A New Trend for the Highly Efficient Transformation of the Microalga Chlorella Vulgaris by Agrobacterium Tumefaciens
Abstract
The microscopic green microalga Chlorella Vulgaris is an important member of photosynthetic microorganisms, producing a wide range of natural high-value compounds. Recently, C. Vulgaris has been gained a great attention as a green bioreactor for the production of valuable biologicals ranging from therapeutic proteins to biofuels. Because of the lack of robust, efficient and low-cost transformation techniques, the significant potential of this expression system is often limited. The Agrobacterium-mediated genetic transformation as one of the most efficient method for the gene transformation can be an ideal solution for the microalgae genetic engineering. The transformation of C. Vulgaris was evaluated using A. tumifaciens strain EHA101, carrying pCAMBIA1304 binary vector. Integration and expression of mgfp:uidA and hptII genes in the transformed cells were determined by Polymerase Chain Reaction (PCR) and Reverse Transcription Polymerase Chain Reaction (RT-PCR), facs flow cytometry and β-Glucuronidase (GUS) activity assays. PCR data confirmed the successful integration of the mgfp and hptII genes into the C. Vulgaris genome. RT-PCR analysis showed the transcripts of the genes were present in the transcriptome of the transformants. The expression of mgfp and uidA genes were successfully evaluated through the FL-1 channel in the facs flow cytometry and GUS activity assays. The strain EHA101 of A. tumifaciens could be used as a more ideal platform for the transformation of Chlorella in order to the high-efficiency production of valuable recombinant proteins.
Keywords:
Agrobacterium-mediated transformation, Chlorella vulgaris, Gene expression, Microalgae, Recombinant proteinReferences
- [1] Yang, B., Liu, J., Jiang, Y., & Chen, F. (2016). Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective. Biotechnology journal, 11(10), 1244–1261. https://doi.org/10.1002/biot.201500617
- [2] Kalhor, A. X., Movafeghi, A., Mohammadi-Nassab, A. D., Abedi, E., & Bahrami, A. (2017). Potential of the green alga Chlorella vulgaris for biodegradation of crude oil hydrocarbons. Marine pollution bulletin, 123(1–2), 286–290. https://doi.org/10.1016/j.marpolbul.2017.08.045
- [3] Gong, Y., Hu, H., Gao, Y., Xu, X., & Gao, H. (2011). Microalgae as platforms for production of recombinant proteins and valuable compounds: Progress and prospects. Journal of industrial microbiology and biotechnology, 38(12), 1879–1890. https://doi.org/10.1007/s10295-011-1032-6
- [4] Dehghani, J., Adibkia, K., Movafeghi, A., Barzegari, A., Pourseif, M. M., Maleki Kakelar, H., ... & Omidi, Y. (2018). Stable transformation of Spirulina (Arthrospira) platensis: A promising microalga for production of edible vaccines. Applied microbiology and biotechnology, 102(21), 9267-9278. https://doi.org/10.1007/s00253-018-9296-7
- [5] Dehghani, J., Movafeghi, A., Barzegari, A., & Barar, J. (2017). Efficient and stable transformation of Dunaliella pseudosalina by 3 strains of Agrobacterium tumefaciens. BioImpacts: Bi, 7(4), 247. https://doi.org/10.15171/bi.2017.29
- [6] Rasala, B. A., Muto, M., Lee, P. A., Jager, M., Cardoso, R. M., Behnke, C. A., ... & Mayfield, S. P. (2010). Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant biotechnology journal, 8(6), 719-733. https://doi.org/10.1111/j.1467-7652.2010.00503.x
- [7] Dawson, H. N., Burlingame, R., & Cannons, A. C. (1997). Stable transformation of Chlorella: Rescue of nitrate reductase-deficient mutants with the nitrate reductase gene. Current microbiology, 35(6), 356–362. https://doi.org/10.1007/s002849900268
- [8] Wang, C., Wang, Y., Su, Q., & Gao, X. (2007). Transient expression of the GUS gene in a unicellular marine green alga, Chlorella sp. MACC/C95, via electroporation. Biotechnology and bioprocess engineering, 12(2), 180–183. https://doi.org/10.1007/BF03028646
- [9] Kim, D. H., Kim, Y. T., Cho, J. J., Bae, J. H., Hur, S. B., Hwang, I., & Choi, T. J. (2002). Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea. Marine biotechnology, 4(1), 63-73. https://doi.org/10.1007/s1012601-0070-x
- [10] Cha, T. S., Yee, W., & Aziz, A. (2012). Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris. World journal of microbiology and biotechnology, 28(4), 1771–1779. https://doi.org/10.1007/s11274-011-0991-0
- [11] Sanitha, M., Radha, S., Fatima, A. A., Devi, S. G., & Ramya, M. (2014). Agrobacterium-mediated transformation of three freshwater microalgal strains. Polish journal of microbiology, 63(4), 382–387. https://doi.org/10.33073/pjm-2014-052
- [12] Cha, T. S., Chen, C. F., Yee, W., Aziz, A., & Loh, S. H. (2011). Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp. Journal of microbiological methods, 84(3), 430-434. https://doi.org/10.1016/j.mimet.2011.01.005
- [13] Gelvin, S. B. (2000). Agrobacterium and plant genes involved in T-DNA transfer and integration. Annual review of plant biology, 51(1), 223–256. https://doi.org/10.1146/annurev.arplant.51.1.223
- [14] Hamilton, C. M., Frary, A., Lewis, C., & Tanksley, S. D. (1996). Stable transfer of intact high molecular weight DNA into plant chromosomes. Proceedings of the national academy of sciences, 93(18), 9975–9979. https://doi.org/10.1073/pnas.93.18.9975
- [15] Bundock, P., den Dulk-Ras, A., Beijersbergen, A., & Hooykaas, P. (1995). Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. The embo journal, 14(13), 3206–3214. https://doi.org/10.1002/j.1460-2075.1995.tb07323.x
- [16] De Groot, M. J. A., Bundock, P., Hooykaas, P. J. J., & Beijersbergen, A. G. M. (1998). Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature biotechnology, 16(9), 839–842. https://doi.org/10.1038/nbt0998-839
- [17] Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C., & Citovsky, V. (2001). Genetic transformation of HeLa cells by Agrobacterium. Proceedings of the national academy of sciences, 98(4), 1871–1876. https://doi.org/10.1073/pnas.98.4.1871
- [18] Kumar, S. V., Misquitta, R. W., Reddy, V. S., Rao, B. J., & Rajam, M. V. (2004). Genetic transformation of the green alga—Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant science, 166(3), 731–738. https://doi.org/10.1016/j.plantsci.2003.11.012
- [19] Chen, Y., Wang, Y., Sun, Y., Zhang, L., & Li, W. (2001). Highly efficient expression of rabbit neutrophil peptide-1 gene in Chlorella ellipsoidea cells. Current genetics, 39(5), 365–370. https://doi.org/10.1007/s002940100205
- [20] Koo, J., Park, D., & Kim, H. (2013). Expression of bovine lactoferrin N-lobe by the green alga, Chlorella vulgaris. Algae, 28(4), 379–387. https://doi.org/10.4490/algae.2013.28.4.379
- [21] Stanier, R. Y., Kunisawa, R., Mandel, M., & Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriological reviews, 35(2), 171–205. https://doi.org/10.1128/br.35.2.171-205.1971
- [22] Holsters, M., De Waele, D., Depicker, A., Messens, E., Van Montagu, M., & Schell, J. (1978). Transfection and transformation of Agrobacterium tumefaciens. Molecular and general genetics (MGG), 163(2), 181–187. https://doi.org/10.1007/BF00267408
- [23] Coll, J. M. (2006). Methodologies for transferring DNA into eukaryotic microalgae: A review. Spanish journal of agricultural research, 4(4), 316–330. https://doi.org/10.5424/sjar/2006044-209
- [24] Luo, D., & Saltzman, W. M. (2000). Synthetic DNA delivery systems. Nature biotechnology, 18(1), 33–37. https://doi.org/10.1038/71889
- [25] Gelvin, S. B. (2003). Agrobacterium-mediated plant transformation: The biology behind the “gene-jockeying” tool. Microbiology and molecular biology reviews, 67(1), 16–37. https://doi.org/10.1128/mmbr.67.1.16-37.2003
- [26] Kathiresan, S., Chandrashekar, A., Ravishankar, G. A., & Sarada, R. (2009). Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales) 1. Journal of phycology, 45(3), 642–649. https://doi.org/10.1111/j.1529-8817.2009.00688.x
- [27] Anila, N., Chandrashekar, A., Ravishankar, G. A., & Sarada, R. (2011). Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil. European journal of phycology, 46(1), 36–44. https://doi.org/10.1080/09670262.2010.550386
- [28] Cheng, R., Ma, R., Li, K., Rong, H., Lin, X., Wang, Z., ... & Ma, Y. (2012). Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium. Microbiological research, 167(3), 179-186. https://doi.org/10.1016/j.micres.2011.05.003
- [29] Pratheesh, P. T., Vineetha, M., & Kurup, G. M. (2014). An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Molecular biotechnology, 56(6), 507–515. https://doi.org/10.1007/s12033-013-9720-2
- [30] Rajam, M. V, & Kumar, S. V. (2006). Green alga (Chlamydomonas reinhardtii). In Agrobacterium protocols (pp. 421–433). Springer. https://doi.org/10.1385/1-59745-131-2:421