Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 533-538
doi:10.1111/j.1745-7270.2008.00425.x
Quantitative analysis of
cytoplasmic actin gene promoter and nuclear polyhedrosis virus immediate-early
promoter activities in various tissues of silkworm Bombyx mori using
recombinant Autographa californica nuclear polyhedrosis virus as vector
Yi Zhang1,2, Xue Zhang2,3, Huanzhang Xia1, Yuegui Xue3, Jianyang Wang2, Baozhong Tian2,4, Zhenguo Wei2,4, and Changde Lu2*
1 School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Shenyang 110016, China
2 State Key Laboratory of Molecular Biology,
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200031, China
3 College of Life Science, Guangxi Normal
University, Nanning 541004, China
4 State Key Laboratory of Modern Silk, Soochow
University, Zhenjiang 215006, China
Received: March 4, 2008
Accepted: April 21,
2008
This work was
supported by a grant from the National Natural Science Foundation of China (No.
30470350)
*Corresponding
author: Tel, 86-21-54921234; Fax, 86-21-54921011; E-mail, [email protected]
Cassettes
harboring luciferase reporter driven by Bombyx mori cytoplasmic actin
gene promoter (A3) (671 bp) and B. mori nuclear polyhedrosis virus
immediate-early promoter (IE-1) (580 bp) were transferred to the bacmid AcDEGT to
generate the recombinant Autographa californica nuclear polyhedrosis
viruses, AcNPVA3Luc and AcNPVIELuc, respectively. Recombinant baculoviruses
were injected into the hemocoele of newly ecdysed 5th instar larvae. The
activities of the A3 and IE-1 promoters in various tissues were measured by
luciferase activity assay and normalized by the copy number of recombinant
virus. Results showed that the activity of the A3 promoter was approximately 10-fold
higher than the IE-1 promoter. The promoter activities of A3 and IE-1 were
highest in the silk gland, followed by fat body, middle gut, malpighian tubule, and hemocyte. In
silk gland, activity of the two promoters was highest in posterior silk gland,
followed by middle and anterior silk glands. The difference in promoter
activities reflects the growth speed of tissue in silkworm larvae. The activity
of the A3 promoter remained unchanged and was not inhibited significantly by
viral factors at least 3–4 d post injection of rAcNPV.
Keywords promoter activity; A3; IE-1; silkworm tissue; rAcNPV
The silkworm Bombyx mori is a domestic insect. Due to its
large size and high protein synthesis ability, as well as its expediency in the
mass culture of silkworm, it is considered a good candidate for producing
recombinant proteins. Baculovirus-based systems have been developed as powerful
expression systems in insect and cultured insect cells [1]. Since the human a-interferon was
produced in silkworm using B. mori nuclear polyhedrosis virus
(BmNPV)-based system in 1985 [2], more than 100 recombinant proteins have been
produced in the hemolymph of silkworm larvae or pupae. In addition to BmNPV, a
host range extended hybrid of BmNPV and Autographa californica nuclear
polyhedrosis virus (AcNPV), HyNPV, has been used as an expression vector in B.
mori [3,4,5]. AcNPV has also been used in permissive strains of B.
mori [6]. To construct a silkworm bioreactor, it is important to know the
activity of different promoters in different silkworm tissues and the influence
of virus infection on the expression of host genes. The activity of a promoter
in different tissues is usually compared semiquantitatively by reverse
transcription-polymerase chain reaction or Northern blot analysis, but has not
been quantitatively compared in silkworm. Recently, we studied the spread of recombinant AcNPV (rAcNPV) in
various tissues of silkworm and the results showed that the budded form of
rAcNPV can enter all kinds of silkworm tissues from hemolymph efficiently, but
the replication levels of recombinant virus in various silkworm tissues are
different [7]. The high efficiency of infection and replicable ability of
rAcNPV in permissive silkworm facilitate promoter investigation, but because
the copies of foreign genes delivered by rAcNPV are different in different
tissues of silkworm, the activity of the promoter should be normalized to each
viral copy.In this study, two recombinant viruses, AcNPVA3Luc and AcNPVIELuc,
containing the luciferase reporter cassette driven by B. mori cytoplasmic
actin gene promoter (A3) and B. mori nuclear polyhedrosis virus
immediate-early promoter (IE-1), respectively, were constructed and injected
into the hemocele of newly ecdysed 5th instar larvae of silkworm. The
activities of the A3 and IE-1 promoters in different silkworm tissues were
measured by activity of luciferase and normalized by the copy number of
recombinant virus. The activities of the A3 and IE-1 promoters and their
relationship to cell growth rate in different silkworm tissues are discussed.
Materials and methods
Recombinant AcNPVs and cell
culture
Recombinant virus AcNPVA3Luc was constructed in our previous work
[7]. Donor plasmid pFNIELuc was constructed as follows. The 580 bp IE-1
promoter of B. mori nucleopolyhedrovirus (position 116399–116978 in BmNPV
complete genome, NC_001962) was amplified by polymerase chain reaction (PCR)
using the primers 5‘–CGGGATCcgatgtctttgtgat-3‘
and 5‘-GGGGTACCACGATCTTGTCGC-3‘. The PCR product was
digested with BamHI and KpnI, then ligated to the same sites of
pFNLuc [7] to form pFNIELuc. After being verified by DNA sequencing and
restriction mapping, donor plasmid pFNIELuc was transferred to the bacmid AcDEGT [8] to
generate the recombinant bacmid pBacAcNPVIELuc. The bacmid was identified with
PCR (data not shown). The purified bacmids were then used to transfect Sf9 culture
cells with Cellfectin (Invitrogen, Carlsbad, USA) to produce budded recombinant
virus AcNPVIELuc. The Sf9 cells were maintained in Grace? medium (Invitrogen)
supplemented with 10% fetal bovine serum (Invitrogen) at 27 ?C. Generation and
large-scale harvest of the recombinant baculovirus and determination of virus
titer were carried out as in our previous study [7].
Silkworm inoculation and
dissection
Silkworm strain 54A was provided by the Sericultural Research
Institute, Chinese Academy of Agricultural Sciences (Zhenjiang, China).
Silkworm larvae were routinely reared on mulberry leaves. An aliquot of 10 ml recombinant
baculovirus (AcNPVA3Luc or AcNPVIELuc approximately 106 p.f.u.)
was injected into the hemocele of each newly ecdysed 5th instar larva. Ten
larvae were randomly taken and dissected at indicated time points [1, 2, 3, or
4 d post infection (d.p.i.)], and different tissue samples were collected,
washed three times, and stored at –70 ?C until use, as in our previous study [7].
Sample preparation from
different tissues
After 300 ml buffer A [10 mM Tris-HCl (pH 8.0) and 50 mM NaCl] was added to the
hemocyte pellets, the samples were homogenized. The supernatant was collected
by centrifugation at 3578 g for 5 min at 4 ?C; 50 ml supernatant
for measuring luciferase activity was stored at –70 ?C until use, and 200 ml supernatant
was used to extract DNA for real-time PCR assay, as in our previous work [7].
Appropriate volumes of buffer A (approximately 2 ml for 1 g tissue) were added
to samples of fat body, silk gland, Malpighian tubule, and middle gut. The
tissues were ground then centrifuged at 3578 g for 5 min at 4 ?C.
Supernatant (50 ml) for measuring luciferase activity was stored at –70 ?C until use,
DNA was extracted from 250 ml supernatant with phenol and chloroform. Ultraviolet absorption for
every DNA extract was measured at a wavelength of 260 nm (DU7400; Beckman
Coulter, Fullerton, USA). Every DNA extract was diluted with ddH2O to a certain concentration and subjected to real-time PCR.
Primer design, real-time PCR
protocol, and analysis of real-time PCR data
Real-time PCR was carried out in a DNA Engine Option 2 thermal
cycler (MJ Research, Waltham, USA) using a SYBR Premix Ex Taq kit (TakaRa,
Dalian, China) with SYBR Green I dye as the binding fluorescent. The design of
primers, real-time PCR protocol, and analysis of real-time PCR data were as
described in our previous report [7].
Detection of luciferase
activity
The supernatant samples of different tissues were diluted with
phosphate-buffered saline [130 mM NaCl, and 10 mM NaH2PO4 (pH 7.2)] to the same copy number as determined by real-time PCR,
and subjected to detection of luciferase activity. The activity of firefly
luciferase was measured in a BG-P luminometer (MGM Instruments, Hamden, USA)
using a Luciferase assay kit (Promega, Madison, USA), following the
manufacturer?
instructions, at 25 ?C [9]. Three measurements were carried out for each
sample. Promoter activities in different silkworm tissues were quantified by
calculating the ratio of photon counts per minute (c.p.m.) for luciferase
activity to the copy number of the luciferase gene.
Results
Kinetic expression of
luciferase gene in various tissues of silkworm
The copy numbers of the firefly luciferase gene and the luciferase
activities in various tissues of silkworm larvae injected with AcNPVA3Luc or
AcNPVIELuc were determined. Results are shown in Tables 1 and 2.To show the kinetic activities of A3 and IE-1 promoters in hemocyte,
fat body, and silk gland, the time curves of the luciferase activity were drawn
together with the time curves of the copy numbers of firefly luciferase gene (Fig.
1). According to the following mathematical equation, if promoter activity
is constant during the measuring period, the difference log(c.p.m.)–log(Luc) will be
unchanged, and the two time curves will be parallel.
Eq.
The profiles of two time curves showed that the luciferase
activities increased along with the increase in viral copy numbers in different
tissues post injection of rAcNPV. With AcNPVA3Luc, the time curve of
log(c.p.m.) is above the time curve of log(copy) in silk gland, the time curve
of log(c.p.m.) is almost overlapping with the time curve of log(copy) in fat
body, whereas the time curve of log(c.p.m.) is below the time curve of
log(copy) in hemocyte, indicating that the A3 promoter is the highest in silk
gland, followed by fat body, then hemocyte. With AcNPVIELuc, all the time
curves of log(c.p.m.) are below the time curves of log(copy), and the data show
the activity of the IE-1 promoter was lower than the A3 promoter in these three
tissues. The log(c.p.m.)–log(copy) also shows the activities of the IE-1 promoter is the
highest in silk gland, followed by fat body, then hemocyte. The results in Fig.
1 show that the time curves of A3 promoter activity are parallel to the
time curves of viral copy numbers in three tissues, indicating that the
activity of the A3 promoter remained unchanged during the 4 d.p.i.. The time
curves of IE-1 promoter activity are parallel to the time curves of viral copy
numbers in fat body and silk gland, but inclines to fall in the 3–4 d.p.i. in
hemocyte.
Normalized A3 and IE-1 promoter
activities in various tissues of B. mori larvae
The copy numbers of AcNPVA3Luc and AcNPVIELuc and the luciferase
activities in Malpighian tubule and middle gut were determined at 4 d.p.i. The
luciferase activity was divided by the copy numbers of the luciferase gene.
Results are shown in Table 3 and Fig. 2. The activities of A3 and IE-1 promoters in different parts of silk
gland (anterior, middle, and posterior silk gland) were determined at 4 d.p.i.
and results showed that the activities of the two promoters were highest in the
posterior part of silk gland, followed by the middle and anterior parts (data
not shown). In our previous work, the enhanced green fluorescent protein (EGFP)
gene driven by the A3 promoter was expressed in fat body, hemocyte, silk gland,
and middle gut, but was not detectable in Malpighian tubule. It was assayed by
Western blot analysis and was shown as the content of EGFP in a certain
amount of total protein [8]. Results of the present work show that the activity
of the A3 promoter is highest in silk gland, followed by fat body, middle gut,
Malpighian tubule, and hemocyte. Although the activity of the A3 promoter was
lowest in hemocyte, because of the very high copy numbers of the EGFP
expression cassette, EGFP was still largely synthesized in hemocyte.
However, the synthesis of other proteins is less in hemocyte. This could
explain why the content of EGFP in a certain amount of total protein is
high in hemocyte. In this experiment, the photon c.p.m. was higher in hemocytes
than that in Malpighian tubule. According to this work, the copy number of
AcNPVA3Luc in Malpighian tubule is lower than that in fat body and close to
that in middle gut, whereas the activity of the A3 promoter was several times
lower than that in middle gut. This could explain why EGFP was detected
at a low level in middle gut but not detectable in Malpighian tubule in our
previous work.
Discussion
Results of the present work showed that both the A3 (671 bp,
position 1764–2432 in B. mori cytoplasmic actin gene BMU49854) and IE-1
(580 bp, position 116399–116978 in BmNPV complete genome, Genbank accession No. NC_001962) promoter activities are the
highest in silk gland, followed by fat body, middle gut, Malpighian tubule, and
hemocyte, and the activity of the A3 promoter is higher than that of the IE-1
promoter. Activities of the two promoters in different regions of silk gland
show the same orderliness, with the highest activities detected in posterior
silk gland and the lowest in anterior silk gland. As the IE-1 promoter is an
immediately early expression gene of BmNPV, only host transcriptional factors
are required for its efficient expression [10]. The transactivating factors for
both the A3 and IE-1 promoters are coded by the host genome and their copy
numbers did not change with the multiplication of the virus, so the activities
of the A3 and IE-1 promoters in various silkworm tissues are comparable. As discussed in our previous paper [7], the genomic DNA of silk
gland cells is amplified dramatically in 5th instar larvae along with the
enlargement of silk gland cells. The actin 3 protein is the major component of
cellular cytoskeleton and involved in many cellular events. Although expression
of the A3 gene is regulated at the level of transcription during the larval
life of silkworm [11–13], it is greatly synthesized during the 5th instar in those
rapidly enlarged tissues of silkworm larva, especially in microfilament-rich,
non-muscular organs [14]. The silk gland, especially its posterior part, grows
the fastest during the 5th instar, and the differences in A3 promoter activity
in different silkworm tissues seems to be associated with the enlarging rate of
tissues. The differences in IE-1 promoter activity in different silkworm
tissues showed similar trends as the A3 promoter, therefore, the IE-1 promoter
might be activated by some of the transcriptional factors for constitutional
genes of silkworm.Viruses affect host transcription with three possible mechanisms.
First, viral factor(s) directly bind to the regulatory region of the host gene
and affect its transcription. Second, the extremely high expression of viral
factor(s) competes with the cellular protein synthesis machinery in the late
phase of viral infection. Finally, viral factor(s) induce the apoptosis of host
cells, as also happens in the late phase of viral infection. Results of this
work show that the activity of the A3 promoter was unchanged during the 4
d.p.i. of rAcNPV in the three tissues we analyzed. It indicates that there is
no interaction between the A3 promoter and the viral factors, and the apoptosis
status of host cells was not established during the first 3–4 d.p.i. of
rAcNPV. In our work, the B. mori larvae could still grow 7–8 d after rAcNPV
was injected into the hemocele of newly ecdysed 5th instar larvae. So, the
promoter activity could be studied in different tissues of B. mori in
vivo during the first 3–4 d.p.i. using rAcNPV as the gene delivery vector, unless the
promoter can interact with viral factors directly. Rahman and Gopinathan showed
that co-infection with AcMNPV and BmNPV, even infection with AcMNPV alone, was
sufficient to cause inhibition of protein synthesis in cultured Bm-N cells
[15]. In their experiment, may be the infection of virus was serious already or
it is due to the different assay methods to be used.The time curves of luciferase activity of AcNPVIELuc is parallel to
the time curves of the viral copy numbers in fat body and silk gland but
inclined to fall in hemocyte at 34 d.p.i. It indicates that the IE-1 promoter
was depressed to some extent in hemocytes at 3–4 d.p.i., which appears to
be negative feedback. Our previous work showed that rAcNPV replicates much
faster in hemocyte than in fat body or silk gland [7], so repression of the
IE-1 promoter appeared earlier in hemocyte than in fat body or silk gland. The
molecular mechanism of depression of IE-1 expression needs to be further
investigated.This work has raised a quantitative method for measuring promoter
activities in different silkworm tissues when baculovirus is used as the
vector. The luciferase reporter gene is very sensitive and can be detected in a
wide range with good linearity. The background was less than 100 photon/10 s;
more than 1108 photon/10 s can be counted using this instrument.
In quantification of promoter activity, the mean error for measuring luciferase
activity was 4%, less than the error in determination of copy numbers by
real-time PCR. Although the dual-luciferase system has been used widely in
research, the activity of the reference promoter is not consistent in different
tissues, so it is only suitable for measuring the activity of two promoters in
same tissue. For measuring the promoter activity in different tissues,
researchers could use the activity of the luciferase divided by its copy
number.
Acknowledgement
We thank Dr. Muwang Li from the Sericultural Research Institute,
Chinese Academy of Agricultural Sciences (Zhenjiang, China) for kindly
providing silkworm larvae.
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