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Marine Genomics
journal homepage: www.elsevier.com/locate/margen
3Q1
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
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a b s t r a c t
Article history:
Received 9 September 2013
Received in revised form 10 October 2013
Accepted 27 October 2013
Available online xxxx
We established a high-efciency nuclear transformation method for the diatom Phaeodactylum tricornutum using
an electroporation system. Based on a universal electroporation protocol, the conditions for the introduction of
exogenous DNA including electric eld strength and plasmid form were optimized. Following optimization,
the diatom cells could be transformed with exogenous gene easily, the maximum transformation frequency
obtained was 2.8 105 cells. The cotransformation of P. tricornutum with a non-selective GUS gene together
with the selectable resistance gene has also been achieved using our new method and found to be very efcient
(up to 60%). The electroporation procedure described in this article offers a number of advantages, including
simplicity, general utility, low-cost and high efciency. The described method also provides some clue for developing electroporation transformation system in other eukaryotic microalgae.
2013 Published by Elsevier B.V.
Keywords:
Electroporation
Diatom transformation
Cotransformation
eGFP
GUS
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1. Introduction
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Please cite this article as: Zhang, C., Hu, H., High-efciency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation,
Mar. Genomics (2013), http://dx.doi.org/10.1016/j.margen.2013.10.003
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Please cite this article as: Zhang, C., Hu, H., High-efciency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation,
Mar. Genomics (2013), http://dx.doi.org/10.1016/j.margen.2013.10.003
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DNA isolated from wild type cells. The eGFP uorescence was observed
under a uorescent microscope. Fluorescence was particularly bright in
a central cellular area (Fig. 3B), which showed that the eGFP gene was
successfully integrated into the genome.
To introduce GUS gene into P. tricornutum cells by electroporation,
we developed a system to cotransform P. tricornutum with two different
plasmids, one containing a selectable gene conferring resistance
to zeocin (pPha-T1) and the other containing a nonselectable uidA
gene (pFcpBp-GUS). Among 20 randomly picked transformants, 12
transformants contained not only sh ble but also uidA, which showed
that the efciency of cotransformation was 60%. Fig. 4A showed the
presence of sh ble as well as uidA in 4 transgenic cells from 6 resistant
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enzymes digested sample, while they contained 34 hybridizing fragments with EcoRI or HindIII restriction enzyme digested DNA. No
bands were detected in DNA extracted from wild type. The result of
the Southern blot analysis conrmed successful incorporation of the
transgene into the nuclear genomes of the transformants. The Southern
blot analysis also indicated multiple insertions of the transgene and
suggested that integration into the genome were random.
In addition, gene transfer ability was also determined by introducing
two reporter plasmids peGFP and pFcpBp-GUS through electroporation.
The presence of the introduced eGFP was detected by genomic PCR
(Fig. 3A). The housekeeping gene histone H4 was used as a control for
PCR amplication. No amplied products were recovered from genomic
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Fig. 1. Transformation efciency as a function of electric eld voltage (A) and DNA forms (B) used during the electroporation of P. tricornutum with 4 g pPha-T1 (CP: circular vector;
LP: linearized vector; CP&SS: circular vector with carrier DNA; LP&SS: linearized vector with carrier DNA) in a 2-mm electroporation cuvette (Bio-Rad). Error bars denote SD, n = 3.
Fig. 3. PCR detection (A) and expression of eGFP gene (B, C) in transgenic P. tricornutum
cells with wild type as the negative control (D, E). PCR products were amplied using
primers specic for the histone H4 gene and the egfp reporter gene from genomic DNAs
of ve zeocin resistant clones (G1G5) and wild type (wt). Fluorescent (B) and light
(C) image of transgenic P. tricornutum cells (G2) expressing eGFP.
Please cite this article as: Zhang, C., Hu, H., High-efciency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation,
Mar. Genomics (2013), http://dx.doi.org/10.1016/j.margen.2013.10.003
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References
247
Apt, K.E., Kroth-Pancic, P.G., Grossman, A., 1996. Stable nuclear transformation of the
diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 252, 572579.
Armbrust, E.V., 2009. The life of diatoms in the world's oceans. Nature 459, 185192.
Bowler, C., Vardi, A., Allen, A.E., 2010. Oceanographic and biogeochemical insights from
diatom genomes. Annu. Rev. Mar. Sci. 2, 333365.
Dnahay, T.G., Jarvis, E.E., Roessler, P.G., 1995. Genetic transformation of the diatoms
Cyclotella cryptica and Navicula saprophila. J. Phycol. 31, 10041012.
Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C., Bowler, C., 1999. Transformation of
nonselectable reporter genes in marine diatoms. Mar. Biotechnol. 1, 239251.
Falkowski, P.G., Barber, R.T., Smetacek, V., 1998. Biogeochemical controls and feedbacks
on ocean primary production. Science 281, 200206.
Field, C.B., Behrenfeld, M.J., Randerson, J.T., Falkowski, P., 1998. Primary production of the
biosphere: integrating terrestrial and oceanic components. Science 281, 237240.
Fischer, H., Robl, I., Sumper, M., Krger, N., 1999. Targeting and covalent modication of
cell wall and membrane proteins heterologously expressed in the diatom
Cylindrotheca fusiformis (Bacillariophyceae). J. Phycol. 35, 113120.
Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In:
Smith, W.L., Canley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum
Press, New York, pp. 2960.
Guo, S.-L., Zhao, X.-Q., Tang, Y., Wan, C., Alam, M.A., Ho, S.-H., Bai, F.-W., Chang, J.-S., 2013.
Establishment of an efcient genetic transformation system in Scenedesmus obliquus.
J. Biotechnol. 163, 6168.
Harrison, P.J., Waters, R.E., Taylor, F.J.R., 1980. A broad spectrum articial seawater medium
for coastal and open ocean phytoplankton. J. Phycol. 16, 2835.
Jefferson, R.A., Kavanagh, T.A., Bevan, M.W., 1987. GUS fusions: -glucuronidase as a
sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 39013907.
Kilian, O., Benemann, C.S.E., Niyogi, K.K., Vick, B., 2011. High-efciency homologous
recombination in the oil-producing alga Nannochloropsis sp. Proc. Natl. Acad. Sci. U.
S. A. 108, 2126521269.
Miyagawa, A., Okami, T., Kira, N., Yamaguchi, H., Ohnishi, K., Adachi, M., 2009. High efciency transformation of the diatom Phaeodactylum tricornutum with a promoter
from the diatom Cylindrotheca fusiformis pre. Phycol. Res. 57, 142146.
Miyagawa-Yamaguchi, A., Okami, T., Kira, N., Yamaguchi, H., Ohnishi, K., Adachi, M., 2011.
Stable nuclear transformation of the diatom Chaetoceros sp. Phycol. Res. 59, 113119.
Miyahara, M., Aoi, M., Inoue-Kashino, N., Kashino, Y., Ifuku, K., 2013. Highly efcient transformation of the diatom Phaeodactylum tricornutum by multi-pulse electroporation.
Biosci. Biotechnol. Biochem. 77, 874876.
Muto, M., Fukuda, Y., Nemoto, M., Yoshino, T., Matsunaga, T., Tanaka, T., 2013. Establishment
of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain
JPCC DA0580a high triglyceride producer. Mar. Biotechnol. 15, 4855.
Niu, Y.-F., Yang, Z.-K., Zhang, M.-H., Zhu, C.-C., Yang, W.-D., Liu, J.-S., Li, H.-Y., 2012. Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker. BioTechniques 13.
Poulsen, N., Krger, N., 2005. A new molecular tool for transgenic diatomscontrol of
mRNA and protein biosynthesis by an inducible promoterterminator cassette.
FEBS J. 272, 34133423.
Poulsen, N., Chesley, P.M., Krger, N., 2006. Molecular genetic manipulation of the diatom
Thalassiosira pseudonana (Bacillariophyceae). J. Phycol. 42, 10591065.
Qin, S., Lin, H., Jiang, P., 2012. Advances in genetic engineering of marine algae. Biotechnol.
Adv. 30, 16021613.
Radakovits, R., Jinkerson, R.E., Fuerstenberg, S.I., Tae, H., Settlage, R.E., Boore, J.L., Posewitz,
M.C., 2012. Draft genome sequence and genetic transformation of the oleaginous alga
Nannochloropis gaditana. Nat. Commun. 3, 686.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, 2nd
edn. Cold Spring Harbor Laboratory Press, NY.
Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998. A Look Back at the U.S. Department of Energy's Aquatic Species ProgramBiodiesel from Algae. National Renewable
Energy Laboratory, Colorado.
Shimogawara, K., Fujiwara, S., Grossman, A., Usuda, H., 1998. High-efciency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148, 18211828.
Smetacek, V., 1999. Diatoms and the ocean carbon cycle. Protist 150, 2532.
Zaslavskaia, L.A., Lippmeier, J.C., Kroth, P.G., Grossman, A.R., Apt, K.E., 2000. Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of
selectable marker and reporter genes. J. Phycol. 36, 379386.
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Fig. 4. PCR detection (A) and expression of GUS gene (B, C) in transgenic P. tricornutum
cells with wild type as the negative control (D, E). PCR products were amplied using
primers specic for sh ble resistance gene and uidA reporter gene from genomic DNAs of
six zeocin resistant clones (U1U6 for cotransformed cells). GUS histochemical assay of
transformed cells without washing with ethanol (B) and washed with 70% ethanol (C).
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Acknowledgments
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Please cite this article as: Zhang, C., Hu, H., High-efciency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation,
Mar. Genomics (2013), http://dx.doi.org/10.1016/j.margen.2013.10.003
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