Google Scholar PubMed
Grabherr, M.G., B.J. Haas, M. Yassour, J.Z. Levin, D.A. Thompson, I. Amit, X. Adiconis, L. Fan, R. Raychowdhury, Q. Zeng, Z. Chen, E. Mauceli, N. Hacohen, A. Gnirke, N. Rhind, F. di Palma, B.W. Birren, C. Nusbaum, K. Lindblad-Toh, N. Friedman and A. Regev. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29: 644–652.10.1038/nbt.1883Search in Google Scholar PubMed PubMed Central
Iseli, C., C.V. Jongeneel and P. Bucher. 1999. ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. Proc. Int. Conf. Intell Syst. Mol. Biol.7: 138–148.Search in Google Scholar
Li, L., M.L. Fu, Y.H. Zhao and Y.T. Zhu. 2012. Characterization of carbonic anhydrase II from Chlorella vulgaris in bio-CO2 capture. Environ. Sci. Pollut. Res. 19: 4227–4232.10.1007/s11356-012-1077-8Search in Google Scholar PubMed
Liu, W., Y. Ming, P. Li and Z. Huang. 2012. Inhibitory effects of hypo-osmotic stress on extracellular carbonic anhydrase and photosynthetic efficiency of green alga Dunaliella salina possibly through reactive oxygen species formation. Plant Physiol. Biochem. 54: 43–48.10.1016/j.plaphy.2012.01.018Search in Google Scholar PubMed
Liu, Z.Y., G.C. Wang and B.C. Zhou. 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99: 4717–4722.10.1016/j.biortech.2007.09.073Search in Google Scholar PubMed
Livak, K.J. and T.D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods25: 402–408.10.1006/meth.2001.1262Search in Google Scholar PubMed
Mitra, M., S.M. Lato, R.A. Ynalvez, Y. Xiao and J.V. Moroney. 2004. Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol. 135: 173–182.10.1104/pp.103.037283Search in Google Scholar PubMed PubMed Central
Moroney, J.V. and R.A. Ynalvez. 2007. Proposed carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii. Eukaryot. Cell6: 1251–1259.10.1128/EC.00064-07Search in Google Scholar PubMed PubMed Central
Moroney, J.V., Y. Ma, W.D. Frey, K.A. Fusilier, T.T. Pham, T.A. Simms, R.J. Dimario, J. Yang and B. Mukherjee. 2011. The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles. Photosynth Res. 109: 133–149.Search in Google Scholar
Ochiai, T., B. Colman and Y. Matsuda. 2007. Acclimation of wild-type cells and CO2-insensitive mutants of the green alga Chlorella ellipsoidea to elevated [CO2]. Plant Cell Environ. 30: 944–951.10.1111/j.1365-3040.2007.01684.xSearch in Google Scholar PubMed
Qiao, H.J., G.C. Wang and X.J. Zhang. 2009. Isolation and characterization of Chlorella sorokiniana GXNN01 (Chlorophyta) with the properties of heterotrophic and microaerobic growth. J. Phycol.45: 1153–1162.10.1111/j.1529-8817.2009.00736.xSearch in Google Scholar PubMed
Premkumar, L., H.M. Greenblatt, U.K. Bageshwar, T. Savchenko, I. Gokhman, A. Zamir and J.L. Sussman. 2003. Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from Dunaliella salina. Acta Crystallogr D Biol Crystallogr. 59: 1084–1086.Search in Google Scholar
Price, G.D., M.R. Badger, F.J. Woodger and B.M. Long. 2008. Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. J. Exp. Bot. 59: 1441–1461.Search in Google Scholar
So, A.K., G.S. Espie, E.B. Williams, J.M. Shively, S. Heinhorst and G.C. Cannon. 2004. A novel evolutionary lineage of carbonic anhydrase (ε class) is a component of the carboxysome shell. J. Bacteriol. 186: 623–630.10.1128/JB.186.3.623-630.2004Search in Google Scholar PubMed PubMed Central
Tachibana, M., A. E. Allen, S. Kikutani, Y. Endo, C. Bowler and Y. Matsuda. 2011. Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. Photosynth. Res.109: 205–221.Search in Google Scholar
Tripp, B.C., K. Smith and J.G. Ferry. 2001. Carbonic anhydrase: new insights for an ancient enzyme. J. Biol. Chem. 276: 48615–48618.10.1074/jbc.R100045200Search in Google Scholar PubMed
Xu, J.F., X. Fan, X.W. Zhang, D. Xu, S.L. Mou, S.N. Cao, Z. Zheng, J.L. Miao and N.H. Ye. 2012. Evidence of coexistence of C3 and C4 photosynthetic pathways in a green-tide-forming alga, Ulva prolifera. PLoS One 7: e37438.10.1371/journal.pone.0037438Search in Google Scholar PubMed PubMed Central
Yamano, T. and H. Fukuzawa. 2009. Carbon-concentrating mechanism in a green alga, Chlamydomonas reinhardtii, revealed by transcriptome analyses. J. Basic Microbiol. 49: 42–51.Search in Google Scholar
Yamano, T., K. Miura and H. Fukuzawa. 2008. Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol. 147: 340–354.10.1104/pp.107.114652Search in Google Scholar PubMed PubMed Central
Ye, J., L. Fang, H. Zheng, Y. Zhang, J. Chen, Z.J. Zhang, J. Wang, S.T. Li, R.Q. Li, L. Bolund and J. Wang. 2006. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34: 293–297.10.1093/nar/gkl031Search in Google Scholar PubMed PubMed Central
Yoshioka, S., F. Taniguchi, K. Miura, T. Inoue, T. Yamano and H. Fukuzawa. 2004. The novel Myb transcription factor LCR1 regulates the CO2-responsive gene Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Cell 16: 1466–1477.Search in Google Scholar
Yu, J.L., J.R. Xia and Y.D. Zou. 2011. Response of carbon anhydrase activity and photosynthesis to high salinity stress in Nitzschia closterium f. minutissima. J. Fisheries China 35: 515–523 [in Chinese].Search in Google Scholar
Zhang, B.Y., F. Yang, G.C. Wang and G. Peng. 2010. Cloning and quantitative analysis of the carbonic anhydrase gene from Porphyra yezoensis. J. Phycol.46: 290–296.10.1111/j.1529-8817.2009.00801.xSearch in Google Scholar
©2014 by De Gruyter
