全 文 ：Received 20 May 2003 Accepted 16 Oct. 2003
* Author for correspondence. Tel: +86 (0)10 64889358; E-mail: < yrsun@genetics.ac.cn >.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 342-346
Construction of a System for the Stable Expression of Foreign
Genes in Dunaliella salina
GENG De-Gui1, HAN Yan2, WANG Yi-Qin1, WANG Peng1, ZHANG Li-Ming1, LI Wen-Bin1, SUN Yong-Ru1*
(1. Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100101, China;
2. Xuzhou Bioengineering School of Jiangsu Province, Xuzhou 221006, China)
Abstract: A stable transformation system for the expression of foreign genes in the unicellular green
marine alga (Dunaliella salina Teod.) was established. Using electroporation, the alga was transformed with
a plasmid containing the hepatitis B surface antigen (HBsAg) gene and the chloramphenicol acetyltransferase
(CAT) gene as a selectable gene. PCR and Southern blotting analysis indicated that the HBsAg gene was
integrated into the D. salina genome. Northern dotting analysis showed that the HBsAg gene was expressed
at the mRNA level. The stable expression of HBsAg protein in transformants was confirmed by HBsAg
enzyme-linked immunosorbent assay (HBsAg ELISA) and Western blotting analysis. Also, PCR and Southern
blotting analyses showed that the CAT gene was integrated into the D. salina genome, and CAT ELISA
indicated that CAT protein was stably expressed in the cells. The introduced HBsAg DNA and HBsAg
protein expression were stably maintained for at least 60 generations in media devoid of chloramphenicol.
This is the first report of the stable expression of foreign genes in D. salina .
Key words: Dunaliella salina ; hepatitis B surface antigen; chloramphenicol acetyltransferase
The unicellular eukaryotic green marine alga (Dunaliella
salina) is an ideal organism for the study of salt-resistance
mechanisms, because it can grow in salt media ranging from
less than 0.5 mol/L to saturated salt solutions (Ben-Amotz
et al., 1982). D. salina may also be used as a good candi-
date bioreactor for the production of foreign proteins, be-
cause of its characteristics. Firstly, it is one of the most
halotolerant eukaryotes and can grow in media with very
high salt concentrations. Under natural and cultural
conditions, there is less chance that D. salina is contami-
nated by less halotolerant microorganisms, algae and alga-
eating animals. Secondly, D. salina lacks a rigid polysac-
charide cell wall (Sadka et al., 1991) and is a natural
protoplast. Foreign genes can be easily introduced into the
cells and foreign proteins can also be easily purified to
meet the demands of safety and efficiency. Thirdly, D. salina
is easily and rapidly cultured in an inexpensive medium
containing simple salts. The cost of producing foreign pro-
teins in this organism is very low. These advantages make
D. salina a good bioreactor for producing foreign proteins.
We have introduced the b-glucuronidase (GUS) gene
into D. salina under the control of five promoters. The
GUS gene was expressed transiently and the Ubiquitin-W
(Ubil-W) promoter was the most efficient (Geng et al., 2002).
Here, we report the stable expression of the HBsAg gene
and the CAT gene in D. salina. A system for the stable
expression of foreign genes in D. salina was established.
1 Materials and Methods
1.1 Strains and media
Dunaliella salina Teod. was obtained from Prof. HUA
Dong (Department of Biology, Xuzhou Normal University,
Xuzhou, China). Cells were grown in liquid-modified
Johnsons medium (J1) (Borowitzka and Borowitzka, 1988)
or on agar plates (Johnsons medium containing 1.5% agar)
at 20 – 25 ℃.
1.2 Construction of plasmids
Plasmids used included plasmid pCAT-control and plas-
mid pD3-HBsAg. The plasmid pCAT-control was obtained
from Prof. QIN Song (Institute of Oceanology, The Chi-
nese Academy of Sciences, Qingdao, China), and the plas-
mid pD3-HBsAg was provided by Dr. Hans J. Netter
(SASVRC, Royal Children’s Hospital, Brisbane, Australia).
The recombinant plasmid pUWHBsAg-CAT used for trans-
formation was constructed starting from pUWGUS. The
GUS gene was excised from pUWGUS, and the HBsAg gene
was ligated into the digested pUWGUS to yield
pUWHBsAg. The HBsAg gene was under the control of
the Ubil-W promoter. The CAT gene under the control of
the simian virus 40 (SV40) promoter from the plasmid pCAT-
control was inserted into pUWHBsAg to complete
pUWHBsAg-CAT.
GENG De-Gui et al.: Construction of a System for the Stable Expression of Foreign Genes in Dunaliella salina 343
1.3 Transformation protocols
Cells in the early-logarithmic phase were collected by
centrifugation and resuspended in electroporation buffer
(per litre: 500 mmol NaCl, 5 mmol KCl, 5 mmol CaCl2, 20
mmol Hepes, 200 mmol mannitol, 200 mmol sorbitol, pH 7.2).
The final cell density was 1×106 cells/mL. Fifteen µL of
plasmid DNA (200 µg/mL) was added to the cell suspen-
sion (0.4 mL) and kept on ice for 5 – 10 min. The resus-
pended cells were transferred into a small electroporation
chamber and electroporated with a Baekon 2000
electroporation system. The pulse voltage was 6.0 kV, the
pulse duration was 0.05 s and the total number of pulses
was 26 – 210.
1.4 DNA isolation, PCR and Southern blotting analysis
Cells in the late-logarithmic phase (approximately
1×107 cells/mL) were collected and DNA was isolated as
described (Chen et al., 2001). According to the coding re-
gion of the HBsAg gene, two primers were designed for
PCR analysis of the HBsAg gene as follows: P1: 5-
GCTGAACATGGAGAACATCACA-3 and P2: 5-
CCATCTCTTTGTTTTGTTAGGG-3. Also, two primers were
designed for PCR analysis of the CAT gene as follows: P3:
5-AGGAAGCTAAAATGGAGAAA-3 and P4: 5-
TTACGCCCCGCCCTGCCACT-3. PCR products were elec-
trophoresed to detect the integration of the HBsAg gene
into the D. salina genome.
For Southern analysis, genomic DNA and plasmid DNA
from pUWHBsAg-CAT were digested with different restric-
tion endonucleases. Five µg of each sample was electro-
phoresed in a 0.8% agarose gel and transferred to a nylon
membrane. Blots were hybridized with a 32P-labeled
HBsAg or CAT DNA probe as described (Sambrook et
al., 1989), followed by exposing to X-ray film for 3 – 5 d at
– 80 ℃.
1.5 RNA isolation and Northern dotting analysis
Cells in the mid-logarithmic phase (approximate 1×106
cells/mL) were collected and RNA was isolated with the
TRIZOL Reagent (Gibco, BRL). For Northern dotting
analysis, total RNA was denatured with formaldehyde, five
µg of each sample was dotted to nylon and hybridized with
the 32P-labelled HBsAg DNA probe.
1.6 Protein extracts, ELISA and Western blotting analy-
sis
Cells in the late-logarithmic phase (20 mL) were collected
and resuspended in 1 mL of PBS buffer (per litre: 137 mmol
NaCl, 2.7 mmol KCl, 10 mmol Na2HPO4, 1.8 mmol KH2PO4,
20 mmol EDTA, 2 mmol b-mercaptoethanol, pH 7.4). The
suspension was ultrasonicated and centrifuged. The su-
pernatant was stored at 4 ℃ and the concentration of the
total proteins was measured as described (Bradford, 1976).
For HBsAg enzyme-linked immunosorbent assay
(HBsAg ELISA), fifty µL of the supernatant was added to
an ELISA plate, incubated at 37 ℃ overnight and washed
with TBS buffer (per litre: 150 mmol NaCl, 10 mmol Tris, pH
7.4). Fifty µL of rabbit anti-HBsAg IgG (OEM) at 1:500 dilu-
tion in TBS buffer was added, incubated at 37 ℃ for 1 –
1.5 h and washed with TBS buffer. Fifty µL of goat anti-
rabbit IgG conjugated with alkaline phosphatase (OEM) at
1:500 dilution in TBS buffer was added, incubated at 37 ℃
for 1 h and washed with TBS buffer. Fifty µL of disodium
para-nitrophenylphosphate in diethanolamine buffer was
added and reacted at room temperature for 20 min. Absor-
bance was measured using an ELISA scanner at 450 nm.
HBsAg protein derived from human serum was used as a
standard.
CAT ELISA was performed with the CAT ELISA Kit
(Boehringer Mannheim), absorbance was measured at 405
nm.
For Western blotting analysis of HBsAg protein, total
protein was electrophoresed by SDS-PAGE and transferred
to a nitrocellulose membrane. Blots were incubated sequen-
tially with rabbit anti-HBsAg IgG and goat anti-rabbit IgG
conjugated with alkaline phosphatase.
1.7 Nonselective culture
Positive transformants were grown in liquid media de-
void of chloramphenicol. The number of cells was counted
under a light microscope. Genomic DNA was isolated and
analyzed by PCR and Southern blotting analysis, protein
extracts were analyzed by ELISA and Western blotting
analysis.
2 Results
2.1 Transformation of D. salina with the plasmid
pUWHBsAg-CAT
D. salina was transformed with the plasmid
pUWHBsAg-CAT containing a foreign gene (the HBsAg
gene) and an antibiotic resistance gene (the CAT gene).
The CAT gene produces chloramphenicol acetyltransferase
which is resistant to chloramphenicol. After transformation,
cells were immediately spread on agar plates containing 60
µg/mL chloramphenicol as a selective antibiotic. Sixty µg
chloramphenicol/mL completely inhibited the growth of
wild-type D. salina (Geng et al., 2001). Small green colo-
nies appeared at a frequency of 10 – 60 colonies/plate after
two weeks. Some colonies stopped growing, bleached and
died after three weeks, but most colonies have been
maintained. Colonies were picked up and grown in liquid
media containing 60 µg/mL chloramphenicol.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004344
2.2 PCR and Southern analysis of transformants
Genomic DNA was isolated from transformants which
were grown in liquid selective media and analyzed by PCR
to check for the presence of the HBsAg gene and the CAT
gene. A 700-bp PCR fragment amplified from the coding
region of the HBsAg gene was found in 14 out of 40 colonies.
Also, a 700-bp PCR fragment amplified from the coding
region of the CAT gene was found.
PCR-positive transformants 1 – 4 were further analyzed
by Southern blotting analysis. When genomic DNA was
digested with HindⅢ and SacⅠ simultaneously, and hy-
bridized with the 700-bp probe of the HBsAg gene, a band
of approximately 2.88 kb with the same size as the pro-
moter-HBsAg fragment was found in transformants.
However, this result is not convincing enough for the inte-
gration of the HBsAg gene into the D. salina genome.
Therefore, genomic DNA was digested with just one
enzyme, SacⅠ, and hybridized. One or two hybridizing
bands of different sizes were found in transformants 1 – 4,
while a 7.88-kb band was visible in the plasmid pUWHBsAg-
CAT (Fig.1). Also, genomic DNA was digested with BanⅠ
and hybridized with the 700-bp probe of the CAT gene, a
hybridizing band was found in transformants. The results
showed that the HBsAg gene and the CAT gene were sta-
bly integrated into the D. salina genome.
2.3 Northern analysis of transformants
Total RNA from transformed D. salina was hybridized
with the probe of the HBsAg gene. A hybridizing dot was
found in transformants, suggesting the expression of the
HBsAg gene at the mRNA level.
2.4 Expression of the HBsAg gene and the CAT gene
The level of HBsAg protein in transformants 1 – 4 was
determined using HBsAg ELISA. Protein extracts from
transformants were stained and measured with an ELISA
scanner. The level of HBsAg protein observed in
transformants 1 – 4 were 3.11 ± 0.50, 1.64 ± 0.18, 2.05 ± 0.37
and 2.32 ± 0.32 ng/mg of soluble protein (Fig.2). Also, the
level of CAT protein in transformants was determined us-
ing CAT ELISA. The level of CAT protein in transformants
1 – 3 were 0.83 ± 0.14, 1.04 ± 0.21 and 0.59 ± 0.07 ng/mg of
soluble protein. Non-transformed alga showed no detect-
able HBsAg protein, suggesting the specificity of antibody.
Protein extracts from transformants 1 – 4 were further
analyzed by Western blotting analysis. After hybridization,
a band at about 24 kD in transformants was found, which
was at the similar position to standard HBsAg protein
(Fig.3). The results showed that the HBsAg gene and the
CAT gene had been successfully transcribed and trans-
lated into proteins in D. salina.
2.5 Maintenance of the HBsAg gene under nonselective
culture
Positive transformants were grown in nonselective liq-
uid media for at least 60 generations. PCR and Southern
analysis showed that the HBsAg gene was stably
Fig.1. Southern blotting analysis of the HBsAg gene in trans-
formed Dunaliella salina. Genomic DNA and plasmid DNA from
pUWHBsAg-CAT were digested with different restriction endo-
nucleases and hybridized. No hybridizing bands were visible in
wild-type alga. Lane 1, plasmid pUWHBsAg-CAT, XhoⅠ and
SacⅠ; lane 2, plasmid pUWHBsAg-CAT, SacⅠ; lane 3, wild-
type D. salina, SacⅠ; lanes 4 – 7, transformants 1 – 4 respectively,
SacⅠ.
Fig.2. HBsAg protein levels in transformed Dunaliella salina.
Protein extracts from wild type and transformants were mea-
sured for HBsAg protein using HBsAg ELISA. Wild-type alga
showed no detectable HBsAg protein. Results are expressed as
mean ± SE. Wt, wild-type D. salina; numbers 1 – 4, transformants
1 – 4 respectively.
Fig.3. Western blotting analysis of HBsAg protein from trans-
formed Dunaliella salina. Total protein from wild type and
transformants was immunoblotted using standard techniques. Wild-
type alga showed no detectable HBsAg protein. Lane 1, standard
HBsAg protein; lane 2, wild-type D. salina; lanes 3 – 6,
transformants 1 – 4 respectively.
GENG De-Gui et al.: Construction of a System for the Stable Expression of Foreign Genes in Dunaliella salina 345
maintained in the D. salina genome. ELISA and Western
analysis showed that HBsAg protein was stably expressed
in transformed cells under nonselective growth conditions.
3 Discussion
In recent years, some unicellular and multicellular eu-
karyotic algae have been transformed successfully. Among
unicellular eukaryotic algae, Chlorella is very successful,
in which Chlamydomonas reinhardtii (Rochaix and van
Dillewijn, 1982; Hasnain et al., 1985; Kindle, 1990; Dunahay,
1992; Hall et al., 1993; Fuhrmann et al., 1999), C. ellipsoidea
(Jarvis and Brown, 1991; Chen et al. , 2001), C.
saccharophila (Maruyama et al., 1994), Acetabularia
mediterranea (Neuhaus et al., 1984), C. vulgaris and C.
sorokiniana (Hawkins and Nakamura, 1999). Of multicellu-
lar eukaryotic algae, Porpyhra miniata (Kuble, 1994), Vol-
vox carteri (Schiedlmeier et al., 1994), Cyclotella cryptica
and Navicula saprophila (Dunahay et al., 1995) have been
reported. Biotechnological investigation of marine algae is
less advanced than that of freshwater algae, however, and
the transformation of Porpyhra miniata (Kuble, 1994),
Thalassiosira weissflogii (Falciatore et al., 1999) and
Phaeodactylum tricornutum (Apt et al., 1996; Falciatore et
al., 1999) has been reported.
The methods used to transform algal cells are particle
bombardment (Schiedlmeier et al., 1994), electroporation
(Chen et al., 2001), microinjection (Neuhaus et al., 1984),
protoplasting (Jarvis and Brown, 1991), agitation with glass
beads (Hall et al., 1993) and silicon fibers (Dunahay, 1992).
Among them, particle bombardment and electroporation
are often used. We report here the successful transforma-
tion of the unicellular green marine alga (D. salina) with the
HBsAg gene and the CAT gene by using an efficient and
reproducible method—electroporation. This is the first re-
port of the stable expression of foreign genes in the cells.
We found a hybridizing band at about 24 kD in transformed
D. salina which was at the similar position to standard
HBsAg protein using Western blotting analysis. This means
that complete HBsAg protein is produced in transformed
D. salina. However, the level of HBsAg protein was obvi-
ously varying in different transformants. The reason is
uncertain. Firstly, the HBsAg gene is inserted randomly
into the D. salina genome at one or more sites. Therefore,
the copy number of the HBsAg gene is varying in different
transformants. Southern blotting analysis indicated that
one or two hybridizing bands of different sizes were found
in transformants 1 – 4, when genomic DNA was digested
with SacⅠ and hybridized with the probe of the HBsAg
gene. This may result in the varying amount of HBsAg
protein in transformants. Secondly, the integration event is
random. Therefore, the HBsAg gene is inserted at different
sites in different transformants. The HBsAg gene near ac-
tively transcribed genes can be strongly transcribed, oth-
erwise it is weakly transcribed or is silent. Also, this may
cause the varying level of HBsAg protein in different trans-
formed D. salina. More detailed work should be done to
produce useful foreign proteins in D. salina.
Acknowledgements: We thank Prof. HUA Dong for kindly
providing D. salina. We are also grateful to Prof. QIN Song
for kindly providing plasmid pCAT-control and Dr. Hans J.
Netter for kindly providing plasmid pD3-HBsAg.
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