遗 传 学 报 Acta Genetica Sinica, January 2006, 33 (1)：56–62
ISSN 0379-4172
Expression of Human BMP-2 Gene in Different Tissues of
Tobacco Plants
GAO Yuan1,2, SUO Guang-Li1, HAN Jin1, HE Zheng-Quan2, YAO Wei2, DAI Jian-Wu1,①
1. Center of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
Beijing 100080, China;
2. Biotechnology Research Center, China Three Gorges University, Yichang 443002, China
Abstract: The bone morphogenetic proteins（BMPs）are a family of growth factors that regulate the development of bone. BMP-2
is the most effective in the induction of bone tissue. A large amount of BMP-2 is needed for both bone tissue engineering research
and clinical application. Thus, an effective way is necessary to produce sufficient BMP-2 protein. With the advance in plant biotechnology, transgenic plants have been targeted as a bioreactor to produce desired recombinant proteins. Here, the expression of
recombinant human bmp-2 gene (rhbmp-2) was studied in tobacco plants using gus as a reporter gene. The difference of expression
levels in root, stem and leaf tissues was analyzed by GUS activity assay, semi-quantitive RT-PCR and western blotting.The results
indicated that the expression levels of fusion protein in root and stem tissues were significantly higher than those in leaf tissue. For
the protein compositions in root and stem tissues were simpler than those in leaf tissue,this suggested that the purification process
with root and stem tissues would potentially be easier.
Key words: bone morphogenetic protein-2 (BMP-2); gus gene; protein expression; transgenic tobacco plant
The bone morphogenetic proteins (BMPs)
are growth factors in the β-TGF family. They
play important roles in the early stage of embryonic development in vertebrates and have
significant impact on the development of bone
tissue [1] . BMPs can accelerate the ossification
of extensive bone lesions [2] and facilitate bone
repair. In vivo, BMPs can also induce parenchyma such as muscle to form bone tissue.
Among BMP family, BMP-2 is the best characterized molecule and it has the strongest
bone-inducing activity. BMP-2 has excellent
potential in tissue engineering and medical
clinic. BMP-2 mainly exists in animal bone tissues
and is very difficult to extract and purify. Great
efforts have been made to obtain adequate amount
of BMP-2 protein. With the development of gene
engineering, the expression of recombinant BMP-2
has been reported in many cells such as E.coli [3],
CHO cell line[4], COS cell line[5] and silkworm
larvae[6]. However, these systems have numbers of
disadvantages including high-cost, low yield, low
bioactivity, and the risk of contamination by animal
viruses. Compared with other expression systems,
transgenic plants have many advantages including
low cost, proper folding and modification of the
protein, easy scale-up of production and storing
heterologous protein in storage organs (such as
tubers and seeds). Various kinds of important
pharmaceutical proteins (such as some human
biopharmaceuticals, recombinant subunit vaccines
and recombinant antibodies) have been success-
Received: 2005-06-02; Accepted: 2005-09-23
This Work Was Supported by Chinese Academy of Sciences “100 Talented Scholar Program” and Chinese Academy of Sciences
“Knowledge Innovation Program” Grants (No.KSCX2-SW-205; KSCW2-SW-218), and also Supported by the Scientific Research
Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. The authors also gratefully acknowledge the
support of K.C. Wong Education Foundation, Hong Kong.
①
Corresponding Author. E-mail: [email protected]; Tel & Fax: +86-10-8261 4426
GAO Yuan et al.: Expression of Human BMP-2 Gene in Different Tissues of Tobacco Plants
fully expressed in different plants[7,8]. In this report,
rhbmp-2 transgenic tobacco plants were made to
express rhbmp-2 gene in different tissues using gus
as a reporter gene. The result would be helpful to
the future protein purification step.
1
that harbored the vector. Transformed callus tissues
were selected by kanamycin resistance. Tobacco
shoots were regenerated following the method described elsewhere [11]. Only the rooted shoots were
used for further analysis.
1. 3
Materials and Methods
57
Genomic DNA analysis
Genomic DNA was isolated from 2-month-old
1. 1 Construction of expression vector pBI-BMP2
transgenic plants following the method described by
The construction was based on binary plant ex-
Edwards [12]. Root, leaf and stem tissues of each plant
pression vector pBI121. The rhbmp-2 gene (provided
were treated separately. These genomic DNA samples
by Professor XUE Yong-Biao) was amplified by
were used as templates for Polymerase Chain Reac-
polymerase chain reaction (PCR) with primers: 5′-CG-
tion (PCR) using sense primer 5′-ATCATCATCATAG-
TCTAGTAAACAATGGCTCCATCATCATCATCAT-
CTGTAAGAGACACCC-3′ (corresponding to rhbmp-2
AGCTGTAAGAGACAC-3′ (sense primer, containing
gene) and anti-sense primer 5′-GTTGGGGTTTCTA-
XbaⅠrestriction site) and 5′-GCGGATCCCTTGTCA-
CAGGAC-3′ (corresponding to the upstream of gus
TCGTCATCCGACACC-CACAACCC-3′ (anti-sense
gene). Genomic DNA from non-transformed tobacco
primer, containing BamH Ⅰ restriction site). The
tissues served as the negative control. The PCR prod-
PCR reaction was carried out as follows: 94℃ for 30 s,
ucts were electrophoresed on the 1% agarose gel
45℃ for 45 s, 72℃ for 45 s for 30 s cycles; an addi-
containing ethidium bromide. The positive samples
tional extension step of 10 min at 72℃ was added at
the end of 30 cycles. PCR product was separated by
agarose gel electrophoresis and it was purified with
the gel extraction kit (Omega). Kozak sequence
defined as TAAACAATGGCT[9], and a His6-tag
was appended to the 5′-terminal of rhbmp-2, and an
enterokinase site was appended to the 3′-terminal.
The heparin binding site at the 5′ region of native
hbmp-2 gene was eliminated because it would reduce
its bioactivity as reported [3]. The modified rhbmp-2
was cloned into the pBI121 vector and controlled by
CaMV35S promoter. The original CaMV35S promoter was replaced with double 35S promoter plus
AMV enhancer. Gus gene was fused to the
3′-terminal of rhbmp-2 in pBI121 and was used as a
reporter gene.
1. 2
Plant transformation
yielded a 382 bp band. Eight random chosen positive
samples were purified from the gel and sequenced.
1. 4
Semi-quantitive RT-PCR
Semiquantitative RT-PCR was performed according
to the method described by Schenone[13]. It was used to
evaluate the amount of rhbmp-2 mRNA in transgenic
tobacco plants. Total RNA was isolated according to the
manufacturer’s protocol of RNeasy Plant Mini Kit
(Qiagen), and it was quantified with spectrophotometery
at 260 nm. Before RT-PCR, contaminating genomic DNA
was removed with RNase-free DNaseⅠ(TaKaRa).
First-strand cDNA was synthesized from 150 ng of total
RNA in a 25 μL final volume reaction. PCR was performed by coamplification of rhbmp-2 and tublin as an
endogenous standard sequence. Amplification was carried
out for 25 cycles consisting of 1 min at 94℃, 1 min at
55℃ and 1 min at 72℃; an additional extension step of
[10]
According to the method described by Horsch ,
Agrobacterium tumefaciens strain LBA4404 was
transformed with the pBI-BMP2 vector; then the leaf
discs of tobacco (Nicotiana tobacum L. cv. NC89)
were infected with such Agrobacterium tumefaciens
10 min at 72℃ was added at the end of 25 cycles. PCR
products were analyzed on 1% agarose gel and the band
intensity was measured directly on the gel on a UVP system. The intensity of bands was expressed as the ratio
between target sequence and tublin. The experiment
遗传学报
58
was performed three times, and the statistical analysis
was done on the average value of the three reactions.
1. 5
Histochemical assay
X-Gluc substrate was used for histochemical assay
of GUS in tissues and cells. It would form a blue precipitate at the GUS active site. Fresh leaves, roots and
stems of transgenic and normal tobacco plants were
harvested. The non-tranformed tobacco tissues served
as negative control. Leaves were cut into 1 cm ×1 cm
pieces, and the roots and stems were cut into 2 cm
length segments. Tissues were stained with about
200 μL of GUS staining buffer (0.5 mg/mL X-Gluc,
0.05 mol/L Na2HPO4, 0.05 mol/L NaH2PO4, 5 mol/L
K3[Fe(CN)6], 5 mol/L K4[Fe(CN)6], 10 mol/L Na2EDTA)
at room temperature for 24 h. Then the tissues were
cleared with 75% ethanol for three times until the control tissues were completely white. Blue spots on the
tissues are the sites where GUS expresses.
1. 6
Protein extraction
The total proteins of transgenic tobacco plants
were extracted from root, stem and leaf tissue. About
0.3 g fresh tissue was ground under liquid nitrogen.
the powders were collected in microfuge tubes and
suspended in 500 μL extraction buffer (50 mol/L
phosphate buffer pH 7.0, 10 mol/L β-mercaptoethanol,
10 mol/L Na2EDTA, 0.1% Sarcosyl, 0.1% Triton X-100).
The samples were centrifuged at 4℃, 12 000 r/min
for 5 min. The supernatant was collected for GUS
activity assay and western blotting. The protein
concentration in the supernatant was determined by
Bradford assay with bovine serum albumin as standard.
1. 7
Fluorometric GUS assay
GUS activity assay was performed according
to the method described by Jefferson[14]. And the
unit to measure GUS activity was pmol metylumbelliferone (4-MU)/mg protein/min. First, the
signal generated by the fluorometer was standardized with a 5 pmol/L concentration of 4-MU. A
volume of 90 μL of assay buffer (1 mmol/L MUG,
50 mmol/L phosphate buffer pH 7.0, 10 mmol/L
Acta Genetica Sinica
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2006
β-mercaptoethanol, 10 mmol/L Na2EDTA, 0.1%
Sarcosyl, 0.1% Triton X-100) was added to 10 μL
of supernatant in a microfuge tube. The solution
was incubated at 37 ℃ for 30 min and then
stopped with 900 μL of 0.2 mol/L Na2CO3. The
fluorescence was measured immediately. GUS
activity of root, stem and leaf tissue of eight
plants were measured. Consider these eight plants
as a population, GUS activity of different tissues
were shown in mean±SD form. The data was statistically analyzed and P < 0.05 was considered
significantly different in t test.
1. 8
SDS-PAGE and Western blotting
Total protein extracts of transgenic tobacco was
fractionated by SDS-PAGE on 10% (w/v) polyacrylamide gels. Load the same amount of protein of each
sample to the gel. The separated proteins on the gel were
transferred onto the nitrocellulose membrane. Hybridize
this membrane with monoclonal mouse anti-hBMP-2
IgG (1:1 000 Sigma), and then blot it with anti-mouse
antibody supplied in WesternBreeze kit (Invitrogen, Cat.
No. WB7104). All experiment steps were performed
according to the manufacturer’s protocol. Another nitrocellulose membrane was prepared under the same
conditions except using mouse anti-tublin antibody as
the primary antibody for an endogenous standard control. The experiments were repeated three times. Band
intensities were measured directly on the membrane on
a UVP system and statistically analyzed.
2
Results
2. 1 Construction of the expression vector pBI-BMP2
The rhbmp-2 gene in binary plant expression
vector pBI-BMP2 was confirmed by sequencing. The
rbmp-2 gene in pBI-BMP2 had a His6-tag and Kozak
sequence appended to its 5′-terminal, and gus gene
fused with its 3′-terminal. There was an enterokinase
site between rhbmp-2 gene and the gus gene. The
fusion gene was controlled by double-35S promoter
plus AMV enhancer. The map of vector pBI-BMP2
was shown in Fig.1.
GAO Yuan et al.: Expression of Human BMP-2 Gene in Different Tissues of Tobacco Plants
59
Fig. 1 Plant expression vector pBI-BMP2
The rhbmp-2 gene was added to the XbaⅠ-BamHⅠsite. ‘EK’ represents enterokinase site between rhbmp-2 and gus.
2. 2
Plant transformation
Leaf discs of tobacco (Nicotiana tobacum L. cv.
sues such as root tips, cambium of stem and buds
have deeper blue color than old tissues.
NC89) were infected with Agrobacterium tumefaciens strain LBA4404 which harbored the pBI-BMP2
vector .We obtained 45 lines of transgenic tobacco
plants.
2. 3
Genome DNA analysis
The result of genome DNA assay is shown in
Fig.2. A 382 bp positive result indicates that the target
gene is successfully integerated into the genome of
tobacco.
Fig. 2 Genomic DNA analysis
Lane ‘M’ is 100 bp DNA marker; ‘NC’ is negative control
using the genomic DNA from non-transformed plant; the other
18 lanes are PCR samples from 18 different transgenic plants.
The predicted PCR DNA fragment is 382 bp.
2. 4
Fig. 3 Semiquantitive RT-PCR
First-strand cDNA was synthesized from 150 ng of total
mRNA. A: RT samples using primers corresponding to bmp-2
in PCR. Lane ‘M’ is 100 bp DNA marker, ‘NC’ is negative
control of non-transformed plant, ‘1-6’ represent six transgenic plants, ‘r’ means root tissue, ‘s’ means stem and ‘l’
means leaf. The predicted PCR fragment is 382 bp. B: The
tubulin gene was used as an endogenous control in RT-PCR.
Semi-quantitive RT-PCR
Semi-quantitive RT-PCR results (Fig.3) dem-
onstrated that rhbmp-2 had higher expression levels
in root than in stem and leaf. The band intensities of
root (134.2±21.6) and stem (125.1±19.4) were
significantly higher than that of leaf (87.7±17.5),
but the difference between root and stem was not
significant.
2. 5
Histochemical assay
GUS expresses in the blue colored tissues
(Fig.4). The results indicate that the fast growing tis-
Fig. 4 Histochemical assay with staining buffer containing 0.5 mg/mL X-Gluc
The blue colored root, stem and leaf tissues indicate the GUS
expression sites.
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2. 6
Acta Genetica Sinica
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2006
Fluorometric GUS assay
Eight PCR-reaction-positive plants were
chosen and the total proteins were extracted from
their root, stem and leaf tissues separately. GUS
activity assay of each sample was performed.
GUS activities of root, stem and leaf were
14642 ± 3448, 10640 ± 2380 and 5079 ± 1040
respectively (Fig.5). The GUS activities were almost doubled in stem and tripled in root compared with those in leaf. Significant difference
was noted between root and leaf, stem and leaf,
and root and stem respectively (P<0.05 in t test).
Fig. 6 Western blotting
The total soluble proteins (150 µg/lane) from root, stem and
leaf tissues were loaded as same amount for SDS-PAGE and
performed western-blotting. A: Probed with anti-BMP-2 antibody. ‘1-3’ represent three transgenic plants, ‘R’ means root
tissue, ‘S’ means stem and ‘L’ means leaf. ‘NC’ is negative
control of non-transformed plant. B: Probed with anti-tublin
antibody as the endogenous control.
3
Discussion
BMP-2 is needed for medical therapeutic appli-
cations and tissue engineering research. Plant expression system has its special benefits, such as low cost,
high bioactivity of the protein, convenient to transport.
The goal of our research is to express rhBMP-2 in
Fig. 5 Fluorometric GUS assay
GUS activity assay of root, stem and leaf tissues. Root has
higher GUS activity than stem (P<0.05), and stem has higher
GUS activity than leaf (P<0.05). The unit of GUS activity is
‘pmol MU/mg protein/min’.
2. 7
SDS-PAGE and Western blotting
The protein extracts from root, stem and
leaf of three transgenic plants were used in
SDS-PAGE and Western blotting. An approximately 82 kDa band was detected (the MW of
GUS was 68 kDa), indicating that the fusion
protein rhBMP-2-GUS had been expressed and
the highest expression level was seen in root.
The band intensity of root (204.4±58.9) was
significantly stronger than that of stem (52.0±
11.2) and leaf (23.8±7.2). But the difference
between stem and leaf was not significant
(Fig.6). The result was consistent with that of
Semi-quantitive RT-PCR.
tobacco plants and study its expression in different
tissues.
It is widely known that the structural features of
the 5′ UTR can significantly affect the rate of translation initiation of a gene. It has been proved that
translation efficiency of eukaryotic mRNAs depends
on the nucleotide sequence flanking the translation
start codon, namely, start codon context or Kozak
sequence. In this work, Kozak sequence defined as
UAAACAAUGGCU[9] is used to increase the efficiency of translation initiation. Promoter is considered as the crucial factor that affects gene expression
level. So the CaMV35S promoter is replaced by a
stronger promoter double CaMV35S plus AMV enhancer. A reporter gene will simplify the analysis of
the BMP-2 in transgenic tobacco. E. coli-originated
β-glucuronidase (GUS) is a widely used marker gene
and has been engineered for expression in a variety of
organisms[15]. Using gus as a reporter gene in plants
has many advantages: absence of endogenous GUS
activity in higher plants, sensitive assays available
GAO Yuan et al.: Expression of Human BMP-2 Gene in Different Tissues of Tobacco Plants
and long half-life.
The results of genomic DNA analysis,
RT-PCR analysis, histochemical assay, GUS
activity assay and Western blotting suggested that
the fusion gene rhbmp-2-gus has been integrated
into tobacco genome and expressed successfully.
From the results of the fluorometric GUS assay,
root and stem tissues had significantly higher
expression level than leaf. This difference was also
detected by semi-quantitive RT-PCR and western
blotting. Another interesting observation by
histochemical assay was that the fusion gene was
mainly expressed in young tissues, namely, the root
tips, cambium of stem, buds and young leaves. In
contrast, in old tissues and fiber tissues, the
expression level of the recombinant gene was much
lower. A possible explanation is that root is an
organ where cells divide actively. There are many
sites in root where genome DNA replicates and
transcripts actively. It is believed that such sites in
genome can help the integration and expression of
a recombinant gene. Another explanation could be
that the protein compositions of different tissues
are different; root and stem may have relatively
simple compositions and low levels of total
proteins, whereas in leaf, various types of proteins
are expressed and proteins such as RUBISCO may
have very high expression levels. Thus in root and
stem tissues the hetrogenious protein accounts for a
larger percent.
Traditionally, leaf is usually the only organ
used for protein extraction and purification, while
root and stem, which tend to contain more target
proteins are discarded. Our results suggested that
stem and root tissues will likely produce higher protein yield. Furthermore, by using tissues with simpler protein compositions, the purification process
will potentially be easier.
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重组人 BMP-2 在烟草不同组织中的表达
高 原1,2，索广力1，韩 津1，何正权2，姚 伟2，戴建武1
1. 中国科学院遗传与发育生物学研究所发育分子生物学研究中心，北京 100080；
2. 中国三峡大学生物技术中心，宜昌 443002
摘 要：骨形态发生蛋白（BMPs）是一类调节骨组织发育的生长因子。BMP-2 是 BMP 家族中诱骨活性最强的。在骨组织
工程研究和临床应用中需要大量的 BMP-2。因此，研究出一种能够有效地大量生产 BMP-2 的方法是十分必要的。随着植
物分子生物学的进展，转基因植物被用作一种生物反应器来生产目的蛋白。以 gus 作为报告基因，研究了重组人 bmp-2 基
因在烟草中的表达。通过 GUS 活性检测、半定量 PCR 和 Western blotting 分析了根、茎、叶组织中基因表达的水平，结果
显示融合蛋白在根和茎组织中表达量显著高于叶组织。由于根和茎组织中蛋白组成与叶组织相比相对简单，提示其更易于
进行目的蛋白的纯化。
关键词：骨形态发生蛋白(BMP-2)；gus 基因；蛋白表达；转基因植物
作者简介：高原（1984-），女，辽宁大连人，研究生，研究方向：再生医学