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The analysis of differentially expressed novel transcripts in diapausing and diapause-activated eggs of Bombyx mori.

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Archives of Insect Biochemistry and Physiology 59:197–201 (2005)
The Analysis of Differentially Expressed Novel
Transcripts in Diapausing and Diapause-Activated
Eggs of Bombyx mori
Jae-Sam Hwang,1* Hyun-Jeong Go,2 Tae-Won Goo,1 Eun-Young Yun,1 Kwang-Ho Choi,1
Su-Il Seong,2 Sang-Mong Lee,3 Bong-Hee Lee,4 Iksoo Kim,1 Taehoon Chun,5 and
Seok-Woo Kang1
As an initial step to define the molecular mechanism of initiation and termination of diapause during the embryogenesis of
silkworms, Bombyx mori, mRNA transcripts from maintained and activated diapause eggs were compared with differential
expression using cDNA array. Twenty-four individual cDNA transcripts were expressed differentially in a total of 1,468 different
cDNAs. Among those clones, mRNA transcript from cytochrome oxidase subunit I (COI), which was detected to be 2-kb
transcripts, gradually increased in diapause-activated eggs during early embryogenesis. Further analysis revealed that mRNA
transcripts from silkworm COI were highly expressed in testis, fat body, and midgut during the larval stage. These results may
indicate that the expression of silkworm COI mRNA is regulated developmentally as well as tissue-specifically. Arch. Insect
Biochem. Physiol. 59:197–201, 2005. © 2005 Wiley-Liss, Inc.
KEYWORDS: DNA array; cytochrome oxidase subunit I; diapause; embryogenesis; insects; silkworm
Most insects have evolved to enter a diapause
at some stage of development to survive under unfavorable environmental conditions. Environmental factors such as temperature and photoperiod
determine the proper moment of a diapause program (Denlinger, 1985). Also, depending on insect species, the embryonic diapause occurs either
at an early embryonic stage or at a much later stage
(Chapman, 1998). The silkworm, Bombyx mori, enters diapause at an early embryonic stage, before
dermal differentiation is completed. Diapause of
the silkworms continues as long as the eggs are
kept at 25°C. Diapuse is terminated due to chilling at 5°C for about 3 months and when the eggs
are transferred back to 25°C, embryonic development restarts. Alternatively, diapause is blocked
when the eggs undergo HCl treatment at 47°C for
5 min after keeping them at 25°C for 20 h after
Diapause hormone (DH), one of the neurohormones, has been identified as a major factor of
inducing diapause in the resulting embryos. The
expression of DH mRNA in the early pupal stage
correlates to the incidence of diapause (Sato et al.,
Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA, Suwon, South Korea
Department of Life Science, College of Natural Science, University of Suwon, Hwaseong, South Korea
Department of Sericulture and Entomology, Miryang National University, Miryang, Korea
Graduate School of Biotechnology, Korea University, Seoul, Korea
Department of Microbiology, School of Medicine, Hanyang University, Seoul, South Korea
Abbreviations used: CO = cytochrome c oxidase; COI = cytochrome oxidase subunit I; DH = diapause hormone.
Contract grant sponsor: Biogreen 21 program, rural development administration, South Korea.
*Correspondence to: Jae-Sam Hwang, Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA, Suwon 441-100, South
Korea. E-mail:
Received 20 May 2004; Accepted 19 February 2005
© 2005 Wiley-Liss, Inc.
DOI: 10.1002/arch.20057
Published online in Wiley InterScience (
Hwang et al.
1993; Xu et al., 1995). Although these findings clearly
show that this hormone regulates in the induction
of embryonic diapause, it is still unknown if the individual gene expression profile may regulate the
stage of initiation or termination of diapause.
Genomics, the study of genes and their function, holds the potential to resolve long-standing
genetic questions. The systematic determination of
the cDNA array by screening specific developmental stages of the insects is required to understand
biological processes.
As the first step to define the molecular mechanism of diapause during the embryogenesis of the
silkworms, Bombyx mori, we carried out differential screening using cDNA expression array and
Northern blot analysis.
Experimental Animals
Silkworm hybrids between Japanese strain 123
and Chinese strain 124 were used in this study. The
eggs after oviposition were preserved at 25°C for 2
months to obtain eggs in diapause stage. To obtain
diapause-activated eggs, the eggs after oviposition
were incubated at 25°C for 3 months and then were
incubated at 5°C for 3 months, inducing the termination of diapause. After the termination of diapause, the eggs restarted their development by being
incubated at 25°C.
Construction of the Full-Length Enriched
cDNA Library
A cDNA Library from B. mori diapausing eggs
and diapause-activated eggs was constructed by using a modification of Maruyama and Sugano’s
method (Maruyama and Sugano, 1994). Briefly,
100 µg of total RNA was treated with 3 U of bacterial alkaline phosphatase (TaKaRa) in 100 µl of 100
mM Tris-HCl (pH 7.5), 2 mM DTT, and 80 U of
RNasin (Promega, Madison, WI) at 37°C for 60
min. After phenol extraction and ethanol precipitation, the total RNA was treated with 100 U of
tobacco acid pyrophosphatase (Waco) in 100 µl
of 50 mM sodium acetate (pH 5.5), 5 mM EDTA,
10 mM 2-mercaptoethanol, and 80 U of RNasin
at 37°C for 60 min. The pre-treated total RNA was
then ligated with 0.4 µg of 5-oligoribonucleotide
GCC UAC UGG-3) using 250 U of RNA ligase
(TaKaRa) in 100 µl of 50 mM Tris-HCl (pH 7.5), 5
mM MgCl2, 5 mM 2-mercaptoethanol, 0.5 mM
ATP, 25% PEG 8000, and 100 U of RNasin at 20°C
for 3 h. After completing these oligo-capping reactions, mRNA was isolated using a commercial kit,
QIAGEN Oligotex™. The synthesis of first-strand
cDNA from the purified mRNA and cDNA amplification was performed as described by Maruyama
and Sugano (1994). The amplified PCR Products
were then digested with SfiI, and cDNAs longer
than 1.3 kb were ligated into DraIII-digested pCNSD2 in an orientation-defined manner. The pCNSD2 vector contains 5 EcoRI -DraIII- EcoRV- DraIII
sites at multi-cloning sites, which were achieved
by modifying pCNS vector (GenBank accession no.
AF416744). The ligated cDNA was then transformed into Escherichia coli Top10F’ (Invitrogen, La
Jolla, CA) by electroporation (Gene Pulser II,
BioRad, Hercules, CA). Then, cDNA sequences were
determined by the dideoxy-mediated chain termination method using a 377 automatic sequencer
(ABI) and were analyzed by BLAST databases
( As a result,
a total of 1,468 different cDNAs were obtained and
used for cDNA expression arrays.
cDNA Expression Array
A total of 1,468 different cDNAs were arrayed
onto Hybond-N membranes (Amersham Biosciences, Sweden) using 96-well format dot blotter (Bio-Rad) after denaturation. To prepare probes,
mRNAs were isolated from diapausing eggs and
diapause-activated eggs. Then, 32P-labeled cDNA
probes were generated by reverse transcription of
0.5–1.0 µg of each poly(A)+ RNA sample in the
presence of [α-32P]dATP. Each cDNA probe was
then hybridized with the membrane at 65°C. A hybridization solution containing 50% formamide,
5 × SSC, 10 × Denhardt’s solution (0.2% each of
bovine serum albumin, Ficoll, and polyvinylpyrArchives of Insect Biochemistry and Physiology
cDNA Array in Silkworm Diapausing Eggs
rolidone), 25 µg/ml sonicated salmon sperm DNA,
and 50 mM sodium phosphate (pH 7.0) was used.
After hybridization, membranes were washed for
30 min with increasing stringency, from 2 × SSC
and 0.1% SDS to 0.1 × SSC and 0.1% SDS. After a
high-stringency wash, membranes were then exposed to X-ray film (AGFA, Germany) for 1 or 3
days at –70°C.
Northern Blot Analysis
Total RNAs were extracted from developing eggs
and five tissues from the 5th instar larva using SV
total RNA isolation system (Promega). To obtain
the developing eggs, diapause-programmed eggs
were treated with hot HCl for 20 h after oviposition. Ten microgram of total RNA per sample was
separated on 1.2% agarose/3% formaldehyde denaturing gel, and transferred onto a nylon membrane (Amersham, Arlington Heights, IL). The cDNA
probe was labeled with [α-32P]dCTP using Prime-It
II random primer labeling kit (Stratagene, La Jolla,
CA), according to the manufacturer’s instructions.
Membrane was hybridized with a cDNA probe at
65°C overnight, and analyzed by autoradiography.
Differentially Expressed cDNAs From Silkworm
Diapausing Eggs and Diapause-Activated Eggs
High-quality mRNAs were isolated from silkworm diapausing eggs and diapause-activated eggs.
The mRNAs were reverse-transcribed to 32P-labeled
cDNAs and hybridized to a cDNA array blot that
contains 1,468 Expressed Sequence Tag (EST)
cDNA clones. After stringent washes and exposure
to X-ray film, the expression profiles of 1,468 genes
in diapausing eggs and diapause-activated eggs
were obtained. By comparing duplicated hybridized blots, we identified 24 genes whose expression patterns were changed as summarized in
Tables 1 and 2. Only genes with expression levels
that were altered more than twofold in comparisons of diapausing eggs and diapause-activated eggs
are included. Ten genes showed increased expression in diapause-activated eggs. Among them, four
August 2005
TABLE 1. Summary of Highly Expressed Genes in Diapause-Activated
Eggs of Silkworm*
Clone no.
Gene bank no.
Putative function
Alcohol dehydrogenase
Cytochrome oxides subunit 1
18 wheeler
D. rerio
D. melanogaster
B. mori
B. mori
*NI, no identity.
genes had functions and the other six genes were
functionally unidentified (Table 1). Four functional
genes were alcohol dehydrogenase, dead-box-1, cytochrome c oxidase subunit I (COI), and 18
wheeler (Bm18w). Bm18w is a Toll-like molecule
that acts as an adhesion molecule during various
processes of morphogenesis in Drosophila melanogaster (Eldon et al., 1994). Most of Bm18w mutants in fruit fly develop up to the third larval instar,
but die prior to pupariation (Williams et al., 1997).
Fourteen genes showed decreased expression
patterns in diapause-activated eggs. Seven out of
14 genes have known functions (Table 2). Among
them, embryonic abnormal visual system gene
(elav) is a gene-specific regulator of alternative premRNA processing, which is important to generate
TABLE 2. Summary of Highly Expressed Genes in Diapausing Eggs of
Clone no.
Gene bank no.
*NI, no identity.
Putative function
ATP synthetase, gamma
Caspase-8/-10 homolog
Ribosomal protein L8
Glycl-tRNA synthetase
tumor protein
Heat shock protein hsp 20.8A
Retinol dehydratase
D. melanogaster
D. melanogaster
S. frugiperda
B. mori
D. rerio
B. mori
S. frugiperda
Hwang et al.
a embryonic neural system in Drosophila (Soller and
Whiter, 2003). Caspase-8/-10 homolog (Dcp2) gene
and translationally controlled tumor protein (tct1)
gene were also down-regulated in diapause-activated eggs. DCP2 contains two N-terminal death
effector domains fused to a caspase-like domain,
which is the mediator of an apoptosis (Inohara et
al., 1997). A protein encoded by tct1 is ubiquitously expressed and is present in evolutionarily
diverse organisms. Recently, TCT1 was reported as
a regulator of translation elongation factor eEF1A
in yeast (Cans et al., 2003).
Silkworm COI Expression
Using cDNA expression array, we found a number of genes that show differential expression in silkworm diapausing eggs and diapause-activated eggs.
We focused on the differential expression of cytochrome oxidase subunit I (COI), whose expression
Fig. 1. Expression analyses of B. mori COI mRNA. A:
Stage-specific expression of the B. mori COI mRNA during
embryogenesis. Northern blot analysis was done with total RNA samples from diapausing eggs and HCl-activated
eggs. The B. mori COI mRNA was detected as an approximately 2.0 kb single mRNA transcript (top). D: diapausing eggs; 0–10: days after HCl treatment. B: Tissue-specific
expression of the B. mori COI mRNA at 5th instar larvae.
was significantly increased in diapause-activated eggs.
To validate this observation, we examined COI mRNA
expression by Northern blot analysis. As shown in
Figure 1A, COI mRNA was expressed at a low level
in diapausing eggs, but began to remarkably increase
the expression level at day 6 in developing eggs.
Cytochrome c oxidase (CO) is a terminal enzyme
in the energy transducing respiratory chain of eukaryotes (Babcock and Wikstrom, 1992). The increased expression of this electron transfer protein
may be associated with oxygen consumption that
accompanies the termination of diapause and the
resumption of development. A silkworm embryo is
completed to larval form between day 6 and 7 after
oviposition. At this point, a large amount of CO
may be required because of the increase in energy
and oxygen consumption that accompanies tissue
formation. COI mRNA was expressed at a low level
in diapausing eggs because of the developmental
Northern analysis was done with total RNA samples from
testis, ovary, fat body, posterior silk gland, and midgut.
Each lane contained 20 µg total RNA. An approximately
2.0-kb single mRNA transcript was detected (top). Lanes
1: Testis; 2: ovary; 3: fat body; 4: posterior silk gland; 5:
midgut. As an internal control, the amount of 18S RNA is
shown (bottom).
Archives of Insect Biochemistry and Physiology
cDNA Array in Silkworm Diapausing Eggs
Previously, rates of oxygen consumption in eggs
were monitored during the silkworm diapause
(Yaginuma and Yamashita, 1999). Oxygen consumption increased slowly in the course of termination of diapause and blastokinesis. Oxygen
consumption then increased rapidly until the stage
of larval hatching. Oxygen consumption also increased in HCl-treated eggs, similar to developing
eggs. In HCl-treated eggs, oxygen consumption increased gradually for 5 days and then increased
markedly toward larval hatching. This is consistent
with our observation that the expression of COI
mRNA was markedly increased on 6 days after HCl
treatment (Fig. 1A).
We further examined tissue expression of COI
mRNA in silkworm larva by Northern blot analysis (Fig. 1B). As a result, silkworm COI mRNA was
expressed highly in testis and midgut of the 5th
instar larva. This result is very similar to a previous report that COI mRNA expression is correlated
to histogenesis and organogenesis in cockroaches
(Martinez-Gonzalez and Hegardt, 1994). Thus, the
COI mRNA expression of silkworm might be correlated to the increase of oxygen consumption during embryogenesis and organogenesis.
This work was supported by a grant from
Biogreen 21 program, Rural Development, Administration, Republic of Korea.
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