Nannochloropsis has been believed to be a promising genus of microalgae, which has the potential as the feed stock for biofuels. To achieve the goal for industrial application, use of genetic modifications to improve oil-producing and growth characters is of great significance in reduction of costs. So, it is necessary to develop an efficient transformation method. Here, a vector pHB4857 that has a sh ble gene derived by Nannochloropsis β-tublin promoter and Phaeodactylum tricornutum fcpA terminator was constructed and transformed in N. gaditana CCAP849/5 by electroporation. Transformants were able to grow on 3 μg·mL-1 zeocin. PCR detection indicated that 100% of the selected colonies were positive transformants. The efficiency was 1.25×10ˉ6. A Southern blot analysis verified that the sh ble gene with single or multiple copies was randomly inserted into the geneome and most of the transformants owned the intact heterologous genes. The transformants were inoculated in fresh zeocin-resistant medium every 10 days for more than seven months. The integration of heterologous gene in the host nuclear genome appeared to be stable since no sh ble gene was lost.
YU Gui-lan
,
HU Han-hua
,
LI Xiu-bo
,
KONG Ren-qiu
. Transformation of marine oleaginous alga Nannochloropsis gaditana CCAP849/5 and heterologous gene integration[J]. Journal of Tropical Oceanography, 2014
, 33(2)
: 72
-77
.
DOI: 10.11978/j.issn.1009-5470.2014.02.010
[1]黄伟超, 胡晗华. 2013. 微拟球藻属对盐度的耐受及其产油特性分析[J]. 水生生物学报, 37(2): 384-387.
[2]李秀波, 徐旭东, 孔任秋. 2010. 五种微绿球藻产油和产多不饱和酸的研究[J]. 水生生物学报, 34 (5): 893-897.
[3]刘飞飞, 李秀波, 方仙桃, 等. 2013. 三角褐指藻产油突变株的筛选[J]. 水生生物学报, 37(4)：799-802.
[4]BRENNAN L, OWENDR P. 2010. Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products[J]. Renewable and Sustainable Energy Reviews, 14: 557-577.
[5]CHEN H L, LI S S, HUANG R, et al. 2008. Conditional production of a functional fish growth hormone in the transgenic line of Nannochloropsis oculata (Eustigmato- phyceae)[J]. J Phycol, 44: 768-776.
[6]GONG Y, HU H, GAO Y, et al. 2011. Microalgae as platforms for production of recombinant proteins and valuable compounds: Progress and prospects[J]. J Ind Microbiol Biotechnol, 38: 1879-1890.
[7]GUILLARD R R L.1975. Culture of phytoplankton for feeding marine invertebrates[C]//SMITH W L, CHANLEY M H. Culture of marine invertebrate animals. Plenum: New York Press: 29-60.
[8]HEMPEL F, BOZARTH A S, LINDENKAMP N, et al. 2011. Microalgae as bioreactors for bioplastic production[J]. Microbial Cell Factories, 10: 81.
[9]HIBBERD D J. 1981. Notes on taxonomy and nomenclature of the algal classes Eustigmatophyceae and Tribophyceae (Synonym Xanthophyceae)[J]. Bot J Linn Soc, 82: 93-119.
[10]JINKERSON R E, RADAKOVITS R, POSEWITZ M C. 2013. Genomic insights from the oleaginous model alga Nannochloropsis gaditana[J]. Bioengineered, 4(1): 37-43.
[11]KILIAN O, BENEMANN C S E, NIYOGI K K, et al. 2011. High-efficiency homologous recombination in the oil- producing alga Nannochloropsis sp[J]. Proc Nat Acad Sci, 108: 20859-20860.
[12]LARKUM A W D, ROSS I L, KRUSE O, et al. 2012. Selection, breeding and engineering of microalgae for bioenergy and biofuel production[J]. Trends in Biotechnology, 30(4): 198-205.
[13]LEON R, FEMANDEZ E. 2007. Nuclear transformation of eukaryotic microalgae[J]. Adv Exp Med Biol, 616: 1-11.
[14]LI J, XUE L, YAN H, et al. 2007. The nitrate reductase gene-switch: A system for regulated expression in transformed cells of Dunaliella salina[J]. Gene, 4031: 32-142.
[15]LI S S, TSAI H J. 2009. Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract[J]. Fish Shellfish Immunol, 26: 316-325.
[16]MURRAY M G, THOMPSON W F. 1980. Rapidisolation of high molecular weight plant DNA[J]. Nucleic Acids Research, 8: 4321-4325.
[17]PAN K, QIN J, LI S, et al. 2011. Nuclear monoploidy and asexual propagation of Nannochloropsis oceanica (Eustigm atophyceae) as revealed by its genome sequence[J]. J Phycol, 47: 1425- 1432.
[18]PAWLOWSKI W P, SOMERS D A. 1998. Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA[J]. Proc Nat Acad Sci, 95: 12106-12110.
[19]RADAKOVITS R, JINKERSON R E, FUERSTENBERG S I, et al. 2012. Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana[J]. Nat Commun, 3: 686.
[20]RODOFI L, CHINI ZITTELLI G, BASSI N, et al. 2009. Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor[J]. Biotech and Bioeng, 102(1): 100-112.
[21]SAMBROOK J, RUSSELL D W. 2001. Molecular cloning: a laboratory manual[M]. 3nd ed. New York: Cold Spring Harbor Laboratory Press: 487-499.
[22]SCHENK P M, THOMAS-HALLl S R, POSTEN C, et al. 2008. Second generation biofuels: High-efficiency microalgae for biodiesel production[J]. Bioenerg Res, 1: 20-43.
[23]SOMERS D A, MAKAREVITC I. 2004. Transgene integration in plants: Poking or patching holes in promiscuous genomes[J]. Curr Opin Biotechnol, 15(2): 126-131.
