close

Вход

Забыли?

вход по аккаунту

?

Two juvenile hormone suppressible storage proteins may play different roles in Hyphantria cunea Drury.

код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 50:157–172 (2002)
Two Juvenile Hormone Suppressible Storage Proteins
May Play Different Roles in Hyphantria cunea Drury
Hyang-Mi Cheon,1 Su-Jeong Hwang,1 Hong-Ja Kim,1 Byung Rae Jin,2 Kwon-Seok Chae,3
Chi-Young Yun,4and Sook-Jae Seo1*
We isolated and sequenced cDNA clones corresponding to two storage proteins (HcSP-1 and HcSP-2) from fall webworm,
Hyphantria cunea. The cDNAs for HcSP-1 (2,337 bp) and HcSP-2 (2,572 bp) code for 753 and 747 residue proteins with
predicted molecular masses of 88.3 and 88.5 kDa, respectively. The calculated isoelectric points are pI = 8.4 (HcSP-1) and
7.6 (HcSP-2). Multiple alignment analysis of the amino acid sequence revealed that HcSP-1 is most similar to SL-1 from S.
litura (73.8% identity) and other methionine-rich hexamers, whereas HcSP-2 is most similar to the SL-2 a subunit from S.
litura (74.8% identity) and other moderately methionine-rich hexamers. The two storage proteins from H. cunea shared only
38.4% identity with one another. According to both phylogenetic analyses and the criteria of amino acid composition, HcSP-1
belongs to the subfamily of Met-rich storage proteins (6% methionine, 10% aromatic amino acid), and HcSP-2 belongs to
the subfamily of moderately methionine-rich storage proteins (3.2% methionine, 12.9% aromatic amino acid). Topical application of the JH analog, methoprene, after head ligation of larvae, suppressed transcription of the SP genes, indicating
hormonal effects at the transcriptional level. The HcSP-1 transcript was detected by Northern blot analysis in Malpighian
tubule, testis, and ovary, in addition to fat body where it was most abundant. The HcSP-2 transcript was detected only in fat
body and Malpighian tubule. The accumulation of HcSP-1 in ovary and HcSP-2 in Malpighian tubule might be related to
differential functions in both organs. Arch. Insect Biochem. Physiol. 50:157–172, 2002. © 2002 Wiley-Liss, Inc.
KEYWORDS: Hyphantria cunea; storage protein; cDNA clone; juvenile hormone suppressible protein; ovary;
Malpighian tubule
INTRODUCTION
Storage proteins are major components in the
hemolymph of immature stages of insects and have
been identified in a wide range of species, both hemimetabolous and holometabolous (Kanost et al., 1990;
Telfer and Kunkel, 1991). They are synthesized by the
fat body and secreted into the hemolymph. Their concentrations in the hemolymph increase markedly in
the final larval instar, and are then partially or wholly
sequestered by the fat body (Levenbook, 1985; Telfer
and Kunkel, 1991).
1
Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, Korea
2
College of Natural Resource and Life Science, Dong-A University, Pusan, Korea
3
Department of Biology, Korea University, Seoul , Korea
4
Department of Biology, Daejon University, Daejon, Korea
The nucleotide sequences reported in this paper have been submitted to the Genbank/EMBL Data Bank with accession number U60988 (HcSP-1) and AF157013
(HcSP-2).
Abbreviations used: BJHSP, basic JH suppressible protein; bp, base pair; JH, juvenile hormone; Met, methionine; SP, storage protein; 20-HE, 20-hydroxyecdysone;
H. cunea, Hyphantria cunea; B. mori, Bombyx mori; M. sexta, Manduca sexta; T. ni, Trichoplusia ni; G. mellonella, Galleria mellonella; S. litura, Spodoptera
litura.
Contract grant sponsor: Brain Korea 21 project.
*Correspondence to: Sook-Jae Seo, Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, 660-701, Korea.
E-mail: sookjae@gshp.gsnu.ac.kr
Received 16 October 2001; Accepted 28 March 2002
© 2002 Wiley-Liss, Inc.
DOI: 10.1002/arch.10040
Published online in Wiley InterScience (www.interscience.wiley.com)
158
Cheon et al.
In Lepidoptera, there are at least two kinds of
storage proteins, namely arylphorins, which are
rich in aromatic amino acids, and methionine-rich
storage proteins (Riddiford and Law, 1983; Telfer
and Kunkel, 1991; Tojo and Yoshiga, 1994). Most
of them have molecular sizes of nearly 500 kDa
and are composed of six identical subunits of 80
kDa each.
The expression of storage proteins is mostly
confined to the last larval instar, a period where
juvenile hormone titers are low, and it has long
been suspected that the hormone prevents the production of storage proteins. Indeed, juvenile hormone has been shown to suppress the expression
of some, but not all, storage proteins (Haunerland,
1996). Although several juvenile hormone suppressible proteins have been described in Lepidopteran insect, little is known about their fate and
function.
In a previous study, we cloned and sequenced
the two forms of storage proteins termed HcSP-1
and HcSP-2 from H. cunea (Cheon et al., 1998;
Hwang et al., 2001). Two cloned cDNAs of storage
proteins from H. cunea were used to demonstrate
the suppression of their mRNA by treatment with
a juvenile hormone analog. We also demonstrated
that HcSP-1 and HcSP-2 were differently localized
in the ovary and Malpighian tubule, respectively.
The possible roles of two HcSPs in both tissues
were discussed.
methoprene (Zoecon Corp. Palo Alto, CA), in 2 ml
acetone. Fat bodies from methoprene-and acetone
(control)-treated larvae were dissected for RNA extraction.
Purification, N-Terminal, and Internal Sequencing of
the Storage Proteins
HcSP-1 was purified from the hemolymph, and
antibody against HcSP-1 was raised in a rabbit according to protocols described previously (Seo et
al., 1998). Dr. Hak Ryul Kim (Korea University,
Korea). graciously provided purified HcSP-2 and
antibody against HcSP-2. Briefly, the purification
protocol involved centrifugation of hemolymph in
a KBr density gradient followed by CM52 ion exchange chromatography (Song et al., 1997). The
N-terminal and internal sequencing of purified
HcSP-1 were accomplished using an Applied
Biosystems 473 sequencer (Laboratoire de Microséquencage des protéines, Institut Pasteur, Paris).
To do internal sequencing, protein was digested
with endoproteinase C (Boehringer-Mannheim
Biochemicals) and recovered sample was injected
onto a DEAE HPLC column linked to a C18 reverse
phase HPLC column. Subsequently, sequencing was
performed with eluted sample. The N-terminal
amino acid sequence of purified HcSP-2 was determined using an Applied Biosystems 476A Sequencer (Biotechnology Laboratory, University of
British Columbia, Vancouver).
MATERIALS AND METHODS
Animals
Fall webworms, H. cunea, were reared on an artificial diet at 27°C and 75% relative humidity under a photoperiod of 16 L:8 D.
Hormone Treatments
Two-day-old last instar larvae were ligated with
thread behind the head capsule in order to isolate
the insect from endocrine organs in the head. At
72 h after ligation, the larvae were topically treated
with 0.1 or 1.0 mg of a juvenile hormone analog,
Isolation of Nucleic Acids
Total RNA was isolated from tissues by lysis buffer,
spin column, and wash buffer according to the protocol recommended by the manufacturer (Qiagen Inc.
Chatsworth, CA). All RNA samples were evaluated
in agarose gels to ensure that they contained intact
rRNA and were free of contaminating DNA.
Primer Synthesis, PCR, and Subcloning
of PCR Products
Oligonucleotide primers were synthesized by
Korea Bioneer, Inc. Degenerate primers between 17
Archives of Insect Biochemistry and Physiology
Two JH Suppressible SPs From H. cunea
and 20 nucleotides in length were designed from
the N-terminal (VVKDDTY) and internal peptide
sequence (NQFINT) for HcSP-1, and N-terminal
(DIKQKQ) and consensus sequence (YEIYPY) for
HcSP-2. The primers and reaction conditions of
PCR amplifications are listed below: numbers in
parentheses refer to nucleotide position as given
in Figure 1, and notation in brackets refers to temperature and duration of the melting/annealing/
synthesis phases of each PCR cycle.
HcSP-1: 5¢-GTNGTNAARGAYGAYACNTA-3¢
(17–23); 5¢-GTRTTDATRAAYTGRTT-3¢ (84–
89); 40 cycles [94°C,30 s/40°C,1 min/72°C,1
min]; template=1st strand cDNA from 5-dayold last instar total RNA.
HcSP-2: 5¢-GAYATHAARCARAARCA-3¢ (27–
32); 5¢-TANGGRTADATYTCRTA-3¢ (157–
162); 35 cycles [94°C,30 s/47°C,1 min/72°C,
1 min]; template=cDNA library from H.
cunea last instar larvae.
The resulting PCR products were separated on
a 1% agarose gel. 0.2-kb (HcSP-1) and 0.4-kb
(HcSP-2) fragments were identified. These fragments were excised from the agarose gel, purified,
ligated into a T-vector, and amplified in XL1 Blue
competent cells.
Screening of the cDNA Library
The cDNA library from the fat body of H. cunea
last instar larvae was kindly provided by Dr. HoYong Park (Korea Research Institute of Bioscience
and Biotechnology, KIST, Korea).
For screening, 50,000 plaques were plated on
15-cm-diameter agar plates. Nitrocellulose filter
(Schleicher & Schuell, Dassel, Germany) blots were
taken from the plates and hybridized at high stringency (65°C and 0.1 ´ SSC) with the 32P-labelled
PCR product. Positive clones were rescreened at low
density on 9-cm diameter agar plates. Positive
plaques were individually isolated in phage buffer
by the plate lysate method followed by lambda
DNA extraction (Sambrook et al., 1989). The insert of the positive clone was subcloned into an
EcoRI site of pBluescript KS(+).
August 2002
159
Subcloning and DNA Sequencing
The cDNA fragments, which hybridized with the
radio-labelled PCR product, were removed from
the gels by electroelution, followed by phenol extraction and ethanol precipitation. Subsequently,
these DNA fragments were ligated into pBluescript
KS(+), followed by electroporation into JM109 cells
(Sambrook et al., 1989).
The sequencing reaction was based on the
dideoxynucleotide chain termination method of
Sanger et al. (1977) and was carried out according
to the thermal cycle DNA sequencing protocol with
DNA polymerase from Thermus thermophilus HB7
(Korea Bioneer, Inc) for HcSP-1. Template-specific
and universal primers derived from pBluescript were
used in the sequence reactions in the presence of
35
S-labelled dATP. Subclones for sequencing were
prepared by ligating the restriction fragments into
pBluescript followed by transformation into JM109
cells. For HcSP-2, both strands were sequenced automatically using an Applied Biosystems model
373A DNA sequencer.
Computer-Assisted Analysis of Sequence Data
The EMBL DataBank was searched with FASTA and
BLAST. Editing and analysis of the DNA sequence
data were performed with DNASTAR software (DNASTAR Inc., Wisconsin). Multiple alignment was carried out using the program PILEUP.
Northern Blot
For tissue specificity of SP expression, tissues
were dissected from 4- and 5-day-old last instar
larvae (fat body, midgut, Malpighian tubule, and
testis) and from 8-day-old pupae (ovary). Ten (fat
body, midgut, Malpighian tubule, and ovary) and
fifteen (testis) insects were pooled for extraction
of RNA. The results from three replicates showed
same patterns (data not shown). Total RNA from
fat body (10 mg) and other tissues (30 mg) were
denatured and subjected to electrophoresis in a
1.2% agarose gel containing 2.2 M formaldehyde.
Following electrophoresis, gels were rinsed in 10 ´
Cheon et al.
Fig. 1.
160
Archives of Insect Biochemistry and Physiology
Two JH Suppressible SPs From H. cunea
SSC and transferred to nitrocellulose (Schleicher
& Schuell) in 10 ´ SSC. Blots were prehybridized
with 1.5 ´ SSPE, 7% SDS, 10% PEG, 0.1 mg/ml
sonicated denatured salmon sperm DNA and 0.25
mg/ml BSA for 4 h at 65°C. Hybridization was performed for 18 h at 65°C in the prehybridization
buffer with 5 ´ 105 cpm/ml of 32P-labelled probes,
prepared according to the method of random priming (Feinberg and Vogelstein, 1983). The filter was
then washed twice with 1 ´ SSC, 0.1% SDS at 65°C
for 15 min, and subsequently two more times for
15 min with 0.1 ´ SSC, 0.1% SDS at 65°C before
exposure to X-ray film at –70°C.
Western Blot
Following SDS-PAGE, proteins in the gel were
electrotransferred to a sheet of nitrocellulose (0.45
mm, Bio-Rad, Hercules, CA) according to the procedure of Towbin et al. (1979). The blots were
blocked in 20 mM Tris-HCl, pH 7.6, 137 mM
NaCl, and 0.2% Tween-20 (buffer A) containing
5% nonfat dry milk, and then incubated with antisera against HcSPs (Song et al., 1997; Seo et al.,
1998) at 1:1,500 dilution in buffer A. After washing in buffer A, the blots were incubated with horseradish-peroxidase- conjugated goat anti-rabbit IgG
(1:3,000) in buffer A for 1 h. Immunoreactivity was
determined using the ECL chemiluminescence reaction (Amersham, Buckinghamshire, UK).
Immunocytochemistry
Immunocytochemistry was performed as previously described (Miller et al., 1990). The tissues
were fixed for 3 h in a mixture (FM) of 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium
Fig. 1. Nucleotide and deduced amino acid sequences
of the cDNAs encoding H. cunea SP-1 (A) and SP-2 (B).
Arrows indicate signal peptide cleavage site. Underlined
amino acids were confirmed by N-terminal amino acid
sequence analysis of SP. An asterisk marks the translation
stop codon and the underlined AATAAA shows the polyadenylation signal. Dashed underlines indicate potential
N-glycosylation sites.
August 2002
161
phosphate buffer (pH 7.5) containing 0.15 mM
CaCl2 and 0.45 M sucrose. Incubating the tissues
overnight in pH 10.4 FM without glutaraldehyde
completed fixation. The tissues were rinsed in 0.1
M sodium phosphate buffer (pH 7.5) and then dehydrated in a graded ethanol series (up to 95%)
and embedded in Lowicryl K4M (Polysciences,
Warrington, PA). Ultrathin sections mounted on
formvar-coated nickel grids were treated for 10 min
with Tris-buffered saline (TBS; 0.02 M Tris-HCl, pH
7.5, containing 0.5 M NaCl). The sections were
etched with 3% H2O2 in double distilled H2O for
5 min and then blocked with 3% bovine serum
albumin (BSA) in TBS for 30 min. The sections
were incubated with 1:200 diluted antisera against
H. cunea SP-1 and SP-2 in TBS plus 1% Tween-20
(TBS/Tween) for 60 min, respectively. Following a
wash in TBS/Tween 3 times for 15 min with gentle
agitation, the sections were exposed to gold-goat
anti-rabbit IgG (20 nm: Zymed, San Francisco, CA)
diluted 1:5 in TBS/Tween for 60 min. The grids
were finally washed with 0.3% BSA in TBS, the sections were poststained with 2% uranyl acetate followed by 0.2% Reynolds’ lead citrate (Reynolds,
1963). Ultrastructural examination was performed
on a Hitachi H-600 transmission electron microscope operating at 75kV.
Gold particles seen in controls were typically
few in number and randomly dispersed. The controls included: (1) substitution of preimmune serum for primary antiserum; (2) use of secondary
antibody in the absence of treatment with primary
antibody; and (3) treatment of thin sections with
colloidal gold alone.
RESULTS AND DISCUSSION
Cloning and Sequencing of HcSP-1 and HcSP-2 cDNAs
The cDNA library from fall webworm mRNA
was screened with PCR-generated 32P-labelled
probes of 215- or 404-bp, complementary to amino
acids 17–89 (HcSP-1) or 27–162 (HcSP-2), respectively. From a total of 50,000 screened plaques, five
positive plaques for HcSP-1 and four positive
plaques for HcSP-2 were isolated. Insert lengths
162
Cheon et al.
were checked after plasmid isolation by digestion
with EcoRI and XhoI. The inserts were about 2.5
kb, consistent with full-length cDNA clones for
each of the two storage proteins. The lengths of
cDNA for HcSP-1 and HcSP-2 were 2,337 and
2,572 nucleotides, respectively (Fig. 1). Within the
cDNA sequences were open reading frames of
2,259 and 2,241 nucleotides encoding proteins of
753 and 747 amino acids with estimated molecular masses of 88.3 and 88.5 kDa, respectively. The
remaining sequences were untranslated regions.
Only 23 (HcSP-1) and 22 nucleotides (HcSP-2)
comprised the 5¢-untranslated sequences, while the
3¢ untranslated region possessed 53 (HcSP-1) and
281 (HcSP-2) nucleotides. In general, 5¢-untranslated regions of hexamerin cDNAs are very short,
but 3¢-untranslated regions are quite variable, ranging in size from 80 to 450 nucleotides (Alonso et
al., 1979; Sato et al., 1982; Delaney et al., 1986;
Sakurai et al., 1988; Fujii et al., 1989; Willott et
al., 1989). The deduced amino acid sequences encoded by the cDNA matching the N-terminal sequences of the storage proteins were preceded by
a sequence encoding 15 (HcSP-1) and 17 (HcSP2) amino acids, lengths typical of a signal peptide.
The signal peptides of other insect storage hexamers
vary between 15 and 18 amino acids (Sakurai et
al., 1988; Willott et al., 1989; Jones et al., 1990;
Memmel et al., 1994; de Kort and Koopmanschap,
1994). One and three potential glycosylation sites
(NXT/S) are present in the HcSP-1 and HcSP-2
cDNAs, respectively (Fig. 1). HcSP-1 and HcSP-2
reacted positively with PAS, Sudan Black B, and
methyl green stains, indicating that they contain
carbohydrates, lipids, and phosphate (Kim et al.,
1989). A translational stop codon TAA was located
at +2260 (HcSP-1) and +3242 (HcSP-2). A single
polyadenylation signal site (AATAAA) was present
35 bp (HcSP-1) and 260 bp (HcSP-2) downstream
from the stop codon.
Sequence Comparisons Among Met-Rich
Storage Proteins
To retrieve protein sequences similar to those
of HcSP-1 and HcSP-2, relevant databases were
searched using the BLAST (Altschul et al., 1990)
and FASTA programs. When the protein sequence
data were compared to those of hexamerins reported for other insects, significant similarities
were found to Met-rich storage proteins [represented by S. litura SL-1 (SL-1) (Zheng et al. 2000),
T. ni basic juvenile hormone suppressible protein
1 (BJHSP1) (Jones et al., 1993), B.mori SP-1
(BmSP-1) (Sakurai et al., 1988), Manduca sexta
SP1 (MsSP1) (Wang et al., 1993)], moderately
Met-rich storage proteins [represented by S. litura
SL-2 a subunit (SL-2 a) (Zheng et al., 2000), T.
ni basic juvenile hormone suppressible protein 2
(BJHSP2) (Jones et al., 1993)] (Fig. 2, Table 1),
and somewhat more distantly to the arylphorins
(data not shown). Reconstruction of the phylogenetic relationships based on amino acid distances revealed two subclades corresponding to
the Met-rich storage protein and moderately Metrich storage protein, respectively (Fig. 3). HcSP-1
is most closely related to the Met-rich storage proteins (65.6–73.8% identity), whereas HcSP-2 is
most closely related to the moderately Met-rich
storage proteins (73.9–74.8% identity) (Table 1).
Sequence identity between HcSP-1 and HcSP-2
(38.4%) is less than that between most members
of their respective hexamerin subfamilies (Table
1, Fig. 3), indicating that the common ancestor
of these two proteins diverged sometime before
the divergence of the Lepidopteran taxa represented in this analysis. This low identity between
the two H. cunea SPs supports numerous different epitopes for antibody recognition. Indeed, antibodies raised against HcSP-1 did not cross-react
with HcSP-2, and antibodies raised against HcSP2 showed no reactivity to HcSP-1, indicating that
the two storage proteins are immunologically distinct (Song et al. 1997).
Isoelectric points calculated from the deduced
amino acid sequences were 8.4 (HcSP-1) and 7.6
(HcSP-2). HcSP-2 methionine content places it at
the low end of the range of Met-rich storage proteins (3.2%), such as the SL-2 (subunit from S.
litura (3.5%), and is a weakly basic protein (pI =
7.6) (Table 2). Thus, it is possible that HcSP-2 has
different characteristics or functions than BJHSPs.
Archives of Insect Biochemistry and Physiology
163
Fig. 2.
Two JH Suppressible SPs From H. cunea
August 2002
164
Fig. 2. Continued.
Fig. 2. Alignment of the amino acid sequences of Met-rich storage proteins. HcSP-1 and HcSP-2, storage protein 1 and 2 from H.
cunea; SL-1 and SL-2 a, storage protein 1 and 2 a subunit from S. litura (Zheng et al., 2000); BJHSP1 and BJHSP2, basic juvenile
hormone suppressible protein 1 and 2 from T. ni (Jones et al.,1993); BmSP1, storage protein 1 from B. mori (Sakurai et al., 1988);
MsSP1, storage protein 1 from M. sexta (Wang et al., 1993).
166
Cheon et al.
TABLE 1. Sequence Identities Among Met-Rich Storage Proteins*
Percent divergence
Percent identiy
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
—
103.8
31.7
91.9
31.7
90.5
45.0
35.9
38.4
—
105.3
29.4
105.1
30.8
106.5
105.1
73.8
37.9
—
96.2
19.2
95.1
41.1
32.5
42.3
74.8
40.7
—
91.8
19.3
99.4
92.6
72.7
37.5
82.0
41.6
—
90.7
42.2
35.3
42.5
73.9
40.9
82.6
41.7
—
99.7
89.9
65.6
38.5
67.6
39.9
65.8
40.2
—
38.0
70.6
38.6
72.6
41.9
69.8
42.7
69.7
—
HcSP-1
HcSP-2
SL-1
SL-2a
BJHSP1
BJHSP2
BmSP1
MsSP1
*Sequences were aligned using the method of Feng and Doolittle (1987). Percent identies (above the diagonal line) and distance score (below the diagonal line) were
calculated by the computer program. Abbreviations and sequence sources as in Figure 2.
Suppression of SP mRNAs by JH
The high degree of similarity between HcSP-1
and T. ni BJHSP1, and between HcSP-2 and T. ni
BJHSP2, suggested to us that the HcSP genes may
be regulated by JH, as has been proposed for other
Met-rich storage proteins (Haunerland, 1996). To
determine if juvenile hormone affects the expression of the storage protein genes from H. cunea, a
juvenile hormone analog, methoprene was topically
applied to ligated last instar larvae. The accumulation of SP transcripts were suppressed in a dosedependent manner (Fig. 4); 0.1 mg of methoprene
per larva suppressed SP transcription to less than
30% of its normal level. When we applied 1.0 mg
methoprene per larva, the appearance of SP transcripts was completely eliminated. Jones et al.
(1993) demonstrated that maintenance of a high
Fig. 3. Distance-based phylogenetic analysis of H. cunea
storage proteins and other Met-rich storage proteins. The
phenogram is based on the alignment shown in Fig. 2
JH titer from the penultimate stadium into the final stadium of T. ni suppressed transcription of the
basic proteins to less than 1% of their normal level
by day 2, which resembles the response observed
in this study for HcSPs. When JH titer begins to decline in M. sexta, female specific storage protein
(FSP) mRNA first appears in female larvae (Riddiford and Hice, 1985). Additionally, allatectomy induces early expression of the female specific protein
gene and JH prevents its normal expression (Webb
and Riddiford, 1988b). Once expression of female
specific protein begins, JH is no longer capable of
suppressing it in M. sexta (Ray et al.,1987a). When
the supply of both JH and ecdysteroid was reduced
by abdominal ligation, fat body from fifth instar
female larvae failed to show an early appearance of
FSP mRNA, indicating that the removal of JH alone
is not sufficient to bring about expression of FSP
and distances are approximate. The names of species, proteins, and its abbreviations are described in Figure 2.
Archives of Insect Biochemistry and Physiology
Two JH Suppressible SPs From H. cunea
Fig. 4. Northern blot analysis of SP mRNAs from normal (C) and JHA-treated (0.1 and 1.0 mg) last instar larvae. Seventy hours after 2-day-old last instar larvae were
ligated, JHA (methoprene) was topically administered. Fat
body was collected for RNA extraction 6 h after hormone
treatment. Ribosomal RNAs (rRNA) are shown as an internal control after staining with ethidium bromide. For
details see Materials and Methods.
mRNA (Corpuz et al., 1991). The expression of
arylphorin genes appears to be regulated by the level
of 20HE and nutrient supply rather than juvenile
hormone level. In M. sexta both the lack of incomTABLE 2. Amino Acid Composition of Two Storage Proteins From H.
cunea and Other Met-Rich Storage Proteins
Residue
H. cunea
SP-1
H. cunea
SP-2
S. litura
SL-1a
S. litura
SL-2aa
T. ni
BJHSP1b
T. ni
BJHSP2b
3.4
1.2
9.1
4.5
4.2
3.9
2.2
8.4
7.1
8.1
6.0
5.0
3.4
1.9
7.2
4.1
7.5
7.1
1.5
4.5
3.0
1.1
10.1
4.0
5.9
3.4
2.2
8.1
8.1
10.0
3.2
6.0
3.0
2.9
5.9
2.5
6.7
7.1
1.1
5.9
3.0
1.2
9.5
3.9
4.6
3.9
2.2
5.6
8.0
9.0
7.7
4.8
3.7
0.8
7.6
2.9
6.5
8.6
1.8
4.8
2.3
1.1
10.4
3.8
6.0
3.5
2.2
6.8
9.3
10.6
3.5
4.9
3.0
1.4
6.5
2.2
5.7
8.2
1.4
7.1
2.5
1.4
8.9
0.7
4.5
4.3
2.4
6.0
8.4
8.7
8.4
3.1
4.0
0.7
7.3
3.4
7.8
7.9
1.5
3.9
1.7
0.8
10.3
4.0
5.9
3.2
2.2
7.0
9.3
11.3
5.3
4.8
3.1
1.4
7.0
1.9
5.8
8.6
1.1
5.2
Ala
Cys
Asp
Glu
Phe
Gly
His
Ile
Lys
Leu
Met
Asn
Pro
Gln
Arg
Ser
Thr
Val
Trp
Tyr
a
From GenBank accession numbers, AJ249470 (SL-1) and AJ249468 (SL-2a subunit).
From Jones et al. (1993) for T. ni basic juvenile hormone suppressible protein.
b
August 2002
167
ing nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts
and at wandering (Webb and Riddiford, 1988b).
Locust “persistent storage hexamer” (PSP) can also
be partially repressed in the last larval instar by a
JH analog, but during the adult stage the response
is reversed and PSP synthesis is stimulated by JH
(Ancsin and Wyatt, 1996). Therefore, different sensitivity of storage protein genes to JH during development suggests stage-specific hormonal regulation
of the appearance of storage protein in insects. It is
conceivable that the regulation of storage protein
expression is complex, involving tissue-specific, sexspecific, stage-specific, hormonal, and nutritional
components (Webb and Riddiford, 1988b).
Tissue-Specific Expression of H. cunea SP Genes
It was of interest to determine whether the two
storage protein genes exhibited similar tissue-specific expression. Total RNA was prepared from fat
body, Malpighian tubules, testes, and midgut of
late stage last instar larvae. Ovary samples were
from 8-day-old pupae. Hybridization of the HcSP1 cDNA insert to total RNA from several tissues
revealed the accumulation of a 2,500 nucleotide
transcript in the ovary as well as in testis, and Malpighian tubule, albeit at much lower levels than
in the fat body (Fig. 5, SP-1). In contrast, the HcSP2 transcript was expressed only in the fat body and
Malpighian tubule (Fig. 5, SP-2).
LHP synthesis in Lepidopterans occurs primarily, but not exclusively, in larval fat body cells
(Kanost et al., 1990). We found HcSP-1 gene expression in the nurse cells and follicular epithelial
cells of the oocyte by in situ hybridization (Cheon
et al., 2001). Locally expressed HcSP-1 in the ovary
contributes to early yolk formation before the
vitellogenic stage. The accumulation of HcSP-1 into
the ovary was also demonstrated by Western blot
(Fig. 6). During vitellogenesis, a substantial amount
of HcSP-1 from the hemolymph is incorporated
into the yolk bodies of the developing oocyte
(Fig.7) and may serve as an amino acid reservoir
for egg formation (Seo et al., 1998).
Synthesis and transcript detection of storage
168
Cheon et al.
Fig. 5. Presence of storage proteins among last instar larval (fat body, midgut, Malpighian tubule, and testis) and
pupal (ovary) tissue types. Northern blot analysis of total
RNA from different tissues using fragments of H. cunea
SP-1 and SP-2 as probes, respectively. For details see Materials and Methods. Fb, fat body; Mg, midgut; Mt, Malpighian tubule; Ov, ovary; Te, testis.
protein in the testes sheath cells were reported for
Heliothis virescens and G. mellonella, respectively
(Miller et al., 1990; Kumaran et al., 1993). Alternative sites of synthesis in Calpodes ethlius included
the epidermis (Palli and Locke, 1987a), midgut
(Palli and Locke, 1987b), and pericardial cells (Fife
et al., 1987). Analogous results were obtained with
M. sexta through the demonstration that arylphorin
genes are transcribed in several larval tissues (Webb
and Riddiford, 1988a). In contrast, the cDNAs encoding two storage proteins in G. mellonella are not
expressed in midgut, silk gland, or Malpighian tubules (Ray et al., 1987b).
Fig. 6. Western blot of protein extract (20 mg) from various tissues in the last instar larvae (hemolymph, fat body,
midgut, testis, and Malpighian tubule) and 8-day-old pupae (ovary). He, hemolymph; other symbols as in Figure 5.
Fig. 7. Accumulation of HcSP-1 in the yolk bodies of
the H. cunea oocyte during vitellogenesis (A). Little labeling is observed in the oocyte by antibody against HcSP-2
(B) or the control (C), which was probed with by preimmune serum. Scale bar = 1 mm.
Archives of Insect Biochemistry and Physiology
Two JH Suppressible SPs From H. cunea
Fig. 8. Immunological labeling of Malpighian tubule
with antibodies against HcSP-1 (A,B) and HcSP-2 (C,D).
August 2002
169
E,F: Controls. A,C,E: Microvilli on the luminal side. B,D,F:
Hemocoel side of Malpighian tubule. Scale bar = 1 mm.
170
Cheon et al.
Using Western blot (Fig. 6) and immunocytochemistry (Fig. 8), we localized HcSP-2 in the
Malpighian tubule at a high concentration (Fig.
8C and D). Though HcSP-1 mRNA is locally expressed in Malpighian tubule (Fig. 5), the accumulation of HcSP-1 in that organ is minimal
(Figs. 6, 8A,B). Miller and Silhacek (1993) observed that a dramatic increase in P82 hexamerin
was accompanied by a marked increase in riboflavin content in the hemolymph, Malpighian tubule, and testes. Direct observations of ligand
binding (Reum et al., 1982) and of transport capability (Haunerland and Bowers, 1986) of storage protein suggest that it may play an excretory
role in the Malpighian tubule. Although several
tissues have been identified as alternate expression sites of storage proteins, the types of tissues
involved are not consistent among species. In general, the relative abundance of HcSP-2 in H. cunea
was considerably less than that of HcSP-1 (Kim
et al., 1989). Thus, HcSP-2 likely makes a lesser
contribution to the storage system than does
HcSP-1. However HcSP-2 seems to play a role in
the Malpighian tubule. The experiments are in
progress for further understanding of physiological function of HcSP-2 in the Malpighian tubule.
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.
Cheon HM, Kim HJ, Chung DH, Kim MO, Park JS, Yun CY,
Seo SJ. 2001. Local expression and distribution of a storage protein in the ovary of Hyphantria cunea. Arch Insect
Biochem Physiol 48:111–120.
Corpuz LM, Choi H, Muthukrishnan S. Kramer KJ. 1991. Sequences of two cDNAs and expression of the genes encoding methionine-rich storage proteins of Manduca sexta.
Insect Biochem 21:265–276.
de Kort CAD, Koopmanschap AB. 1994. Nucleotide and deduced amino acid sequence of a cDNA clone encoding diapause protein 1, an arylphorin-type storage hexamer of the
Colorado potato beetle. J Insect Physiol 40:527–535.
Delaney SJ, Smith DF, McClelland A, Sunkel C, Glover DM.
1986. Sequence conservation around the 5¢ ends of the
larval serum protein 1 genes of Drosophila melanogaster. J
Mol Biol 189:1–11.
Feinberg AP, Vogelstein B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13.
Feng D, Doolittle R. 1987. Progressive sequence alignment
as a prerequisite to correct phylogenetic trees. J Mol Evol
25:351–360.
ACKNOWLEDGMENTS
We are very grateful to Dr. Thomas W. Sappington (USDA-ARS, IFNRRU) for a critical reading of the manuscript.
LITERATURE CITED
Alonso A, Flytzanis CN, Schatzle U, Scheller K, Sekeris CE.
1979. Purification and reverse transcription of the messenger RNA coding for the insect protein, calliphorin, isolated from larvae of the blowfly, Calliphora vicina. Eur J
Biochem 94:601–608.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990.
Basic local alignment search tool. J Mol Biol 215:403–410.
Ancsin JB, Wyatt GR. 1996. Purification and characterization
of two storage proteins from Locusta migratoria showing
distinct developmental and hormonal regulation. Insect
Biochem Mol Biol 26:501–510.
Fife HU, Palli SR, Locke M. 1987. A function for the pericardial cell in an insect. Insect Biochem 17:829–840.
Fujii T, Sakurai H, Izumi S, Tomino S. 1989. Structure of the
gene for the arylphorin-type protein SP2 of Bombyx mori.
J Biol Chem 264:11020–11025.
Haunerland NH. 1996. Insect storage proteins: gene families
and receptors. Insect Biochem Molec Biol 26:755–765.
Haunerland NH, Bowers WS. 1986. Binding of insecticides
to lipophorin and arylphorin, two hemolymph proteins
of Heliothis zea. Arch Insect Biochem Physiol 3:87–96.
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.
Jones G, Brown N, Manczak M, Hiremath S, Kafatos FC. 1990.
Archives of Insect Biochemistry and Physiology
Two JH Suppressible SPs From H. cunea
Molecular cloning, regulation, and complete sequence of
a hemocyanin-related, juvenile hormone-suppressible protein from insect hemolymph. J Biol Chem 265:8596–8602.
Jones G, Manczak M, Horn M. 1993. Hormonal regulation
and properties of a new group of basic hemolymph proteins expressed during insect metamorphosis. J Biol Chem
268:1284–1291.
Kanost MR, Kawooya JK, Ryan RD, Van Heusden MC, Ziegler
R. 1990. Insect hemolymph proteins. Adv Insect Physiol
22:299–366.
Kim HR, Kang CS, Mayer RT. 1989. Storage proteins of the
fall webworm, Hyphantria cunea Drury. Arch Insect Biochem Physiol 10:115–130.
Kumaran AK, Memmel NA, Wang C, Trewitt PM. 1993. Developmental regulation of arylphorin gene activity in fat
body cells and gonadal sheath cells of Galleria mellonella.
Insect Biochem Mol Biol 23:145–151.
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.
Memmel NA, Trewitt PM, Grezlak K, Rajaratnam VS, Kumaran
AK. 1994. Nucleotide sequence, structure and developmental regulation of LHP82, a juvenile hormone suppressible
hexamerin gene from the waxmoth Galleria mellonella. Insect Biochem Mol Biol 24:133–144.
Miller SG, Silhacek DL. 1993. Properties of two haemolymph
riboflavin-binding proteins from Heliothis virescens. Insect
Biochem Mol Biol 23:413.
Miller SG, Leclerc RF, Seo SJ, Malone C. 1990. Synthesis and
transport of storage proteins by testes in Heliothis virescens.
Arch Insect Biochem Physiol 14:151–170.
171
Ray A, Memmel NA, Orchekowski RP, Kumaran AK. 1987b.
Isolation of two cDNA clones coding for larval hemolymph proteins of Galleria mellonella. Insect Biochem
17:603–617.
Reum L, Käuser G, Enderle U, Koolman J. 1982. A steroidbinding protein from insect haemolymph isolated by photoaffinity labelling and immunoadsorption. Z Naturforsch
37c:967–978.
Reynolds ES. 1963. The use of lead citrate at high pH as an
electron opaque stain in electron microscopy. J Cell Biol
17:208–212.
Riddiford LM, Hice RH. 1985. Developmental profiles of the
mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta. Insect
Biochem 15:489–502.
Riddiford IM, Law JH. 1983. Larval serum proteins of Lepidoptera. In: Scheller K, editor. The larval serum proteins
of insects. Stuttgart: Thieme, p 75–85.
Sakurai H, Fujii T, Izumi S, Tomino S. 1988. Complete nucleotide sequence of gene for sex-specific storage protein of
Bombyx mori. Nucleic Acid Res 16:7717–7718.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning:
a laboratory manual. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press.
Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA
74:5463–5467.
Sato JD, Powell DJ, Robert BD. 1982. Purification of the
mRNAs encoding the subunits of larval serum proteins
1 and 2 of Drosophila melanogaster. Eur J Biochem
128:199–207.
Palli SR, Locke M. 1987a. The synthesis of hemolymph proteins by the larval midgut of an insect, Calpodes ethlius
(Lepidoptera: Hesperiidae). Insect Biochem 17:561–572.
Seo SJ, Kang YJ, Cheon HM, Kim HR. 1998. Distribution
and accumulation of storage protein-1 in ovary of
Hyphantria cunea Drury. Arch Insect Biochem Physiol
37:115–128.
Palli SR, Locke M. 1987b. The synthesis of hemolymph proteins by the larval epidermis of an insect, Calpodes ethlius
(Lepidoptera : Hesperiidae) Insect Biochem 17:711–722.
Song JK, Nha JH, Kim HR. 1997. Comparative analysis of
storage proteins of the fall webworm (Hyphantria cunea
Drury). Comp Biochem Physiol 118B:123–129.
Ray A, Memmel NA, Kumaran AK. 1987a. Developmental
regulation of the larval hemolymph protein genes in Galleria mellomella. Wilhelm Roux’s Arch Dev Biol 196:
414–420.
Telfer WH, Kunkel JG. 1991. The function and evolution of
insect storage hexamers. Annu Rev Entomol 36:205–228.
August 2002
Tojo S, Yoshiga T. 1994. Purification and characterization of
172
Cheon et al.
three storage proteins in the common cutworm, Spodoptera
litura. Insect Biochem 24:729–738.
Towbin H, Stachelin 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.
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. 1988a. Synthesis of two storage proteins during larval development of the tobacco hornworm,
Manduca sexta. Dev Biol 130:671–681.
Webb BA, Riddiford LM. 1988b. Regulation of expression
of arylphorin and female-specific protein mRNAs in the
tobacco hornworm, Manduca sexta. Dev Biol 130:
682–692.
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.
Zheng Y, Yoshiga T, Tojo S. 2000. cDNA cloning and deduced
amino acid sequences of three storage proteins in the common cutworm, Spodoptera litura (Lepidoptera:Noctuidae).
Appl Entomol Zool 35:31–39.
Archives of Insect Biochemistry and Physiology
Документ
Категория
Без категории
Просмотров
1
Размер файла
742 Кб
Теги
two, cunea, suppressible, different, play, storage, hyphantria, druri, may, protein, role, juvenile, hormone
1/--страниц
Пожаловаться на содержимое документа