MARGEN-00166; No of Pages 4

Marine Genomics xxx (2013) xxxxxx

Contents lists available at ScienceDirect

Marine Genomics
journal homepage: www.elsevier.com/locate/margen

3Q1

Chunye Zhang a,b, Hanhua Hu a,

a
b

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

i n f o

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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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Diatoms are one of the most common types of phytoplankton. They

contribute at least 20% of annual primary productivity, equivalent to the
tropical rain forests (Falkowski et al., 1998; Field et al., 1998). Diatoms
play an important role in the biogeochemical cycling of carbon, nitrogen
and silicon in the aquatic ecosystems (Armbrust, 2009; Bowler et al.,
2010; Smetacek, 1999). In addition, since the main storage compounds
of diatoms are lipids (triacylglycerol, over 4060%), they are considered
as one of the most suitable feedstock for biodiesel production (Sheehan
et al., 1998). Therefore, to develop an efcient genetic transformation
system is very important for both basic biological research and commercial exploitation of diatoms.
At present, transformation of diatoms has been established for several
diatom species, including Phaeodactylum tricornutum (Apt et al., 1996;
Falciatore et al., 1999; Miyagawa et al., 2009; Zaslavskaia et al., 2000),
Cylindrotheca fusiformis (Fischer et al., 1999; Poulsen and Krger,
2005), Thalassiosira pseudonana (Poulsen et al., 2006), Chaetoceros sp.
(Miyagawa-Yamaguchi et al., 2011), Fistulifera sp. (Muto et al., 2013),
Navicula saprophila and Cyclotella cryptica (Dnahay et al., 1995). These
established techniques for transporting DNA into diatom cells relied on
microprojectile bombardment. However, microprojectile bombardment
is costly and not routinely available, and the transformation efciency is
low thus limiting its application in many laboratories.
Though high efcient electroporation-mediated transformation was
achieved in eukaryotic Chlamydomonas reinhardtii fteen years ago

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High-efciency nuclear transformation of the diatom Phaeodactylum

tricornutum by electroporation

Corresponding author. Tel./fax: +86 27 68780078.

E-mail address: hanhuahu@ihb.ac.cn (H. Hu).

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(Shimogawara et al., 1998), stable gene transfer by electroporation

was established in other eukaryotic microalgae only very recently, including Nannochloropsis sp. (Kilian et al., 2011; Radakovits et al., 2012)
and Scenedesmus obliquus (Guo et al., 2013). Niu et al. (2012) and
Miyahara et al. (2013) reported the transformation of P. tricornutum
by electroporation, however, the former did not achieve higher transformation efciency compared with microprojectile bombardment
while the latter applied a multi-pulse electroporation system dependent on a special apparatus. In this study, we developed a simple
method for high efciency nuclear transformation of P. tricornutum by
electroporation.

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2. Material and methods

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2.1. Strain and culture conditions

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P. tricornutum Bohlin (CCMP2561) was obtained from the culture

collection of the Provasoli-Guillard National Center for Culture of
Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, USA.
Cells were grown axenically in articial seawater (ASW) (Harrison
et al. 1980) enriched with f/2 nutrients (main components: 0.88 mM
NaNO3, 36.3 M NaH2PO4; micronutrients: 0.08 M ZnSO4, 0.9 M
MnCl2, 0.03 M Na2MoO4, 0.05 M CoCl2, 0.04 M CuSO4, 11.7 M
FeCl 3, 11.7 M EDTA; vitamin: 0.5 g cyanocobalamin l 1, 0.5 g
biotin l 1, 100 g thiamineHCl l 1). (Guillard, 1975) at 22 C and
continuously illuminated with 75 mol photons m2s1. For growth
on solid media, cultures were grown on 50% ASW containing f/2
nutrients, 1.2% agar.

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1874-7787/$ see front matter 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.margen.2013.10.003

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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P. tricornutum was grown to a cell density of 45 106 cellsml1.

A total of 2 108 cells were harvested by centrifugation at 1500 g for
10 min at 4 C, and washed three times with 1 ml 375 mM sorbitol
(sterile and ice cold), then resuspended in 100 l 375 mM sorbitol to
a nal density of 2 109 cellsml1. Under standard conditions, a suspension aliquot of 100 l was mixed with 4 g (0.2 gl1) of plasmid
DNA (linearized by ScaI digestion) and 40 g (10 gl1) of salmon
sperm DNA (denatured by boiling for 1 min), and incubated on ice for
10 min, then transferred into a 2-mm electroporation cuvettes. Electroporation was performed with a Bio-Rad Gene Pulser Xcell Electroporation System. The Electroporation System was adjusted to exponential
decay, 0.5 kV eld strength, 25 F capacitance, and 400 Ohm shunt
resistance. After electroporation, cells were immediately transferred to
15-ml conical Falcon tubes containing 10 ml f/2 medium and incubated
in low light (~30 mol photons m2s1) overnight without shaking.
Cells were then collected by centrifugation at 1500 g for 10 min and
resuspended in 0.6 ml f/2 medium, and 0.2 ml of this suspension was
plated onto solid medium containing 75 gml1 zeocin. Colonies appeared after 1012 d and could be further processed after 2 weeks.
For cotransformation, two linearized plasmids were used and both
were 2 g.

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2.4. PCR and Southern blot analysis

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Cells in the late-logarithmic phase were collected and genomic DNA

was isolated as described (Falciatore et al., 1999). Primer pairs 5-AGGT
CCTTCGCGACAATATC-3 and 5-ACGGAATCACGAATGACGTT-3 for histone H4, 5-ATGGCCAAGTTGACCAGTGCCGT-3 and 5-TTAGTCCTGCTC
CTCGGCCACGAA-3 for sh ble, 5-CTGATAGCGCGTGACAAAAA-3 and
5-GAGCGTCGCAGAACATTACA-3 for uidA, 5-ATGGTGAGCAAGGGCG
AGG-3 and 5-TTACTTGTACAGCTCGTCCATGC-3 for egfp were used in
genomic PCR.
For Southern blot analysis, 5 g of genomic DNA from two
transformants and wild type cells of P. tricornutum was digested with
EcoRI, BamHI or HindIII and loaded on 0.7% agarose gels. After electrophoresis, the DNA samples were transferred to an Immobilon Ny +
membrane (Millipore) and cross-linked with UV light as described
(Sambrook et al., 1989). Digoxigenin labeling of the sh ble DNA probe,
hybridization of the probe with membrane bound DNA and detection
of hybridization were performed using the DIG DNA Labeling and
Detection Kit (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions.

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2.5. GFP and GUS assay

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GFP uorescence cell images were acquired using an Eclipse 80i

uorescence microscope equipped with a DS-Ri1 digital camera
(Nikon, Tokyo, Japan), a GFP lter set. GUS staining was performed as
previously described (Jefferson et al., 1987). Briey, 2 ml of mid-

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3. Results and discussion

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In electroporation, transient holes in the cell membrane are formed

when pulses of an electric current are applied. Gene transfer by electroporation has been widely carried out in animal, plant and bacterial cells
for over 30 years because of the simplicity of the procedure and a
high efciency with a small amount of DNA (Qin et al., 2012). High
efciency of transformation by electroporation was achieved in
microalga C. reinhardtii to 2 10 5 transformants per cell and g of
DNA (Shimogawara et al., 1998). Recently, the oleaginous model alga
Nannochloropsis sp. was also successfully genetically transformed by
electroporation with an efciency of 12 105 (Kilian et al., 2011;
Radakovits et al., 2012). Using a protocol similar to these eukaryotic
microalgae, we transformed P. tricornutum via electroporation with
the plasmid pPha-T1.
Transformants were obtained in electric eld strength from 0.45 to
0.75 kV and using a linearized vector or circular vector with carrier
DNA (salmon sperm DNA was used as the carrier). The electric eld
strength for electroporation of P. tricornutum was higher than that of
C. reinhardtii (Shimogawara et al., 1998), but lower than that of
Nannochloropsis sp. (Kilian et al., 2011; Radakovits et al., 2012), which
might be explained by their different cell wall characteristics. As
shown in Fig. 1A, the most transformants were obtained using 0.5 kV
electric eld strength. For an efcient transformation, it is very important to use a linearized vector and add the carrier DNA. In this study,
no colonies were formed by using only the circular vector (Fig. 1B).
A large enhancement of the transformation frequency by the addition
of carrier DNA and using a linearized vector has been reported in
microalgae (Kilian et al., 2011; Miyahara et al., 2013; Shimogawara
et al., 1998; Radakovits et al., 2012). The maximal transformation efciency reached about 2.8 105, at least 10 times higher than reported
in P. tricornutum transformation by microprojectile bombardment
(Miyagawa et al., 2009; Zaslavskaia et al., 2000). Niu et al. (2012) used
similar electroporation parameters but a different buffer for transformation of P. tricornutum and achieved a comparable transformation efciency to that by microprojectile bombardment. However, a detailed
protocol was not presented in their paper.
Miyahara et al. (2013) also reported electroporation transformation
of P. tricornutum, however, the protocol applied a multi-pulse electroporation for the transformation, and the efciency depends on the number
of poring pulses applied. Although the transformation efciency is a
little bit higher than the achieved efciency by our protocol, the
multi-pulse electroporation are dependent on a special apparatus and
it is not routinely available in most laboratories.
To conrm the integration of the selectable marker sh ble gene, PCR
with gene-specic primers was performed to amplify the gene from
total genomic DNA isolated from the selected colonies. Fig. 2A showed
the PCR results of wild type (wt) and ve randomly picked
transformants. All of the resistant cell lines contained a DNA fragment
of the correct size and no amplied product was from wild type
cell lines. This result suggested that the exogenous sh ble gene has integrated into the genome of P. tricornutum. In order to further conrm the
integration and copy number of sh ble gene, Southern blot analysis was
performed for the transformants using the sh ble gene as a probe
(Fig. 2B). The genomic DNA was independently digested with three restriction enzymes EcoRI, BamHI and HindIII. The two resistant clones
contained only 12 hybridizing fragments with BamHI restriction

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2.3. Electroporation protocol

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To generate transgenic P. tricornutum, plasmid pPha-T1 (Zaslavskaia

et al., 2000), which has a bleomycin-resistant gene (sh ble) cassette
with the fcpB promoter, was used. eGFP gene (egfp) was amplied by
PCR from peGFP-N1 (GenBank accession number U55762) using the
sense primer 5-cggGGTACCATGGTGAGCAAGGGCGAGG-3 (KpnI site
underlined) and the antisense primer 5-ctagTCTAGATTACTTGTACAG
CTCGTCCATGC-3 (XbaI site underlined). The PCR product was digested
with KpnI and XbaI and cloned into the KpnIXbaI sites of pPha-T1,
which is located downstream of the fcpA promoter, to generate the
peGFP expression vector. For the cotransformation, plasmid pFcpBpGUS containing the complete GUS (uidA) gene (Falciatore et al., 1999)
and plasmid pPha-T1 were used.

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logarithmically grown cells were pelleted by centrifugation and washed

twice with distilled water to remove salts, then resuspended in 100 l of
staining solution (50 mM Na-phosphate, pH 7.0, 10 mM Na2EDTA, 0.1%
Triton X-100, 0.5 mM K3Fe[CN]6, 0.5 mM K4Fe[CN]6, 2 mM X-Gluc), and
incubated overnight at 37 C. After staining, the samples were washed
twice with 70% ethanol and then immersed in 100% ethanol until
the negative controls (wild type cells) became white. Blue cells were
detected by microscopy.

2.2. Plasmid construction

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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. 2. A schematic map of the linearized pPha-T1 transformation vector digested by

ScaI (A). PCR (B) and Southern blot analysis (C) of P. tricornutum wild type (wt) and resistant cell lines. PCR were performed using primers specic for the histone H4 gene and the
sh ble resistance gene. Southern blot probed with DIG-labeled sh ble DNA. Lane 1, plasmid
pPha-T1 (5 ng) linearized by digestion with EcoRI; lane 2, wt genomic DNA (5 g); lanes
35, genomic DNA (5 g) from clone T2 digested with EcoRI (lane 3), BamHI (lane 4), and
HindIII (lane 5); lanes 68, genomic DNA (5 g) from clone T3 digested with EcoRI (lane
6), BamHI (lane 7), and HindIII (lane 8); lane 9, genomic DNA (5 g) from zeocin resistant
clone T2; lane 10, genomic DNA (5 g) from zeocin resistant clone T3.

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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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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This work was supported by the National Natural Science Foundation

of China (No. 40976079) and the National Key Basic Research Project of
China (2011CB200901).

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clones. Observation under a light microscope showed strong GUS

staining in the cells containing uidA (Fig. 4B, C).
In this study, a universal electroporation protocol was applied in
the transformation of P. tricornutum. Electroporation parameters and introduced exogenous DNA form were optimized for highly efcient
transformation. Successful transformation of P. tricornutum via electroporation requires linear DNA fragment or carrier. Our protocol for the
genetic transformation of P. tricornutum is simpler than reported before
and also provides some clue for establishing electroporation transformation system in other eukaryotic microalgae.

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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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