The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.
References
[1]
Fernández, E.; Schnell, R.; Ranum, L.P.; Hussey, S.C.; Silflow, C.D.; Lefebvre, P.A. Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 1989, 86, 6449–6453, doi:10.1073/pnas.86.17.6449.
[2]
Dunahay, T.G.; Jarvis, E.E.; Roessle, P.G. Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J. Phycol. 1995, 31, 1004–1012.
[3]
Apt, K.E.; Kroth-Pancic, P.G.; Grossman, A.R. Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 1996, 252, 572–579.
[4]
León-Ba？ares, R.; González-Ballester, D.; Galván, A.; Fernández, E. Transgenic microalgae as green cell- factories. Trends Biotechnol. 2004, 22, 45–52.
[5]
Walker, T.L.; Collet, C.; Purton, S. Algal transgenics in the genomic era. J. Phycol. 2005, 41, 1077–1093, doi:10.1111/j.1529-8817.2005.00133.x.
[6]
León, R.; Fernández, E. Nuclear transformation of eukaryotic microalgae: Historical overview, achievementes and problems. Adv. Exp. Med. Biol. 2007, 616, 1–11, doi:10.1007/978-0-387-75532-8_1.
[7]
Eichler-Stahlberg, A.; Weisheit, W.; Ruecker, O.; Heitzer, M. Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 2009, 229, 873–883, doi:10.1007/s00425-008-0879-x.
Harris, E.H. Chlamydomonas as a model organism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 363–406, doi:10.1146/annurev.arplant.52.1.363.
[10]
Kim, D.H.; Kim, Y.T.; Cho, J.J.; Bae, J.H.; Hur, S.B.; Hwang, I.; Choi, T.J. Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea. Mar. Biotechnol. 2002, 4, 63–73, doi:10.1007/s1012601-0070-x.
[11]
Feng, S.; Xue, L.; Liu, H.; Lu, P. Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method. Mol. Biol. Rep. 2009, 36, 1433–1439, doi:10.1007/s11033-008-9333-1.
[12]
Walker, T.L.; Becker, D.K.; Collet, C. Characterisation of the Dunaliella tertiolecta RbcS genes and their promoter activity in Chlamydomonas reinhardtii. Plant Cell. Rep. 2005, 23, 727–735, doi:10.1007/s00299-004-0884-x.
[13]
Neupert, J.; Shao, N.; Lu, Y.; Bock, R. Genetic transformation of the model green alga Chlamydomonas reinhardtii. Methods Mol. Biol. 2012, 847, 35–47.
[14]
Dawson, H.N.; Burlingame, R.; Cannons, A.C. Stable Transformation of Chlorella: Rescue of nitrate reductase-deficient mutants with the nitrate reductase. Gene Curr. Microbiol. 1997, 35, 356–362, doi:10.1007/s002849900268.
[15]
Sun, Y.; Yang, Z.; Gao, X.; Li, Q.; Zhang, Q.; Xu, Z. Expression of foreign genes in Dunaliella by electroporation. Mol. Biotechnol. 2005, 30, 185–192, doi:10.1385/MB:30:3:185.
[16]
Walker, T.L.; Becker, D.K.; Dales, J.L.; Collet, C. Towards the development of a nuclear transformation system for Dunaliella tertiolecta. J. Appl. Phycol. 2005, 17, 363–368, doi:10.1007/s10811-005-4783-5.
[17]
Steinbrenner, J.; Sandmann, G. Transformation of the Green Alga Haematococcus pluvialis with a phytoenedesaturase for accelerated astaxanthin biosynthesis. Appl. Environ. Microbiol. 2006, 72, 7477–7484, doi:10.1128/AEM.01461-06.
[18]
Corellou, F.; Schwartz, C.; Motta, J.P.; Djouani-Tahri, B.; Sanchez, F.; Bouget, F.Y. Clocks in the green lineage: Comparative functional analysis of the circadian architecture of the picoeukaryote Ostreococcus. Plant Cell. 2009, 21, 3436–3449, doi:10.1105/tpc.109.068825.
[19]
Kiliana, O.; Benemann, C.S.E.; Niyogi, K.K.; Vicka, B. High-efficiency homologous recombinationin the oil-producing alga Nannochloropsis sp. Proc. Natl. Acad. Sci. USA 2011, 108, 21265–21269.
Blanvillain, R.; Gallois, P. Promoter trapping system to study embryogenesis. Methods Mol. Biol. 2008, 427, 121–135.
[22]
Springer, P.S. Gene traps tools for plant development and genomics. Plant Cells 2000, 12, 1007–1020.
[23]
Haring, M.A.; Beck, C.F. A promoter trap for Chlamydomonas reinhardtii: Development of a gene cloning method using 5′ RACE-based probes. Plant J. 1997, 11, 1341–1348.
[24]
Auchincloss, A.H.; Lorocha, A.I.; Rochaix, J.D. The argininosuccinatelyase gene of Chlamydomonas reinhardtii: Cloning of the cDNA and its characterization as a selectable shuttle marker. Mol. Gen. Genet. 1999, 261, 21–30, doi:10.1007/s004380050937.
[25]
Ohresser, M.; Matagne, R.F.; Loppes, R. Expression of the arylsuphatase reporter gene under the control of the NIT1 promoter in Chlamydomonas reinhartii. Curr. Genet. 1997, 31, 264–271, doi:10.1007/s002940050204.
[26]
Gónzalez-Ballester, D.; de Montaigu, A.; Higuera, J.J.; Galván, A.; Fernández, E. Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas. Plant Physiol. 2005, 137, 522–533.
[27]
Dent, R.; Haglund, C.; Chin, B.L.; Kobayashi, M.C.; Niyogi, K.K. Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol. 2005, 137, 545–556, doi:10.1104/pp.104.055244.
[28]
Pollock, S.V.; Pootakham, W.; Shibagaki, N.; Moseley, J.L.; Grossman, A.R. Insights into the acclimation of Chlamydomonas reinhardtii to sulfur deprivation. Photosynth. Res. 2005, 86, 475–489.
[29]
Gónzalez-Ballester, D.; Pootakham, W.; Mus, F.; Yang, W.; Catalanotti, C.; Magneschi, L.; de Montaigu, A.; Higuera, J.J.; Prior, M.; Galván, A.; Fernandez, E.; Grossman, A.R. Reverse genetics in Chlamydomonas: A platform for isolating insertional mutants. Plant Methods 2011, 27, 24.
[30]
Sizova, I.; Fuhrmann, M.; Hegemann, P. A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 2001, 277, 221–229.
[31]
Lerche, K.; Hallmann, A. Stable nuclear transformation of Gonium pectoral. BMC Biotechnol. 2009, 9, 64–69, doi:10.1186/1472-6750-9-64.
[32]
Chen, P.Y.; Wang, C.K.; Soong, S.C.; To, K.Y. Complete sequence of binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol. Breed. 2003, 11, 287–293.
[33]
Schroda, M.; Bl？cker, D.; Beck, C.F. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J. 2000, 21, 121–131.
[34]
Chlamydomonas reinhardtii Genome Database. Available online: http://www.phytozome.net/chlamy (accessed on 1 September 2012).
[35]
León, R.; Couso, I.; Fernández, E. Metabolic engineering of ketocarotenoids biosíntesis in the unicelullarmicroalga Chlamydomonas reinhardtii. J. Biotechol. 2007, 130, 143–152.
[36]
Yamamoto, Y.Y.; Tsuhara, Y.; Gohda, K.; Suzuki, K.; Matsui, K.M. Gene trapping of the Arabidopsis genome with a firefly luciferase reporter. Plant J. 2003, 35, 273–283, doi:10.1046/j.1365-313X.2003.01797.x.
[37]
Fuhrmann, M.; Oertel, W.; Hegemann, P. A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J. 1999, 19, 353–361, doi:10.1046/j.1365-313X.1999.00526.x.
[38]
Fuhrmann, M.; Hausherr, A.; Ferbitz, L.; Sch？dl, T.; Heitze, M.; Hegemann, P. Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol. Biol. 2004, 55, 869–881.
[39]
Plesch, G.; Kamann, E.; Mueller-Roeber, B. Cloning of regulatory sequences mediating guard-cell-specific gene expression. Gene 2000, 249, 83–89.
[40]
Liu, Y.G.; Whittier, R.F. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 1995, 25, 674–681, doi:10.1016/0888-7543(95)80010-J.
[41]
González-Ballester, D.; de Montaigu, A.; Galván, A.; Fernández, E. Restriction enzyme site-directed amplification PCR: A tool to identify regions flanking a marker DNA. Anal. Biochem. 2005, 340, 330–335, doi:10.1016/j.ab.2005.01.031.
[42]
Tonooka, Y.; Fujishima, M. Comparison and critical evaluation of PCR-mediated methods to walk along the sequence of genomic DNA. Appl. Microbiol. Biotechnol. 2009, 85, 37–43, doi:10.1007/s00253-009-2211-5.
[43]
De Montaigu, A.; Sanz-Luque, E.; Macias, M.I.; Galván, A.; Fernández, E. Transcriptional regulation of CDP1 and CYG56 is required for proper NH4+ sensing in Chlamydomonas. J. Exp. Bot. 2011, 62, 1425–1437.
[44]
Fischer, N.; Rochaix, J.D. The flanking regions of PsaDdrive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol. Genet. Genomics 2001, 265, 888–894, doi:10.1007/s004380100485.
[45]
Merchant, S.S.; Prochnik, S.E.; Vallon, O.; Harris, E.H.; Karpowicz, S.J.; Witman, G.B.; Terry, A.; Salamov, A.; Fritz-Laylin, L.K.; Maréchal-Drouard, L.; et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 2007, 318, 245–250.
[46]
Bowler, C.; Allen, A.E.; Badger, J.H.; Grimwood, J.; Jabbari, K.; Kuo, A.; Maheswari, U.; Martens, C.; Maumus, F.; Otillar, R.P.; et al. The Phaeodactylum genome reveals the evolutionary history of diatom genome. Nature 2008, 456, 239–244.
[47]
Derelle, E.; Ferraz, C.; Rombauts, S.; Rouzé, P.; Worden, A.Z.; Robbens, S.; Partensky, F.; Degroeve, S.; Echeynié, S.; Cooke, R.; et al. Moreau, genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc. Natl. Acad. Sci. USA 2006, 103, 11647–11652.
[48]
Worden, A.Z.; Lee, J.-H.; Mock, T.; Rouzé, P.; Simmons, M.P.; Aerts, A.L.; Allen, A.E.; Cuvelier, M.L.; Derelle, E.; Everett, M.V.; et al. Green evolution and dynamic adaptations revealed by genomes of the marine Picoeukaryotes Micromonas. Science 2009, 324, 268–272.
[49]
The Nuclear Genome Sequencing Project of Microalgae. Available online: http://genome.jgi-psf.org/programs/plants/plant-projects.jsf (accessed on 1 September 2012).
[50]
Loppes, R.; Radoux, M.; Ohresser, M.C.; Matagne, R.F. Transcriptional regulation of the Nia1 gene encoding nitrate reductase in Chlamydomonas reinhardtii: Effects of various environmental factors on the expression of a reporter gene under the control of the Nia1 promoter. Plant Mol. Biol. 1999, 41, 701–711.
[51]
Harris, E.H. ChlamydomonasSourcebook: Introduction to Chlamydomonas and Its Laboratory Use, 2nd; Stern, D., Witman, G., Eds.; Academic Press: San Diego, CA, USA, 2009; Volume 1.
[52]
Sambrook, J.; Russell, D.W. Molecular Cloning, a Laboratory Manual, 3rd ed.; Cold Spring Harbour Laboratory Press: Cold Spring Harbour, New York, NY, USA, 2001.
[53]
Kindle, K.L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 1990, 87, 1228–1232, doi:10.1073/pnas.87.3.1228.
[54]
Ingham, D.J.; Beer, S.; Money, S.; Hansen, G. Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 2001, 31, 132-134, 136-140.
