Research Paper
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Acta Biochim Biophys Sin
2005,37:709–712
doi:10.1111/j.1745-7270.2005.00092.x
Recombination and Heterologous
Expression of Allophycocyanin Gene in the Chloroplast of Chlamydomonas
reinhardtii
Zhong-Liang SU1,3, Kai-Xian QIAN1*, Cong-Ping TAN2,4, Chun-Xiao MENG2,4, and Song QIN2*
1 Department of
Biotechnology, College of Life Sciences, Zhejiang University, Hangzhou 310027,
China;
2 Institute of
Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
3 Qingdao University of
Science and Technology, Qingdao 266061, China;
4 Graduate
University of Chinese Academy of Sciences, Beijing 100039, China
Received: January
18, 2005
Accepted: June 24,
2005
This work was
supported by a grant from the Key Innovative Project (KZCX-3-SW-215) of the
Chinese Academy of Sciences
*Corresponding
authors:
Song QIN: Tel,
86-532-2898500; Fax, 86-532-2880645; E-mail, [email protected]
Kai-Xian
QIAN: Tel, 86-571-8792761; E-mail, [email protected]
Abstract Heterogeneous expression
of multiple genes in the nucleus of transgenic plants requires the introduction
of an individual gene and the subsequent backcross to reconstitute
multi-subunit proteins or metabolic pathways. In order to accomplish the
expression of multiple genes in a single transformation event, we inserted both
large and small subunits of allophycocyanin gene (apcA and apcB)
into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S.
The constructed vector was then introduced into the chloroplast of C.
reinhardtii by micro-particle bombardment. Polymerase chain reaction and
Southern blot analysis revealed that the two genes had integrated into the
chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed
that the two genes from the prokaryotic cyanobacteria could be correctly
expressed in the chloroplasts of C. reinhardtii. The expressed foreign
protein in transformants accounted for about 2%–3%
of total soluble proteins. These findings pave the way to the reconstitution of
multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii
chloroplasts in a single transformation event.
Key words Chlamydomonas reinhardtii; chloroplast transformation;
allophycocyanin gene
Expression of multiple genes in the nucleus of transgenic plants is
complicated and time-consuming due to the monocistronic translation of nuclear
mRNAs. For example, in order to express the polyhydroxybutyrate polymer or Guy’s
13 antibody, a single gene was first introduced
into the nuclear genome of an individual transgenic plant, then the plant was backcrossed
to reconstitute the entire pathway or the complete multi-subunit protein
[1,2]. In contrast, most chloroplast genes of plants are co-transcribed. This
provides the possibility of expressing foreign polycistrons using the Chlamydomonas
reinhardtii chloroplast and reconstituting entire metabolic pathways or
multi-subunit proteins in a single transformation event. Similar work was
successful in tobacco chloroplasts [3].
Allophycocyanin is one of the
photosynthetic antenna proteins in cyanobacteria and red algae [4]. The basic
unit of allophycocyanin is a heterodimer composed of an alpha subunit and a beta subunit with molecular mass between 15 kDa and
23 kDa [5]. Our previous studies suggested that the recombinant allophycocyanin
(rAPC) could remarkably inhibit the S-180
carcinoma in mice with an inhibitory rate ranging from 45% to 64%, without any
obvious effect on the thymus index or leukocyte count [6,7]. This indicates
that the expression of APC in the C. reinhardtii chloroplast may
facilitate the production of a new valuable plant-derived protein.
In this study, the allophycocyanin gene apc
(containing the fragments encoding alpha subunit, apcA, and beta
subunit, apcB) was used as a model gene to demonstrate the possibility
of multiple genes co-expression in the C. reinhardtii chloroplast.
Experimental Procedures
The wild-type C. reinhardtii strain 137cc was kindly provided
by the Biotechnology Research Institute, Chinese Academy of Agricultural
Sciences (Beijing, China). This alga was cultured in Tris-acetate-phosphate (TAP)
medium [8] with a cycle of 16 h light:8 h dark (30 mmol.m?C2.s?C1) at 25 ?C.
Then it was cultured on solid medium by adding 2% agar.pUC18 and pBluescriptII SK (+) were kept in our laboratory. apcA
and apcB were cloned from the cyanobacterium Spirulina maxima and
then subcloned into pUC18 to obtain plasmid pUC18-apc. Plasmid p64D containing
the chlL homologous fragment of the C. reinhardtii chloroplast
and aminoglycoside adenine transferase gene (aadA) cassette (including
the atpA promoter and aadA-rbcL terminator) was obtained from
the Biotechnology Research Institute. All restriction enzymes, Taq DNA
polymerase for polymerase chain reaction (PCR), T4 DNA ligase and Klenow
fragment were purchased from TaKaRa (Dalian, China).
The aadA cassette was cleaved from p64D with EcoRV and
SacI, and ligated to pBluescriptII SK (+) digested with the same enzymes
to create the plasmid pSK-aadA. The plasmid pSK-apc was
constructed by inserting apc cleaved from pUC18-apc with SmaI
and SphI into pSK-aadA to replace aadA. Consequently, apc
was driven by the atpA promoter and terminated by the rbcL
terminator of the C. reinhardtii chloroplast. To obtain the plasmid pSK-apc-aadA, the aadA cassette, which was cleaved from p64D
as described above, was inserted into pSK-apc, following the apc
cassette. Both apc and aadA cassettes were cut from pSK-apc–aadA
and used to replace the aadA cassette in the plasmid p64D to
make a C. reinhardtii chloroplast homologous integration vector papc-S.
DNA sequencing was used to check the open reading frame (ORF) of apc to
ensure its correct expression.
Gold particles coated with plasmid papc-S (containing apcA
and apcB) were bombarded into C. reinhardtii using the biolistic
bombardment equipment PDS1000/He (Bio-Rad, Hercules, USA) as described by
Kindle et al. [9]. After transformation, cells were incubated at 21 ?C
in dim light for 24 h, then washed with TAP liquid medium before transferring
them onto fresh TAP plates containing 100 mg/ml spectinomycin (Sigma,
St. Louis, USA). After a two-week culturing period at 25 ?C, colonies were
picked out and inoculated in 50 ml liquid TAP selective medium containing 100 mg/ml
spectinomycin for 7 d on a gyratory shaker at 160 rpm. In order to improve
homogeneity, the solid-liquid selection procedure was repeated twice.Total DNA of C. reinhardtii was isolated as described
by Goldschmidt-Clermont [10]. In order to verify the integration of apc into
chloroplast genomes of C. reinhardtii transformants, two PCR primers
were designed according to the 5‘ downstream sequence of chlL and
the 3‘ upstream sequence of apc: primer chlL-F, 5‘-GTTTTTATTCCTGGAGTTTG-3‘;
and primer apc-R, 5‘-TATGCATGCTTGGAAGCTTAG-3‘. The
protocol for PCR was: 30 cycles of 95 ?C for 1 min, 50 ?C for 1 min and 72 ?C
for 2 min. The PCR products were visualized on 1% agarose gel.
For Southern blot analysis, C. reinhardtii total DNA was
digested with EcoRV and SacI, then loaded onto 1% agarose gel.
The gel was transferred to a nylon filter using the Mini protean II cell
blotter system (Bio-Rad) Southern blot was carried out with intact apc,
which was cleaved from pUC18-apc, as the probe using the DIG DNA
labeling and detection kit (Roche, Basel, Switzerland).
The standard APC antigen was prepared as described by Zhang and Chen
[11]. Rabbit anti-APC polyclonal antibodies were obtained according to the
method of Krakauer et al. [12]. Crude protein was extracted from C.
reinhardtii as described by Goldschmidt-Clermont [10]. The concentration of
total soluble proteins (TSP) from the C. reinhardtii transformants was
quantified according to Bradford [13]. The content of recombinant APC in
transgenic C. reinhardtii was determined using quantitative
enzyme-linked immunosorbent assays (ELISA) as described by Sun et al.
[14].
After centrifugation at 6600 g for 2 min, the supernatant of
crude protein was subjected to 20% sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), then transferred onto nitrocellulose membrane by electro-blotting
(Bio-Rad). Rabbit anti-APC (1:1000) was used as the primary antibody and
alkaline phosphatase-conjugated goat anti-rabbit IgG (1:500) was used as the
secondary antibody. The antigen-antibody complexes were visualized by 3,3‘-diaminobenzidine
(DAB; Amresco, Solon, USA).
Results and Discussion
DNA sequencing showed that the ORF of apc in papc–S
was in the right orientation. Fifteen colonies were obtained after the
first round of spectinomycin selection of the transformants. When the
transformants and wild-type C. reinhardtii were incubated in dim light,
all the transformants became yellow; in contrast, the wild-type strain showed distinct green phenotype. According to the report of
Suzuki and Bauer [15], the transformants will become yellow when they are
incubated in dim light if chlL is replaced by a foreign gene. The same
phenomenon was observed in our experiments, which suggested that the target
gene cassettes had been integrated into the directed
site of the C. reinhardtii chloroplast genome through homologous
recombination.
After three rounds of spectinomycin selection, two colonies were
picked out randomly. The result of PCR amplification using the pair of primers
(chlL-F and apc-R) showed that an expected 1.7 kb band covering
the atpA promoter and apc fragment was amplified in these two transformants, but no band was obtained in wild-type C. reinhardtii [Fig. 1(A,B)].
After digestion and Southern blot analysis, a band of approximately 3.8 kb
representing the apc and aadA
cassettes was visualized in the total DNA of both transformants (Fig. 2).
The result of PCR and Southern analysis suggested that fragments of the
apc and aadA cassettes had been integrated into the chloroplast genome of C.
reinhardtii.
In Western blot analysis, two bands of approximately 21 kDa and 17
kDa were detected (Fig. 3), corresponding to the alpha
and beta subunits of rAPC in molecular weight, respectively. This indicates
that the two foreign genes (apcA and apcB) were correctly expressed
in the chloroplast of C. reinhardtii. Determination of expressed
APC in the two transformants using quantitative ELISA techniques revealed
that they constitute 23.6±0.1 mg and 26.0±0.2 mg per milligram of TSP, respectively (Fig. 4).In this research work, we successfully expressed prokaryotic apcA
and apcB genes using a single atpA promoter in the C.
reinhardtii chloroplast, indicating that prokaryotic cyanobacteria
polycistrons can be correctly translated in eukaryotic chloroplasts. This
result provides a foundation for the expression of foreign pathways or
pharmaceutical proteins involving multiple genes in the C. reinhardtii chloroplast.
The expression level of APC in the C. reinhardtii transformants
accounted for 2%–3% (W/W) of TSP, showing the feasibility of using
transgenic C. reinhardtii chloroplasts as a kind of bioreactor to
produce functional proteins. Transgenic plants, as the recombinant functional
protein source, have several advantages. For example, they are relatively
inexpensive and safe [16]. The recombinant protein expressed in the algal
chloroplast will be further tested for its biological activity and its
potential application in pharmacology.
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