Accessible Requires Authentication Published by De Gruyter September 29, 2014

Transcriptome sequencing of the marine microalga, Chlorella pyrenoidosa (Chlorophyta), and analysis of carbonic anhydrase expression under salt stress

Xue Sun, Weiwei Wang, Jia Shen and Nianjun Xu
From the journal Botanica Marina

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.


Corresponding author: Nianjun Xu, Key Laboratory of Marine Biotechnology of Zhejiang Province, School of Marine Sciences, Ningbo University, Zhejiang Ningbo 315211, P.R. China, e-mail:

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search 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. Search 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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

Moroney, J.V. and R.A. Ynalvez. 2007. Proposed carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii. Eukaryot. Cell6: 1251–1259. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

Received: 2014-3-11
Accepted: 2014-9-2
Published Online: 2014-9-29
Published in Print: 2014-10-1

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