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Molecular cloning expression and characterization of a cDNA encoding the arylphorin-like hexameric storage protein from the mulberry longicorn beetle Apriona germari.

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Archives of Insect Biochemistry and Physiology 53:49–65 (2003)
Molecular Cloning, Expression, and Characterization
of a cDNA Encoding the Arylphorin-Like Hexameric
Storage Protein From the Mulberry Longicorn Beetle,
Apriona germari
Seong Ryul Kim,1 Hyung Joo Yoon,2 Nam Sook Park1 Sang Mong Lee,3 Jae Yu Moon,4
Sook Jae Seo,5 Byung Rae Jin,1 and Hung Dae Sohn1*
We describe here the cloning, expression, and characterization of a cDNA encoding the arylphorin-like hexameric storage
protein from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The complete cDNA sequence of A.
germari hexamerin (AgeHex) is comprised of 2,160 bp with 720 amino acid residues. The deduced protein sequence of
AgeHex is most similar to Tenebrio molitor hexamerin2 (65.3%). Phylogenetic analysis further confirmed the AgeHex is more
closely related to T. molitor hexmerin2 and T. molitor early-staged encapsulation inducing protein than to the other insect
storage proteins. Southern blot analysis suggested the presence of A. germari hexamerin gene as a single copy and Northern
blot analysis confirmed fat body-specific expression at the transcriptional level. The cDNA encoding AgeHex was expressed as a
80-kDa protein in the baculovirus-infected insect cells. Western blot analysis using the polyclonal antiserum against recombinant AgeHex indicated that the AgeHex corresponds to storage protein 2 (SP2) present in the A. germari larval hemolymph.
Arch. Insect Biochem. Physiol. 53:49–65, 2003. © 2003 Wiley-Liss, Inc.
KEYWORDS: insect; mulberry longicorn beetle; Apriona germari; hexamerin; cDNA sequence; storage protein; phylogeny; insect cells; baculovirus
INTRODUCTION
Insect specific larval hemolymph proteins, larval storage proteins (LSPs), are hexameric proteins
(~500 kDa), consisted of 80 kDa subunits (Telfer
and Kunkel, 1991; Haunerland, 1996). They are
generally synthesized during larval development in
the fat body, released into the hemolymph, and
also sequestered in the fat body where they serve
as sources of amino acids for utilization by pupae
and adults during metamorphosis and reproduction (Levenbook, 1985; Kanost et al., 1990; Telfer
and Kunkel, 1991). These proteins are generally
classified into two groups, aromatic amino acidrich arylphorins and methionine-rich storage proteins (Haunerland, 1996; Burmester, 1999).
1
College of Natural Resources and Life Science, Dong-A University, Busan, Korea
2
Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, RDA, Suwon, Korea
3
Department of Sericultural and Entomological Biology, Miryang National University, Miryang, Korea
4
College of Agriculture and Life Sciences, Seoul National University, Suwon, Korea
5
Division of Life Science, Gyeongsang National University, Chinju, Korea
Contract grant sponsor: Dong-A University Research Fund, 2001.
*Correspondence to: Hung Dae Sohn, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
E-mail: hdsohn@mail.donga.ac.kr
Received 16 September 2002; Accepted 1 March 2003
© 2003 Wiley-Liss, Inc.
DOI: 10.1002/arch.10085
Published online in Wiley InterScience (www.interscience.wiley.com)
50
Kim et al.
The larval storage proteins are important for insect development, therefore, cDNA sequence information for the hexameric storage proteins has been
reported from diverse insects. Furthermore, arylphorin-like hexameric storage protein genes have
been sequenced in two coleoptran species, the yellow mealworm, Tenebrio molitor (Cho et al., 1999)
and Colorado potato beetle, Leptinotarsa decemlineata (Koopmanschap et al., 1995).
The Cerambycidae, commonly known as longhorned beetles, is one of the largest groups in Coleoptera. The family has about 20,000 species
throughout the world and most species of the family are wood-borers (Crowson, 1981; Daly et al.,
1998). One of the long-horned beetles, the mulberry longicorn beetle, Apriona germari, is widely
distributed in eastern Asia, but not much information on this species is available. Thus, our objective in initiating this study is to illustrate the
structure of the storage protein gene in the longhorned beetle.
In a previous study, we reported on the purification and characterization of three hexameric
storage proteins (SPs) (designated as SP1, SP2,
and SP3) in the larval hemolymph of the mulberry longicorn beetle, A. germari (Yoon et al.,
2001). These SP1, SP2, and SP3 have a native molecular weight of 480, 440, and 420 kDa, respectively. We isolated and newly sequenced the
hexameric storage protein (SP2) gene of A. germari
and compared the sequences with that of the
known storage protein genes. This study describes
the cloning, expression, and characterization of a
cDNA encoding the hexameric storage protein
(SP2) from the mulberry longicorn beetle, A.
germari.
MATERIALS AND METHODS
Animals
The larvae of the mulberry longicorn beetle,
Apriona germari (Coleoptera, Cerambycidae), were
reared on an artificial diet as described previously
(Yoon and Mah, 1999).
cDNA Library Screening, Nucleotide Sequencing, and
Data Analysis
A cDNA library (Kim et al., 2001) was constructed from the whole body of A. germari larvae.
The sequencing of randomly selected clones harboring cDNA inserts was performed to generate the
expressed sequence tags (ESTs). For DNA sequencing, plasmid DNA was extracted by Wizard minipreparation kit (Promega, Madison, WI). Sequence
of each cDNA clone was determined using an automatic sequencer (model 310 Genetic Analyzer;
Perkin-Elmer Applied Biosystems, Foster City, CA).
The sequences were compared using the DNASIS
and BLAST programs provided by the NCBI.
GenBank, EMBL, and SwissProt databases were
searched for sequence homology using a BLAST algorithm program.
MacVector (ver. 6.5) was used to align the
amino acid sequences of hexameric larval storage protein (LSP) gene. Including the 33 GenBankregistered amino acid sequences of LSP genes,
phylogenetic analysis among LSP genes was performed using PAUP (Phylogenetic Analysis using Parsimony) version 3.1 (Swofford, 1990).
The tree was obtained by bootstrap analysis with
the option of heuristic search (1,000 replications). The accession numbers of the sequences
in the GenBank are as follows: A. germari hexamerin (AgeHex, AF509880; this study), T. molitor
hexamerin2 (TmoHex, AAK77560), T. molitor
early-staged encapsulation inducing protein
(TmoESEIP, BAA81665), Leptinotarsa decemlineata
diapause protein1 (LdeDP1, X86074), Bombyx
mori methionine-rich storage protein (BmoMt,
P09179), B. mori arylphorin (BmoAry, AAA27849),
Choristoneura fumiferana diapause associated protein1 (CfuDAP1, AAC35428), C. fumiferana diapause associated protein2 (CfuDAP2, AAC35429),
Galleria mellonella arylphorin (GmeAry,
AAA74229), Hyalophora cecropia methionine-rich
storage protein (HceMt, AAB86646), H. cecropia
arylphorin (HceAry, AAB86644), Hyphantria
cunea storage protein1 (HcuSP1, AAB38773), H.
cunea storage protein2 (HcuSP2, AAD39550),
Manduca sexta methionine-rich storage protein
Archives of Insect Biochemistry and Physiology
Storage Protein cDNA From A. germari
(MseMt, AAA29320), M. sexta arylphorin a
(MseArya, AAA29303), M. sexta arylphorin b
(MseAryb, AAA29304), Plodia interpunctella storage protein1 (PinSP1, AAK71136), P. interpunctella
storage protein2 (PinSP2, AAK71137), Spodoptera
litura methionine-rich storage protein (SliMt,
CAB55604), Trichoplusia ni basic juvenile hormone-suppressible protein1 (TniBJHSP1,
AAA27883), T. ni basic juvenile hormone-suppressible protein2 (TniBJHSP2, AAA27882),
Aedes aegypti hexamerin1 (AaeHex1, AAB46713),
A. aegypti hexamerin2 (AaeHex2, AAB46714), A.
atropalpus hexamerin1.2 (AatHex1.2, AAK77560),
Anopheles gambiae hexamerinA (AgaHexA,
AAA96405), A. merus hexamerinA (AmeHexA,
AAC31879), Calliphora vicina Arylphorin (CviAry,
AAB58985), C. vicina larval serum protein2
(CviLSP2, AAC24157), Drosophila melanogaster
larval serum protein1 (DmeLSP1, AAB58821), D.
melanogaster larval serum protein2 (DmeLSP2,
CAA66371), Musca domestica arylphorin (MdoAry, AAB48820), M. domestica hexamerin (MdoHex, AAF05597), Blaberus discoidalis hexamerin
(BdiHex, AAA74579), and Periplaneta americana
(PamHex, AAB09629).
RNA Isolation and Northern Blot Analysis
Total RNAs were isolated from the whole body,
mid gut, and fat body of the A. germari by using
the Total RNA Extraction Kit (Promega). Total
RNAs (10 mg/lane) from the A. germari were denatured by glyoxalation (McMaster and Carmichael, 1977), transferred onto a nylon blotting
membrane (Schleicher & Schuell, Dassel, Germany) and hybridized at 42°C with a probe in a
buffer containing 2 ´ PIPES, 50% formamide, 1%
sodium dodecyl sulphate (SDS), and blocking
agent (Boehringer Mannheim, Mannheim, Germany). The probe used to detect the hexamerin
gene transcripts was 2,160 bp of AgeHex gene
cloned in this study and labeled with [a-32P] dCTP
(Amersham, Arlington Heights, IL) using the
Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA). After hybridization, the
membrane filter was washed three times for 30
June 2003
51
min each in 0.1% SDS and 0.2 ´ SSC (1 ´ SSC is
0.15 M NaCl and 0.015 M sodium citrate) at
65°C, and finally exposed to X-ray film. For
rehybridization, the membrane was washed for
20 min at room temperature in sterile millipore
water. Then, the membrane was washed overnight
at 65°C in 50 mM Tris-HCl (pH 8.0), 50% dimethylformamide, and 1% SDS in order to remove the hybridized probe. The membrane was
then rehybridized to [a-32P] dCTP-labeled 60S
rRNA probe (Kim et al., 2001). The 60S rRNA
gene was used as an internal loading control.
Genomic DNA Isolation and Southern Blot Analysis
Genomic DNA was extracted from the larvae
of A. germari using a Wizard™ Genomic DNA Purification Kit, according to the manufacturer’s instructions (Promega). Genomic DNA from A.
germari was digested with EcoRI, BamHI, and ApaI,
and electrophoresed through 1.0% agarose gel.
The DNA from the gel was transferred onto a nylon blotting membrane (Schleicher & Schuell)
and hybridized at 42°C with a probe in a hybridization buffer containing 5 ´ SSC, 50% formamide, 0.1% (W/V) N-lauroylsarcosine, 0.02%
SDS, and 2% blocking agent (Boehringer Mannheim). The probe used to detect the DNA fragment containing hexamerin gene was 2,160 bp
of AgeHex gene cloned in this study and labeled
with [a-32P] dCTP (Amersham) using the PrimeIt II Random Primer Labeling Kit (Stratagene). The
other procedures for washing of the membrane
filter and exposing to X-ray film were performed
as in the Northern blot analysis.
Construction of Baculovirus Transfer Vector
The 2,160 bp AgeHex gene from pBlueScriptAgeHex was digested with XbaI and XhoI, and then
inserted into the XbaI and XhoI sites of pBacPAK9
(Clontech, Palo Alto, CA) to produce transfer vector pBacPAK9-AgeHex. In this transfer vector, the
hexamerin gene is under the control of the polyhedrin promoter of Autographa californica nuclear
polyhedrosis virus.
52
Kim et al.
Cell Culture and Virus
The Spodoptera frugiperda IPLB Sf21-AE (Vaughn
et al., 1977) clone 9 (Sf9) cells were maintained
at 27°C in TC100 medium (Gibco BRL Life Technologies, Gaithersburg, MD), supplemented with
10% fetal bovine serum (FBS; Gibco BRL Life Technologies) as described by standard methods
(O’Reilly et al., 1992). Wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant AcNPV were propagated in Sf9 cells.
The titer was expressed as plaque forming units
(PFU) per ml (O’Reilly et al., 1992).
Construction of Recombinant Virus
Cell culture dishes (35 mm) were seeded with
1.0–1.5 ´ 106 cells and incubated at 27°C for 1 h
to allow cell attachment. One microgram of BacPAK6 viral DNA (Clontech), 5 mg of pBacPAK9AgeHex in 20 mM HEPES buffer and sterile water
to make a total volume of 50 ml were mixed in a
polystyrene tube. Fifty microliters of 100 mg/ml
Lipofectin™ (Gibco BRL Life Technologies) were
gently mixed with the DNA solution and the mixture was incubated at room temperature for 30
min. The cells were washed twice with 2 ml serum-free TC100 medium and fed with 1.5 ml serum-free TC100 medium. The Lipofectin-DNA
complexes were added dropwise to the medium
covering the cells while the dish was gently swirled.
After incubation at 27°C for 5 h, TC100 medium
containing antibiotics and 10% FBS was added to
each dish and incubation at 27°C was continued.
At 5 days of postinfection (p.i.), the supernatant
was harvested, clarified by centrifugation at 2,000
rpm for 5 min, and stored at 4°C before plaquing
on Sf9 cells. Recombinant AcNPV was plaque purified on 6-well plates seeded with 1.5 ´ 106 Sf9
cells as described by O’Reilly et al. (1992). Cells
were visualized under the inverted phase contrast
microscope (Olympus, Tokyo, Japan).
SDS-Polyacrylamide Gel Electrophoresis (PAGE)
Insect Sf9 cells were mock-infected or infected
with the wild-type AcNPV and recombinant
AcNPV in a 35-mm diameter dish (1 ´ 106 cells)
at a multiplicity of infection (MOI) of 5 PFU
per cell. After incubation at 27°C, cells were harvested at 1, 2, and 3 days p.i. For SDS-PAGE
(Laemmli, 1970) of cell lysates, uninfected Sf9
cells and cells infected with virus were washed
twice with PBS and mixed with protein sample
buffer and boiled. The total cellular lysates were
subjected to 10% SDS-PAGE. After electrophoresis, gel was fixed and stained with 0.1% Coomassie brilliant blue R-250.
N-Terminal Amino Acid Sequencing
To determine the N-terminal amino acid sequence of the A. germari hexamerin, the recombinant A. germari hexamerin expressed in the
baculovirus-infected insect cells was electroeluted from the SDS-PAGE gels. The eluted
recombinant A. germari hexamerin was subsequently subjected to 10% SDS-PAGE. The protein band of the A. germari hexamerin was
blotted onto a polyvinylidene difluoride (PVDF)
membrane (ProBlott™, Applied Biosystems), cut
out from the membrane, and subjected to automated Edman degradation using an Applied
Biosystems sequencer.
Preparation of Polyclonal Antibody and
Western Blot Analysis
The recombinant A. germari hexamerin was
electroeluted from the gel, mixed with equal volume of Freund’s complete adjuvant (a total of 200
ml), and injected into Balb/c mice, respectively.
Three successive injections were performed with a
one-week interval beginning a week after the first
injection with antigens mixed with equal volume
of Freund’s incomplete adjuvant (a total of 200
ml). Blood was collected 3 days after the last injection and centrifuged at 13,000 rpm for 5 min.
The supernatant antibodies were stored at –70°C
until use.
For Western blot analysis, SDS-PAGE was carried out as described above. Proteins were blotted
to a sheet of nitrocellulose membrane (Sigma,
Archives of Insect Biochemistry and Physiology
Storage Protein cDNA From A. germari
0.45 mm of pore size) (Towbin et al., 1979). The
blotting was performed in transfer buffer (25
mM Tris and 192 mM glycine in 20% methanol) at 30 volts overnight at 4°C. After blotting,
the membrane was blocked by incubation in 1%
BSA solution for 2 h at room temperature. The
blocked membrane was incubated with antiserum solution (1:1,000 v/v) for 1 h at room temperature and washed in TBST (10 mM Tris-HCl,
pH 8.0, 100 mM NaCl, 0.05% Tween 20). Subsequently, the membrane was incubated with
goat anti-mouse IgG alkaline phosphatase conjugate (1:10,000 v/v, Sigma) for 30 min at room
temperature. After repeated washing, substrate
solution (10 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 5 mM MgCl2) containing NBT (nitro-blue
tetrazolium) and BCIP (5-bromo-4-chloroindolyl
phosphate) was added. The reaction was quenched
with distilled water.
RESULTS AND DISCUSSION
Cloning, Sequencing and Phylogenetic Analysis of a
cDNA Encoding the Hexamerin From A. germari
To identify a cDNA encoding the storage protein of the mulberry longicorn beetle, A. germari,
we selected a cDNA clone showing similarity to
the reported insect storage proteins. The nucleotide sequence of the cDNA clone was analyzed
and its amino acid sequence was deduced. The
nucleotide and deduced amino acid sequences
of a cDNA encoding the A. germari hexameric
storage protein (AgeHex) is presented in Figure
1. The complete cDNA sequence of AgeHex comprised 2,233 bp with an initiation codon at positions 1–3 and a termination codon at positions
2,161–2,163. A polyadenylation signal AATAAA
occurred in the 3¢-untranslated region at positions 2,191–2,196. The AgeHex cDNA sequence
had an open reading frame of 2,160 nucleotides,
encoding a protein of 720 amino acid residues.
Hydropathy profile of the deduced amino acid
sequence of AgeHex suggests that the N-terminal amino acid sequence of AgeHex is followed
by a cluster of 18 hydrophobic amino acids inJune 2003
53
dicating a signal peptide for transmembrane
transport (data not shown).
A multiple sequence alignment of the deduced protein sequence of AgeHex gene with
other coleopteran hexamerin sequences, including T. molitor hexamerin2, T. molitor early-staged
encapsulation inducing protein (Cho et al.,
1999), and L. decemlineata diapause protein1
(Koopmanschap et al., 1995), is shown in Figure 2. One potential N-glycosylation site (AsnX-Ser/Thr) is conserved at position Asn-202
among the species aligned. The AgeHex and three
coleopteran hexamerin genes also contained the
highly conserved two larval storage protein (LSP)
signature motifs (Burmester, 1999; Zhu et al.,
2002).
The LSP signature-1 motif of Y(F/Y/W)-ED(L/
I/V/M)—N———H—P is highly conserved in the
coleopteran, lepidopteran, and dipteran LSPs
(Table 1). In AgeHex a corresponding sequence
was YYLEDVGINAFYYYFNLYYP at positions 218–
237. The conserved histidine residue in the insect LSP signature-1 motifs was substituted for
an asparagine residue (underlined). This substitution also appeared in the known coleopteran
hexamerins (Koopmanschap et al., 1995; Cho et
al., 1999), C. vicina LSP2 (Burmester et al.,
1998), and D. melanogaster LSP2 (Mousseron et
al., 1997). The LSP signature-2 motif of T—
RDP-(F/Y)(F/Y/W) is conserved in most insect
LSPs. The corresponding sequence in AgeHex
was TSMRDPVFF at positions 421–429 (Table 2).
Based on homology searching using the
DNASIS and BLAST programs provided by the
NCBI, the AgeHex sequence was closely related to
insect LSPs such as hexamerins, arylphorins, diapause proteins, methionine-rich storage proteins,
and juvenile hormone-suppressible proteins (Table
3). Furthermore, the AgeHex showed a high protein sequence identity to the three coleopteran
hexamerins. The AgeHex had 65.3% protein sequence identity with T. molitor hexamerin2, 58.9%
identity with T. molitor early-staged encapsulation
inducing protein, and 61.0% identity with L.
decemlineata diapause protein1. The AgeHex also
was homologous with dictyopteran hexamerin,
54
Kim et al.
Figure 1.
Archives of Insect Biochemistry and Physiology
Storage Protein cDNA From A. germari
55
Fig. 1. The nucleotide and deduced amino acid sequences
of A. germari hexamerin gene. The start codon of ATG is
boxed and the termination codon is asterisk. The polyadenylation signal AATAAA is underlined. The putative
glycosylation site is indicated by closed diamond. The arrowhead shows the end of the signal peptides. The
GenBank accession number of A. germari hexamerin gene
is AF509880.
Blaberus discoidalis hexamerin (Jamroz et al., 1996)
with 49.9% amino acid sequence identity and
Periplaneta americana hexamerin (Wu et al., 1996)
with 46.1% identity. However, the AgeHex showed
relatively lower protein sequence identity (33–
48%) to lepidopeteran and dipteran LSPs.
Phylogenetic relationships among the deduced
amino acid sequences of AgeHex and 33 insect
LSPs were inferred on the basis of maximum par-
simony analysis (Fig. 3). The dendrogram showed
that those of insect LSPs were clustered largely into
four separated groups on the basis of their taxonomic relationships of insect order. The AgeHex
formed a subgroup only with coleopteran hexamerins (100% of bootstrap value).
The amino acid composition of AgeHex was
compared with other insect hexameric storage proteins, including coleopteran hexamerins, aryl-
June 2003
56
Kim et al.
Fig. 2. Multiple sequence alignment of the deduced protein sequence of the A. germari hexamerin gene with other
coleopteran storage protein sequences. In shaded boxes
are the residues that are identical to those in A. germari
hexamerin (AgeHex). LSP signature sequences are in unshaded boxes.
Archives of Insect Biochemistry and Physiology
Storage Protein cDNA From A. germari
57
TABLE 1. Amino Acid Sequence Comparison of LSP Signature-1 Motif of A. germari Hexamerin and 18 Other
Insect Larval Storage Proteins
Order
Coleoptera
Lepidoptera
Diptera
a
LSP signature-1 motif a
Species
AgeHex
TmoHex2
TmoESEIP
LdeDP1
SliMt
PinSP1
MseMt
MseArya
MseAryb
HcuSP1
HcuSP2
HceMt
GmeAry
BmoMt
BmoAry
AaeHex2
CvilSP2
DmeLSP2
218
210
214
216
229
227
231
230
227
231
222
225
227
229
227
197
219
220
Reference
YYLEDVGINAFYYYFNLYYP
YYMEDVGLNSFYYYYNLYYP
YFTEDIGVNSFYYYYNLYYP
YFLEDIDMNSLYYYYNLYYP
YFTEDIDLNTYLYYLHMSYP
YFTEDIDLNTYYYYFHVDYP
YFTEDIDLNTYMYYLHMNYP
YFYEDIGLNSYYYYFHMHLP
YFTEDIGLNSYYYYFHSHLP
YFTEDVDLNTYMYYLHMSYP
YFTEDIDLNTYYYYFHVDYP
YFTEDIDLNTYYYYFHVDYP
YFIEDIGWNSYYYYFHNRFP
YFMEDVDLNTYMYYLHMNYP
YFTEDIGMNAYYYYFHSHLP
YFTEDIGLNTYYYYFHADYP
YFLEDIGFNAFYYYYNLDYP
YYLEDVGFNAFYYYFNLDYP
237
229
233
235
248
246
250
249
246
250
241
244
246
248
246
216
238
239
This study
GenBank accession number: AAK77560
Cho et al. (1999)
Koopmanschap et al. (1995)
Zheng et al. (2000)
Zhu et al. (2001)
Wang et al. (1993)
Willott et al. (1989)
Willott et al. (1989)
Cheon et al. (1996)
Hwang et al. (2001)
Burmester et al. (1998)
Memmel et al. (1992)
Sakurai et al. (1988)
Fujii et al. (1989)
Gordadze et al. (1999)
Burmester et al. (1998)
Mousseron et al. (1997)
The LSP signature-1 motif if Y,F/Y/W,X,E,D,L/I/V/M,X,X,N,XXXXXXHXXXP.
phorin, and methionine-rich storage proteins
(Table 4). In general, the arylphorin contains 1 to
3% methionine and 16 to 21% aromatic amino
acids, and methionine-rich storage protein contains
4 to 11% methionine and 9 to 13% aromatic
amino acids in the lepidopteran insects (Telfer and
Kungel, 1991). The AgeHex contains 2.36% methionine and 17.78% aromatic amino acids. The
amino acid composition of AgeHex is similar to
that of TmoHex2 with high aromatic amino acids.
Also, the LdeDP1 (Koopmanschap et al., 1995), an
arlyphorin-type storage hexamer of the Colorado
potato beetle, containing 3.13% methionine and
16.52% aromatic amino acids, is similar to that of
AgeHex. These results suggest that AgeHex can be
classified as arylphorin-like storage protein.
TABLE 2. Amino Acid Sequence Comparison of LSP Signature-2 Motif of A. germari Hexamerin and 18
Other Insect Larval Storage Proteins
Order
Coleoptera
Lepidoptera
Diptera
a
June 2003
LSP signature-2 motif a
Species
AgeHex
TmoHex2
TmoESEIP
LdeDP1
SliMt
PinSP1
MseMt
MseArya
MseAryb
HcuSP1
HcuSP2
HceMt
GmeAry
BmoMt
BmoAry
AaeHex2
CvilSP2
DmeLSP2
421
412
416
419
421
424
423
423
420
423
417
420
423
422
420
388
401
402
TSMRDPVFF
TSLRDPAFY
TSMRDPAFY
TCMRDPMFF
TCLRDPVFW
TALRDPVFY
TCLRDPVFW
TSLRDPVFY
TSLRDPMFY
TCLRDPVFW
TALRDPVFY
TALRDPVFY
TSLRDPAFY
TCLRDPVFW
TSLRDPAFY
TSLRDPMFY
TSLRDPLFY
TSMRDPIFY
The LSP signature-2 motif if T,X,X,R,D,PX,F/Y,F/Y/W.
Reference
429
420
424
427
429
432
431
431
428
431
425
428
431
430
428
396
409
410
This study
GenBank accession number: AAK77560
Cho et al. (1999)
Koopmanschap et al. (1995)
Zheng et al. (2000)
Zhu et al. (2001)
Wang et al. (1993)
Willott et al. (1989)
Willott et al. (1989)
Cheon et al. (1996)
Hwang et al. (2001)
Burmester et al. (1998)
Memmel et al. (1992)
Sakurai et al. (1988)
Fujii et al. (1989)
Gordadze et al. (1999)
Burmester et al. (1998)
Mousseron et al. (1997)
1. AgeHex
2. TmoHex2
3. LdeDP1
4. TmoESEIP
5. PamHex
6. BdiHex
7. MseArya
8. MseAryb
9. HceAry
10. GmeAry
11. BmoAry
12. TniBJHSP2
13. HcuSP2
14. HceMt
15. CfuDAP1
16. PinSP1
17. CfuDAP2
18. PinSP2
19. BmoMt
20. MseMt
21. HcuSP1
22. SliMt
23. TniBJHSP1
24. AaeHex1
25. AaeHex2
26. AatHex12
27. AgaHexA
28. AmeHexA
29. CviAry
30. CviLSP2
31. DmeLSP1
32. DmeLSP2
33. MdoAry
34. MdoHex
–
323
363
382
466
501
498
505
513
512
526
521
526
526
534
542
559
550
545
542
555
554
553
544
484
552
527
527
578
538
582
549
602
571
1
0.347
–
400
270
473
483
492
510
507
498
531
539
540
539
546
549
567
563
557
557
566
571
572
543
506
551
543
543
591
527
585
544
601
552
2
0.390
0.430
–
449
489
504
525
530
533
522
549
545
561
550
550
551
565
560
560
550
566
566
563
552
510
558
545
545
614
545
617
572
603
569
3
0.411
0.290
0.483
–
534
549
551
560
558
546
562
558
558
558
563
554
592
588
578
570
575
581
586
586
533
593
583
583
629
584
615
589
655
603
4
0.501
0.509
0.526
0.574
–
363
527
531
541
535
559
530
534
527
547
557
547
543
544
535
544
543
552
536
505
540
526
526
606
546
615
559
594
564
5
0.539
0.519
0.542
0.590
0.390
–
535
535
537
538
566
561
570
563
570
581
579
575
577
564
581
578
584
564
534
564
552
551
622
582
629
594
626
603
6
0.535
0.529
0.565
0.592
0.567
0.575
–
225
221
317
281
513
508
504
512
528
564
537
531
533
538
546
546
536
498
535
515
514
581
532
592
540
588
557
7
0.543
0.548
0.570
0.602
0.571
0.575
0.242
–
178
330
237
515
514
510
516
524
563
544
530
534
542
541
548
530
491
534
522
521
592
539
591
549
583
553
8
0.552
0.545
0.573
0.600
0.582
0.577
0.238
0.191
–
328
230
509
507
502
509
515
562
549
541
536
539
546
550
542
498
544
530
531
588
550
592
554
599
569
9
0.551
0.535
0.561
0.587
0.575
0.578
0.341
0.355
0.353
–
359
531
515
524
528
537
568
547
545
547
555
551
560
547
515
549
536
537
588
545
592
561
594
562
10
0.566
0.571
0.590
0.604
0.601
0.609
0.302
0.255
0.247
0.386
–
529
530
528
534
535
579
555
558
551
556
559
565
563
514
560
550
552
614
553
613
562
592
568
11
TABLE 3. Pairwise Comparison Among Amino Acid Sequences of A. germari Hexamerin and the Known Storage Proteins*
0.560
0.580
0.586
0.600
0.570
0.603
0.552
0.554
0.547
0.571
0.569
–
193
226
194
245
461
448
461
435
443
447
441
543
522
550
546
547
611
590
611
591
641
608
12
0.566
0.581
0.603
0.600
0.574
0.613
0.546
0.553
0.545
0.554
0.570
0.208
–
249
240
274
478
463
469
465
471
469
472
552
529
556
553
554
615
592
611
586
635
603
13
0.566
0.580
0.591
0.600
0.567
0.605
0.542
0.548
0.540
0.563
0.568
0.243
0.268
–
229
261
474
463
467
460
453
458
464
550
523
556
562
563
613
591
598
585
633
604
14
0.574
0.587
0.591
0.605
0.588
0.613
0.551
0.555
0.547
0.568
0.574
0.209
0.258
0.246
–
228
477
463
465
452
454
467
462
545
525
549
560
561
613
590
608
591
633
603
15
16
0.583
0.590
0.592
0.596
0.599
0.625
0.568
0.563
0.554
0.577
0.575
0.263
0.295
0.281
0.245
–
477
453
469
446
447
473
469
558
533
561
560
561
630
605
614
598
650
607
17
0.601
0.610
0.608
0.637
0.588
0.623
0.606
0.605
0.604
0.611
0.623
0.496
0.514
0.510
0.513
0.513
–
299
311
267
275
266
287
574
556
580
582
582
645
612
632
619
641
612
58
Kim et al.
Archives of Insect Biochemistry and Physiology
June 2003
0.591
0.605
0.602
0.632
0.584
0.618
0.577
0.585
0.590
0.588
0.597
0.482
0.498
0.498
0.498
0.487
0.322
–
311
265
287
289
303
569
564
573
563
563
640
606
639
615
630
602
18
0.586
0.599
0.602
0.622
0.585
0.620
0.571
0.570
0.582
0.586
0.600
0.496
0.504
0.502
0.500
0.504
0.334
0.334
–
228
260
245
256
558
545
559
570
570
615
600
619
597
640
609
19
0.583
0.599
0.591
0.613
0.575
0.606
0.573
0.574
0.576
0.588
0.592
0.468
0.500
0.495
0.486
0.480
0.287
0.285
0.245
–
219
208
228
560
543
554
557
557
625
595
625
604
635
608
20
0.597
0.609
0.609
0.618
0.585
0.625
0.578
0.583
0.580
0.597
0.598
0.476
0.506
0.487
0.488
0.481
0.296
0.309
0.280
0.235
–
198
202
560
552
554
562
562
625
602
626
608
642
614
21
0.596
0.614
0.609
0.625
0.584
0.622
0.587
0.582
0.587
0.592
0.601
0.481
0.504
0.492
0.502
0.509
0.286
0.311
0.263
0.224
0.213
–
132
561
560
566
563
563
641
606
634
605
649
616
22
0.595
0.615
0.605
0.630
0.594
0.628
0.587
0.589
0.591
0.602
0.608
0.474
0.508
0.499
0.497
0.504
0.309
0.326
0.275
0.245
0.217
0.142
–
560
554
565
559
559
640
604
629
607
654
621
23
0.585
0.584
0.594
0.630
0.576
0.606
0.576
0.570
0.583
0.588
0.605
0.584
0.594
0.591
0.586
0.600
0.617
0.612
0.600
0.602
0.602
0.603
0.602
–
428
135
271
268
514
496
519
529
552
510
24
0.520
0.544
0.548
0.573
0.543
0.574
0.535
0.528
0.535
0.554
0.553
0.561
0.569
0.562
0.565
0.573
0.598
0.606
0.586
0.584
0.594
0.602
0.596
0.460
–
455
437
438
519
408
516
446
487
438
25
0.594
0.592
0.600
0.638
0.581
0.606
0.575
0.574
0.585
0.590
0.602
0.591
0.598
0.598
0.590
0.603
0.624
0.616
0.601
0.596
0.596
0.609
0.608
0.145
0.489
–
258
256
510
499
520
533
565
542
26
*Numbers above the diagonal are mean distance values; numbers below diagonal are absolute distance values.
1. AgeHex
2. TmoHex2
3. LdeDP1
4. TmoESEIP
5. PamHex
6. BdiHex
7. MseArya
8. MseAryb
9. HceAry
10. GmeAry
11. BmoAry
12. TniBJHSP2
13. HcuSP2
14. HceMt
15. CfuDAP1
16. PinSP1
17. CfuDAP2
18. PinSP2
19. BmoMt
20. MseMt
21. HcuSP1
22. SliMt
23. TniBJHSP1
24. AaeHex1
25. AaeHex2
26. AatHex12
27. AgaHexA
28. AmeHexA
29. CviAry
30. CviLSP2
31. DmeLSP1
32. DmeLSP2
33. MdoAry
34. MdoHex
0.567
0.584
0.586
0.627
0.566
0.594
0.554
0.561
0.570
0.576
0.591
0.587
0.595
0.604
0.602
0.602
0.626
0.605
0.613
0.599
0.604
0.605
0.601
0.291
0.470
0.277
–
8
499
494
508
529
553
525
27
0.567
0.584
0.586
0.627
0.566
0.592
0.553
0.560
0.571
0.577
0.594
0.588
0.596
0.605
0.603
0.603
0.626
0.605
0.613
0.599
0.604
0.605
0.601
0.288
0.471
0.275
0.009
–
498
495
507
529
553
526
28
0.622
0.635
0.660
0.676
0.652
0.669
0.625
0.637
0.632
0.632
0.660
0.657
0.661
0.659
0.659
0.677
0.694
0.688
0.661
0.672
0.672
0.689
0.688
0.553
0.558
0.548
0.537
0.535
–
561
284
563
630
593
29
0.578
0.567
0.586
0.628
0.587
0.626
0.572
0.580
0.591
0.586
0.595
0.634
0.637
0.635
0.634
0.651
0.658
0.652
0.645
0.640
0.647
0.652
0.649
0.533
0.439
0.537
0.531
0.532
0.603
–
561
271
377
303
30
0.626
0.629
0.663
0.661
0.661
0.676
0.637
0.635
0.637
0.637
0.659
0.657
0.657
0.643
0.654
0.660
0.680
0.687
0.666
0.672
0.673
0.682
0.676
0.558
0.555
0.559
0.546
0.545
0.305
0.603
–
574
626
593
31
0.590
0.585
0.615
0.633
0.601
0.639
0.581
0.590
0.596
0.603
0.604
0.635
0.630
0.629
0.635
0.643
0.666
0.661
0.642
0.649
0.654
0.651
0.653
0.569
0.480
0.573
0.569
0.569
0.605
0.291
0.617
–
425
368
32
0.647
0.646
0.648
0.704
0.639
0.673
0.632
0.627
0.644
0.639
0.637
0.689
0.683
0.681
0.681
0.699
0.689
0.677
0.688
0.683
0.690
0.698
0.703
0.594
0.524
0.608
0.595
0.595
0.677
0.405
0.673
0.457
–
239
33
34
0.614
0.594
0.612
0.648
0.606
0.648
0.599
0.595
0.612
0.604
0.611
0.654
0.648
0.649
0.648
0.653
0.658
0.647
0.655
0.654
0.660
0.662
0.668
0.548
0.471
0.583
0.565
0.566
0.638
0.326
0.638
0.396
0.257
–
Storage Protein cDNA From A. germari
59
60
Kim et al.
Fig. 3. Phylogenetic tree for aligned amino acid sequences of AgeHex and the known insect storage proteins.
The tree was obtained by bootstrap analysis with the option of heuristic search and the numbers on the branches
represent bootstrap values for 1,000 replicates. Outgroup
was chosen as Blaberus discoidalis hexamerin (BdiHex) and
Periplaneta americana hexamerin (PamHex) on the basis
of the sequence homology by pairwise comparison. The
abbreviations are given in Materials and Methods.
Archives of Insect Biochemistry and Physiology
Storage Protein cDNA From A. germari
61
TABLE 4. Amino Acid Composition of Hexamerin From A. germari and Other Insect Storage Proteins*
Mol. percent
Amino
acid
AgeHex
TmoHex2
TmoESEIP
LdeDP1
GmeAry
BmoAry
HceAry
MseMt
BmoMt
HceMt
SliMt
Cys
Asp/Asn
Glu/Gln
Ser
Gly
His
Arg
Thr
Ala
Pro
Tyr
Phe
Met
Ile
Leu
Val
Trp
Lys
0.14
10.56
11.53
4.58
5.28
6.25
3.47
3.33
4.58
4.17
10.97
6.81
2.36
4.31
6.94
6.94
0.97
6.81
0.28
9.55
11.11
6.27
5.27
5.98
3.13
3.28
4.70
4.27
13.68
6.70
2.71
3.70
7.12
6.27
1.14
4.84
0.27
10.34
12.07
5.04
5.31
2.12
3.98
4.51
5.44
4.51
14.85
6.37
1.86
3.45
6.10
6.76
1.06
5.97
0.85
11.54
9.67
5.27
5.56
3.42
5.41
3.56
5.13
5.56
8.40
8.12
3.13
4.27
6.84
5.70
1.71
5.84
0.00
12.11
11.82
5.70
4.13
0.85
4.27
3.28
5.13
5.27
11.40
5.98
1.14
5.70
9.26
6.55
1.85
5.56
0.17
11.65
9.94
4.69
3.69
1.99
3.69
4.97
5.68
4.40
7.67
9.23
3.84
4.55
8.24
6.39
0.99
7.67
0.00
11.21
10.79
4.55
4.40
3.41
3.55
4.69
4.55
5.26
9.23
8.95
1.56
4.69
7.39
7.53
0.85
7.39
0.8
19.2
1.6
3.2
3.2
2.8
8.0
8.0
0.4
3.6
2.4
6.0
5.6
5.6
10.1
11.6
1.2
6.8
1.87
11.78
7.09
5.35
4.69
1.20
5.49
6.02
4.02
3.75
4.95
4.28
10.98
4.55
7.90
7.63
1.34
7.10
0.80
12.37
7.98
5.72
3.46
0.93
4.52
4.92
3.86
3.72
6.65
5.32
4.79
5.59
10.64
8.91
0.93
8.91
1.20
13.98
4.66
2.80
3.86
2.13
7.59
6.39
3.73
3.60
4.66
4.53
7.72
5.46
9.32
8.79
1.73
7.86
*TmoHex2, Hexamerin 2 from T. molitor; TmoESEIP, Early-staged encapsulation inducing protein from T. molitor; LdeDP, Diapause protein 1 from
L. decemlineata; GmeAry, Aryphorin from G. mellonella; BmoAry, Aryphorin from B. mori; HceAry, Aryphorin from H. cecropia; MseMt, Methionine rich storage protein from M. sexta; BmoMt, Methionine rich storage protein from B. mori; HceMt, Methionine rich storage protein from H.
cecropia; SliMt, Methionine rich storage protein from S. litura.
Genomic Organization and Expression at
Transcriptional Level of the A. germari
Hexamerin cDNA
trol and fat body. The Northern hybridization revealed that A. germari hexamerin is expressed in
the fat body of A. germari larvae.
To scrutinize the genomic organization of the
hexamerin in A. germari, genomic DNA was digested with several restriction enzymes, which have
no restriction site inside the AgeHex gene and hybridized with the full-length AgeHex gene cloned
in this study as a probe. The Southern blot analysis revealed that the AgeHex gene in A. germari genome was detected as a single band (Fig. 4). This
single hybridization signal suggests that AgeHex
gene exists as a single copy in A. germari, as is true
for L. decemlineata (Koopmanschap et al., 1995).
In general, storage proteins are synthesized by
the fat body tissues of the last instar larva (Webb
and Riddiford, 1988). To confirm the expression
of the A. germari hexamerin gene at the transcriptional level, therefore, the Northern blot analysis
was carried out using the mRNA prepared from
the midgut and fat body, respectively (Fig. 5). A
hybridization signal was detected as a single band
in mRNA from the whole body as a positive con-
Expression of a cDNA Encoding the A. germari
Hexamerin in the Baculovirus-Infected Insect Cells
June 2003
To assess A. germari hexamerin, the 2,260-bp for
AgeHex gene was inserted into the baculovirus transfer vector. The baculovirus transfer vector was used
to generate a recombinant virus expressing A. germari
hexamerin. Transfer vector pBacPAK9-AgeHex was
constructed by insertion of AgeHex gene under the
control of AcNPV polyhedrin promoter of pBacPAK9
(data not shown). Recombinant AcNPV, which we
designated AcNPV-AgeHex, was produced in insect
Sf9 cells by cotransfection with wild-type AcNPV
DNA and the transfer vector.
To examine the expression of the A. germari
hexamerin gene by recombinant virus in insect
cells, the protein synthesis in Sf9 cells infected with
the recombinant virus was analyzed by SDS-PAGE
(Fig. 6). The A. germari hexamerin expressed by
the AgeHex gene was present as a single protein of
62
Kim et al.
Fig. 4. Southern blot analysis of A. germari genomic DNA
for the hexamerin gene. Genomic DNAs were digested with
restriction enzymes, EcoRI, BamH I, and ApaI, and hybridized with the radiolabelled A. germari hexamerin gene. Size
markers are shown on the left.
about 80 kDa in the cells infected with recombinant virus, but not in the cells infected with wildtype AcNPV or mock-infected cells.
To verify removal of the leader peptide of
AgeHex, furthermore, N-terminal amino acid sequencing was employed on the recombinant
AgeHex by a protein sequencer. Cleavage of the signal peptide occurred between Ala18 and Val19 in
AgeHex, as represented in Figure 1. The mature
AgeHex is predicted to be 702 amino acid residues.
Identification of A. germari Hexameric
Storage Protein
In a previous study, we reported for purification and characterization of three storage proteins
(SP1, SP2 and SP3) of the mulberry longicorn
beetle, A. germari (Yoon et al., 2001). To determine
Fig. 5. Northern blot analysis of the A. germari hexamerin
gene. Total RNAs were isolated from the whole body (lane
1), fat body (lane 2), and midgut (lane 3). The RNAs
were separated by 1.0% formaldehyde agarose gel electrophoresis (A), transferred on to a nylon membrane, and
hybridized with the radiolabelled A. germari hexamerin
gene. Transcripts of the AgeHex (B) gene are indicated on
the right by arrows. The 60S rRNA gene was used as an
internal loading control (C).
the relationship between the AgeHex and three
storage proteins, therefore, the A. germari hexmerin
expressed in the baculovirus-infected Sf9 cells was
electroeluted from the gel and injected into mice
for the preparation of polyclonal antiserum. Western blot analysis using the polyclonal antiserum
against recombinant A. germari hexamerin showed
that the antiserum reacted with storage protein 2
(SP2) in the larval hemolymph (Fig. 7). The electrophoretic mobility of SP2 present in the A.
germari larval hemolymph in Western blot analysis was identical to the corresponding protein band
of the recombinant A. germari hexamerin expressed
in the baculovirus-infected insect cells. Therefore,
this result strongly suggests that the AgeHex is the
Archives of Insect Biochemistry and Physiology
Storage Protein cDNA From A. germari
Fig. 6. SDS-PAGE analysis of the A. germari hexamerin
(AgeHex) expression in the recombinant baculovirus-infected insect Sf9 cells. Sf9 cells were mock-infected (lane
2) or infected with wild-type AcNPV (lane 3), recombinant AcNPV-AgeHex (lanes 4–6) at an MOI of 5 PFU per
cell. Cells were collected at 1 (lane 4), 2 (lane 5), and 3
(lane 6) days p.i. The solid arrows on the right indicate
the AgeHex band of 80 kDa. Molecular weight standards
were used as size marker (lane 1).
SP2 subunit present in the larval hemolymph and
has a molecular weight of approximately 80 kDa
on SDS-PAGE analysis.
In conclusion, we report for the first time the
molecular characterization of SP2, arylphorin-like
hexameric storage protein, gene in mulberry longicorn beetle, A. germari. In this study, characterization of AgeHex and its gene will expand our
understanding on the beetle storage protein. Further studies should examine the physiological and
biochemical features of AgeHex during the pupal
and adult stages.
June 2003
63
Fig. 7. Western blot analysis of A. germari larval hemolymph protein using antiserum against recombinant
AgeHex hexamerin. The hemolymph protein was collected
from the 6th instar A. germari larva (lane 1). Cell lysates
were collected from the Sf9 cells infected with recombinant AcNPV-AgeHex (lane 2) at 3 days p.i. The protein
samples were subjected to 10% SDS-PAGE (A), electroblotted, and incubated with recombinant AgeHex antibody (B). SDS-PAGE molecular weight standards are
indicated on the left.
LITERATURE CITED
Burmester T. 1999. Evolution and function of the insect
hexamerins. Eur J Entomol 96:213–235.
Burmester T, Kolling C, Schroer B, Scheller K. 1998. Complete
sequence, expression, and evolution of the hexamerin LSP-2
of Calliphora vicina. Insect Biochem Mol Biol 28:11–22.
Cheon HM, Hwang IH, Chung DH, Seo SJ. 1998. Sequence
analysis and expression of Met-rich storage protein SP-1
of Hyphantria cunea. Mol Cells 8:219–225.
Cho MY, Choi HW, Moon GY, Kim MH, Kwon TH, Homma
K, Natori S, Lee BL. 1999. An 86 kDa diapause protein 1like protein is a component of early-staged encapsulationrelating proteins in coleopteran insect, Tenebrio molitor
larvae. FEBS Lett 451:303–307.
Crowson RA. 1981. The biology of the Coleoptera. London:
Academic Press.
64
Kim et al.
Daly HV, Doyen JT, Purcell III AH. 1998. Introduction to insect biology and diversity, 2nd ed. London: Oxford University Press.
double-stranded nucleic acids on polyacryamide and agarose gels by using glyoxal and acridine orange. Proc Natl
Acad Sci USA 74:4835–4838.
Fujii T, Sakurai H, Izumi S, Tomino S. 1989. Structure of the
gene for the arylphorin-type storage protein SP 2 of Bombyx
mori. J Biol Chem 264:11020–11025.
Memmel NA, Trewitt PM, Silhacek DE, Kumaran AK. 1992.
Nucleotide sequence and structure of the arylphorin gene
from Galleria mellonella. Insect Biochem Mol Biol 22:
333–342.
Gordadze AV, Korochkina SE, Zakharkin SO, Norton AL,
Benes H. 1999. Molecular cloning and expression of two
hexamerin cDNA from the mosquito, Aedes aegypti. Insect
Mol Biol 8:55–66.
Haunerland NH. 1996. Insect storage proteins; gene families
and receptors. Insect Biochem Mol Biol 26:755–765.
Hwang SJ, Cheon HM, Kim HJ, Chae KS, Chung DH, Kim
MO, Park JS, Seo SJ. 2001. cDNA sequence and gene expression of storage protein-2, a juvenile hormone-suppressible hexamerin from the fall webworm, Hyphantria
cunea Drury. Comp Biochem Physiol B129:97–107.
Jamroz RC, Beintema JJ, Stam WT, Bradfield JY. 1996. Aromatic hexamerin subunit from adult female cockroaches
(Blaberus discoidalis): molecular cloning, suppression by
juvenile hormone, and evolutionary perspectives. J Insect
Physiol 42:115–124.
Kanost MR, Kawooya JK, Ryan RD, Van Heusden MC, Ziegler
R. 1990. Insect hemolymph proteins. Adv Insect Physiol
22:299–366.
Koopmanschap AB, Lammers JH, de Kort CAD. 1995. The
structure of the gene encoding diapause protein 1 of the
colorado potato beetle (Leptinorarsa decemlineata). J Insect
Physiol 41:509–518.
Kim SR, Yoon HJ, Park NS, Lee SM, Moon JY, Jin BR, Sohn
HD. 2001. Molecular cloning of a cDNA encoding a cathepsin D homologue from the mulberry longicorn beetle,
Apriona germari. Int J Indust Entomol 3:121–126.
Laemmli UK. 1970. Cleavage of structure proteins during the
assembly of the head of bacteriophage T4. Nature 227:
680–685.
Levenbook L. 1985. Insect storage proteins. In: Kerkut GS,
Gilbert LI, editors. Comprehensive insect physiology, biochemistry and pharmacology. New York: Pergamon Press.
p 307–346.
McMaster GK, Carmichael GG. 1977. Analysis of single- and
Mousseron GS, Kejzlarova LJ, Burmester T, Chihara C, Barray
M, Delain E, Pictet R, Lepesant JA. 1997. Sequence, structure and evolution of the ecdysone-inducible Lsp-2 gene
of Drosophila melanogaster. Eur J Biochem 245: 191–198.
O’Reilly DR, Miller LK, Luckow VA. 1992. Baculovirus expression vectors: a laboratory manual. New York: W. H. Freeman & Co.
Sakurai H, Fujii T, Izumi S, Tomino S. 1988. Complete nucleotide sequence of gene for sex-specific storage protein of
Bombyx mori. Nucleic Acids Res 16:7717–7718.
Swofford DL. 1990. PAUP: phylogenetic analysis using parsimony, ver. 3.0. Illinois Natural History Survey, Champaign
(on disk).
Telfer WH, Kunkel JG. 1991. The function and evolution of
insect storage hexamers. Ann Rev Entomol 36:205–228.
Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc Natl Acad
Sci USA 76:4350–4354.
Vaughn JL, Goodwin RH, Thompkins GJ, McCawley P. 1977.
The establishment of two insect cell lines from the insect
Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro
13:213–217.
Wang XY, Frohlich DR, Wells MA. 1993. Polymorphic cDNAs
encode for the methionine-rich storage protein from
Manduca sexta. Insect Mol Biol 2:13–20.
Webb BA, Riddiford LM. 1988. Regulation and expression
of arylphorin and female-specific protein mRNAs in the
tobacco hornworm, Manduca sexta. Dev. Biol 130:682–
691.
Willott E, Wang XY, Wells MA. 1989. cDNA and gene sequence
of Manduca sexta arylphorin, an aromatic amino acid-rich
larval serum protein. Homology to arthropod hemocyanins. J Biol Chem 264:19052–19059.
Archives of Insect Biochemistry and Physiology
Storage Protein cDNA From A. germari
65
Wu CH, Lee MF, Liao SC, Luo SF. 1996. Sequencing analysis
of cDNA clones encoding the American cockroach Cr-Pl
allergens. Homology with insect hemolymph proteins. J
Biol Chem 271:17937–17943.
Zheng Y, Yoshiga T, Tojo S. 2000. cDNA cloning and deduced
amino acid sequences of three storage proteins in the common cutworm, Spodoptera litura. Appl Entomol Zool 35:
31–39.
Yoon HJ, Mah YI. 1999. Life cycle of the mulberry longicorn
beetle, Apriona germari Hope on an artificial diet. J Asia-
Zhu YC, Kramer KJ, Dowdy AK, Baker JE. 2000. Typsinogenlike cDNA and quantitative analysis of mRNA levels from
the Indianmeal moth, Plodia interpunctella. Insect Biochem
Mol Biol 30:1027–1035.
Pacific Entomol 2:169–173.
Yoon HJ, Kim SR, Jin BR, Lee SM, Moon JY, Mah YI, Sohn
HD. 2001. Purification and characterization of storage proteins from the mulberry longicorn beetle, Apriona germari
Hope. Int J Indust Entomol 2:61–66.
June 2003
Zhu YC, Muthukrishnan S, Kramer KJ. 2002. cDNA sequences
and mRNA levels of two hexamerin storage proteins
PinSP1 and PinSP2 from the Indianmeal moth, Plodia
interpunctella. Insect Biochem Mol Biol 32:525–536.
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expressions, molecular, mulberry, beetle, apriona, germari, longicornis, storage, cdna, like, cloning, arylphorin, hexameric, protein, characterization, encoding
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