close

Вход

Забыли?

вход по аккаунту

?

Expression of pheromone biosynthesis activating neuropeptide and its receptor PBANR mRNA in adult female Spodoptera exigua LepidopteraNoctuidae.

код для вставкиСкачать
A r t i c l e
EXPRESSION OF PHEROMONE
BIOSYNTHESIS ACTIVATING
NEUROPEPTIDE AND ITS
RECEPTOR (PBANR) mRNA
IN ADULT FEMALE Spodoptera
exigua (LEPIDOPTERA:
NOCTUIDAE)
Yunxia Cheng, Lizhi Luo, Xingfu Jiang, and Lei Zhang
State Key Laboratory for Plant Diseases and Insect Pests, Institute of
Plant Protection Chinese Academy of Agricultural Sciences,
Beijing, China
Changying Niu
College of Plant Sciences and Technology, Huazhong Agricultural
University, Wuhan, China
The full-length cDNA of pheromone biosynthesis activating neuropeptide
receptor (PBANR) was cloned from the beet armyworm, Spodoptera
exigua (Hübner) (Lepidoptera: Noctuidae); it included an open reading
frame of 1,053 bp encoding 350 amino acids. The PBANR of S. exigua
(SePBANR) was structurally characteristic of G protein–coupled
receptor and its amino acid sequence shared 98% identity with the
PBANR of Spodoptera littoralis. Both pheromone biosynthesis activating
neuropeptide (PBAN) and PBANR mRNA abundance were measured
in the brain-subesophageal ganglion complex, pheromone gland, ventral
nerve cord, and ovary of S. exigua female moths by real-time RT-PCR.
The abundance of PBAN mRNA in brain-subesophageal ganglion
complex and PBANR mRNA in pheromone gland was significantly
greater compared to other tissues, suggesting that the ligand-receptor
Grant sponsor: Programs of Industrial Science and Technology of Ministry of Agriculture of China; Grant
number: 200803007; Grant sponsor: Research Projects of State Key Laboratory for Plant Diseases and Insect
Pests; Grant number: SKL2007SR09SKL2009SR02; Grant sponsor: Natural Science Foundation of Beijing;
Grant number: 6072023.
Correspondence to: Lizhi Luo, State Key Laboratory for Plant Diseases and Insect Pests, Institute of Plant
Protection Chinese Academy of Agricultural Sciences, Beijing 100193, China. E-mail: lzluo@ippcaas.cn
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 75, No. 1, 13–27 (2010)
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2010 Wiley Periodicals, Inc. DOI: 10.1002/arch.20379
14
Archives of Insect Biochemistry and Physiology, September 2010
relationship of PBAN and PBANR exists quantitatively in S. exigua.
Both PBAN and PBANR expression displayed a remarkable diurnal
rhythm, for they were low and stable during the photophase
(07:00–21:00) and increased markedly during the scotophase (with a
maximum abundance at 23:30) in 3-day-old female moths. The
abundance of PBAN and PBANR increased steadily from the 1st day to
the 5th day of the adult female life. The pattern of both diurnal and daily
expression of PBAN and PBANR mRNA were coincident with enhanced
capacity of sex pheromone release and mating of S. exigua moths during
the same period. We infer from these results that pheromone biosynthesis
and release in S. exigua is regulated by PBAN via up-regulating
C 2010 Wiley Periodicals, Inc.
synthesis. Keywords: beet armyworm; sex pheromone; PBAN; PBANR
G protein–coupled receptor
INTRODUCTION
The production and release of sex pheromone, one of the semiochemicals by female
moths to attract conspecific males, are regulated by pheromone biosynthesis activating
neuropeptide (PBAN) (Raina and Klun, 1984; Tang et al., 1989; Jurenka et al., 1991).
PBAN was first identified as a 33–amino acid C-terminal amidated peptide in
Helicoverpa zea (Raina et al., 1989) and is a member of the pyrokinin/PBAN family with
a common C-terminal motif of FXPRLamide (Rafaeli, 2002). This is also the minimal
sequence required for its biological activity (Raina and Kempe, 1990, 1992). This motif
has been identified in pyrokinin/PBAN from a variety of insect orders, and a
crustacean (Torfs et al., 2001). However, pyrokinin/PBAN functions not only as a sex
pheromone biosynthesis regulator but also has additional roles in different species (see
reviews in Rafaeli, 2002, 2005).
The characterization of pheromone biosynthesis activating neuropeptide receptor
(PBANR) in several moth species indicates that PBANR is a member of G
protein–coupled receptors (GPCR) (Choi et al., 2003; Rafaeli et al., 2003; Hull et al.,
2004; Lee and Boo, 2005). PBANR was identified first in H. zea (Choi et al., 2003). It is
related to the mammalian neuropeptide receptor neuromedium U (NmU) and the
homologous receptors in Drosophila melanogaster (Adams et al., 2000). Subsequently,
PBANR was cloned and identified separately from female moths of Helicoverpa
armigera (Rafaeli et al., 2003), B. mori (Hull et al., 2004), and Plutella xylostella (Lee and
Boo, 2005). PBANR had been recorded in larval Spodoptera littoralis, where it mediated
cuticular pigmentation (Zheng et al., 2007). There is an instant influx of calcium and
increased cAMP in the target cell after PBAN binds to PBANR in H. zea (Choi et al.,
2003). PBANR functional identification had been established in many moth species
(Rafaeli et al., 2003; Hull et al., 2004; Zheng et al., 2007). The PBANR gene expressed
mainly in the sex pheromone glands of female moths (Hull et al., 2004; Rafaeli et al.,
2007); however, it also occurs in the neural tissues of adult H. armigera females and in
male aedeagus tissue (Rafaeli et al., 2007). So far, most characterization of PBAN and
PBANR has been completed in H. armigera (Rafaeli et al., 2003) and B. mori (Hull et al.,
2004). The roles of PBAN and PBANR have also been reported for mated Heliothis
virescens and H. subflexa females (Groot et al., 2005). PBANR can occur in other tissue
Archives of Insect Biochemistry and Physiology
PBAN and PBANR Expression in S. exigua
15
types with different functions, due to the ubiquity and multifunctional nature of the
pyrokinin/PBAN family of peptides (Rafaeli et al., 2007). However, because the biology
of PBAN and PBANR is complex among insect species, the structures and roles of
PBANR should be investigated in other species. Whether the relative expression
patterns of both PBAN and PBANR are synchronous with the release of sex
pheromone should also be investigated because their relationship has been
documented only in a few species.
The beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), males and
females mate multiple times with sexual activity lasting up to 7 days of adult life (Chu
and Wu, 1992; Dong and Du, 2001; Dong et al., 2001; Luo et al., 2003). As recorded
for a variety of moth species, the calling and mating behavior of S. exigua occurs in a
diurnal rhythm. Their sexual activities are most likely regulated by the production and
release of sex pheromone by female moths in scotophase (Dong and Du, 2001). PBAN
and PBANR play an important role in regulation of sex pheromone production in
female moths (Choi et al., 2003; Rafaeli et al., 2003). We posed the hypothesis that the
expression pattern of S. exigua PBAN (SePBAN) and PBANR (SePBANR) is related to
the release of sex pheromone. In this study, we report the outcomes of experiments
designed to test our hypothesis.
MATERIALS AND METHODS
S. exigua were reared on an artificial diet (Jiang et al., 1999) at a constant temperature
of 27711C and photoperiod of 14L:10D (light period was from 07:00 to 21:00).
Pupae were sexed on the third day and adult males and females were allowed to
emerge separately in transparent containers. The adults were maintained under the
same conditions as juveniles and fed with a 5% honey solution until use.
PBANR Cloning
The pheromone glands of the 2- and 3-day-old females were isolated and frozen in
liquid nitrogen immediately and kept at 801C until use. Total RNA was isolated from
pheromone glands with Trizol (Invitrogen, Carlsbad, CA). The quality and quantity of
the RNA were assessed by using a spectrophotometer (NanoDrop, Wilmington, DE).
First-strand cDNA synthesis was performed using Quantscript RT kit (Tiangen, Beijing,
China) following the manufacturer’s instructions. Primer pairs used to amplifying the
specific fragment of SePBANR were designed according to the sequence of S. littoralis
PBANR and the highly homology regions of H. zea, H. virscens, H. armigera, and
S. littoralis PBANR gene. By using the primer pairs (sense primer 50 -ATGACATTGTCAGCGCCCCCGA-30 and anti-sense primer 50 -CTCCTCTGTGATGCTCCATTCCT-30 ),
a SePBANR gene fragment of 791 bp was amplified with Ex Taq (Takara, Dalian,
China). The PCR protocol consisted of 30 cycles at 941C for 30 s, 581C for 30 s,
and 721C for 1min. This fragment was cloned into pEASY-T3 Vector (TransGen,
Beijing, China) and sequenced. To obtain the complete cDNA sequence of the
SePBANR gene, gene-specific primers (30 -RACE gene-specific primers: outer primer
50 -GTGCACAGATTATGGAATCCGGACA-30 , inner primer 50 -GTGAGCGCGTGTACAGTGAAGGGTG-30 ; 50 -RACE gene-specific primers: outer primer 50 -AGGCACCACGAAGAACACGAAACTA-30 , inner primer 50 - CACCCTTCACTGTACACGCGCTCAC-30 )
matching the primers in the 30 - and 50 -Full RACE kit (Takara, Dalian, China) were
designed again. The 30 -RACE outer and inner PCR was carried out with 20 cycles at
Archives of Insect Biochemistry and Physiology
16
Archives of Insect Biochemistry and Physiology, September 2010
941C for 30 s, 581C for 30 s, and 721C for 1 min, then at the condition of 30 cycles at
941C for 30 s, 601C for 30 s, and 721C for 1 min. 50 -RACE outer and inner PCR
amplification condition was the same as the 30 -RACE outer and inner protocols. The
amplified products of 30 -RACE and 50 -RACE were cloned into pEASY-T3 Vector
(TransGen, Beijing, China) and sequenced. Primer pairs (sense primer 50 -CGGCAGAAGGTAAAATGACATTGTC-30 , anti-sense primer 50 -CAAGGAAATCAATCATAAATGTAAC-30 ) were used to amplify the predicted gene including the open
reading frame of 1,053 bp. The PCR products were cloned into the pEasy-T3 vector
(Takara, Dalian, China) and sequenced.
PBAN and PBANR Real-Time Quantitative RT-PCR (qRT-PCR)
Material preparation. Ovaries (O), brain-subesophageal ganglion complexes (B-SEG),
ventral nerve cords (VNC), and sex pheromone glands (PG) were dissected from the
1- to 3-day virgin female moths. Newly emerged 3-day-old female moths were
collected every 4 h from 7:30 AM to 03:30 AM. Newly emerged female moths were also
collected at 03:30 AM every day from the first day emergence to the fifth day. The
brain-subesophageal ganglion complexes and sex pheromone glands were isolated
and kept at 801C after freezing in liquid nitrogen. Three independent biological
replicates were prepared for each tissue.
Total RNA isolation, DNase treatment, and reverse transcription. Total RNA was extracted
using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions and
suspended in 40 ml of RNase-free water. RNA purity and concentration were
determined by using a spectrophotometer (NanoDrop, Wilmington, DE). RNase-free
DNaseI (Takara, Dalian, China) was used to get rid of the potential genomic DNA, and
then the purity and concentration of the new RNA samples were measured by the
same method. First-strand cDNA was synthesized by using Quantscript RT Kit
(Tiangen, Beijing, China) for each sample following the manufacturer’s protocol.
Conventional RT-PCR and real-time qRT-PCR. Conventional RT-PCR for the SePBANR,
SePBAN, and b-actin genes was carried out by using Ex Taq polymerase (Takara,
Dalian, China) and the corresponding primer pairs (SePBAN: sense primer
50 -TGGGAAGGCGATTGTCTGATG-30 , anti-sense primer 50 -AGTCTGGGCGAGAAGTATTTGGT-30 ; SePBANR: sense primer 50 -GTGCACAGATTATGGAATCCGGACA-30 , anti-sense primer 50 -GACAACTTCGACATGGTGTGCGA-30 ; b-actin: sense
primer 50 -TCCAGCCTTCCTTCTTGGGTAT-30 , anti-sense primer 50 -CAGGTCCTTACGGATGTCAACG-30 ) on the same amplification condition: 951C for 1 min
followed by 40 cycles at 951C for 10 s, 601C for 30 s. The PCR amplification products
were analyzed using electrophoresis on 1.5% agarose gels and extracted using
TIANprep Mini Plasmid Kit (Tiangen, Beijing, China) and cloned into pEASY-T3
Cloning Vector (TransGen, Beijing, China) and sequenced (Shanghai Sangon, Beijing,
China). qRT-PCR for PBAN, PBANR, and b-actin genes was carried out on an iCycler
iQ (Bio-Rad, Hercules, CA) using SYBR Premix Ex Taq (Takara, Dalian, China) with
the following conditions: 951C for 10 s, followed by 40 cycles at 951C for 5 s, 601C
for 5 s. To evaluate the specificity of the SYBR Green real-time RT-PCR, a melting
curve temperature was determined by using the dissociation curves obtained from the
PCR products and the derivatives (-dF/dT) of fluorescence values plotted at 0.51C
intervals from 521C to 941C. To make sure it is well repeatable and accurate, intra- and
Archives of Insect Biochemistry and Physiology
PBAN and PBANR Expression in S. exigua
17
inter-assay variation of qRT-PCR was determined by using dilution series for b-actin,
SePBAN, and SePBANR in triplicate tubes for one sample run in one plate and
running PCRs three times on different plates with each sample having three replicates
in each PCR run separately.
Mathematical Model and Data Analysis
After sequencing, the original concentration of recombinant plasmids for SePBAN,
SePBANR, and b-actin was determined by spectrophotometer (NanoDrop, Wilmington,
DE) and each recombinant plasmid was diluted by 10-fold to obtain a six order of
magnitude dilution series used for the standard curve samples. Three technical
replicates were carried out for each sample and three biological replicates for each
testing sample. For all SYBR Green qRT-PCR assays, a no-template control was
included. The reference gene b-actin was used as an endogenous control. Data
analysis model for real-time PCR can be described with Rn 5 R0 (11E)n. (Pfaffl, 2001;
Rasmussen, 2001).
All the numerical data obtained from the quantitative tests of SePBAN and
SePBANR are presented as mean7SE. Statistical analysis was conducted using SPSS V.
16.0 software, applying one-way ANOVA and the LSD test. Significance was set at
Po0.05.
RESULTS
To obtain the full-length cDNA of the SePBANR gene, the initial 791-bp specific gene
fragment of SePBANR was cloned. This fragment shared 92.5% similarity with
sequences from H. zea, H. armigera, B. mori, and S. littoralis (data not shown). The
amplified 50 -RACE fragment of 766 bp comprised 170 bp of 50 -UTR and part of the
ORF including the start codon ATG. The 30 -RACE amplification product was 794 bp
consisting of part of the ORF including stop codon TGA and 317 bp 30 -UTR including
a polyA structure and the polyA signal sequence near the end of the stop codon. To
verify the authenticity of the complete ORF, additional primer pairs were designed
based on these results. A PCR product of 1,077 bp was amplified, containing an ORF of
1,053 bp and part of the 30 - and 50 -UTR. This matched the sequence previously
cloned. The final full-length SePBANR cDNA (GenBank accession no. EU365878)
contained a 50 -UTR of 170 bp, an ORF of 1,053 bp, and a 30 -UTR of 317 bp and
encoded 350 amino acids (Fig. 1).
The SePBANR has seven transmembrane domains predicted from the secondary
structure of the deduced amino acid sequence (Fig. 2) (www.cbs.dtu.dk/services/
TMHMM). SePBANR includes two possible N-linked glycosylation sites (Asn19 and
Asn22) near the N-terminus.
BLAST search showed that the deduced amino acid sequence of SePBANR is very
similar to the functionally verified PBANRs from S. littoralis (98%), H. zea (96%),
H. armigera (95%), H. virescens (95%), B. mori (86%), and P. xylostella (74%). Among these
PBANRs, the major deviation occurs at the N-terminus and C-terminus (Fig. 2). The
highly conserved region occurs in the transmembrane domains and there is only one
amino acid variation in all of the 7 transmembrane domains of S. exigua PBANR: TM1,
Val48 (indicated by arrow in Fig. 2), when compared to the amino acid sequences of
H. zea and S. littoralis.
Archives of Insect Biochemistry and Physiology
18
Archives of Insect Biochemistry and Physiology, September 2010
Figure 1. Nucleotide and deduced amino acid sequence of the S. exigua PBANR. The nucleotide sequence
is numbered in the 50 to 30 direction. The ORF has a length of 1,053 nucleotides flanked by the start codon
ATG and stop codon TGA. In the 30 -terminus, multiple putative poly-adenylation signals are predicted. The
deduced amino acid sequence of the ORF is shown below the nucleotide sequence for each codon. Two
potential N-linked glycosylation sites on the N-terminus of the protein (Asn19 and Asn22) are indicated and
the ERY motif is labeled by light gray shading.
Relative Expression Abundance of SePBAN and SePBANR in Four Tissues
The expression abundance of SePBAN mRNA in four tissues. SePBAN mRNA is present in
brain-subesophageal ganglion complex, ventral nerve cord, ovary, and pheromone
gland of adult females (Fig. 3). The maximal relative expression abundance of SePBAN
mRNA was found in brain-subesophageal ganglion complex, followed by ventral nerve
cord, ovary, and pheromone gland in decreasing order. The relative expression
abundance of SePBAN mRNA in brain-subesophageal ganglion complex was
Archives of Insect Biochemistry and Physiology
PBAN and PBANR Expression in S. exigua
19
Figure 2. Alignment of amino acid sequences of moth PBANRs. Identical amino acids are highlighted in
dark black and conserved amino acids in the lighter color for five sequences. The putative TM domains
(TM1-TM7) for these PBANRs were predicted by software analysis (TMHMM Server v.2.0, www.cbs.dtu.dk/
services/TMHMM/) and are indicated by brackets above the amino acid sequences. Dotted lines indicate
alignment gaps. GenBank accession number: S. exigua EU365878, B. mori AB181298, P. xylostella AY974334,
H. zea AY319852, H. armigera AY792036, and S. littoralis DQ407742. Variation of amino acid residue in
transmembrane domains of H. zea, S. littoralis, and S. exigua PBANRs is indicated by an arrow.
Archives of Insect Biochemistry and Physiology
20
Archives of Insect Biochemistry and Physiology, September 2010
20
PBAN
PBANR
A
Relative expression level
16
12
8
a
B
4
b
b
B
B
b
0
B-SEG
VNC
PG
O
Tissues
Figure 3. Relative expression abundance of the PBAN and PBANR in different tissues of S. exigua. B-SEG,
brain-subesophageal ganglion; VNC, ventral nerve cord; PG, pheromone gland; O, ovary. For PBAN, the
gene expression in the pheromone gland was set as the calibrator. And for PBANR, the gene expression in
the brain-subesophageal ganglion was set as the calibrator. Histograms represent mean1SE. Different letters
indicate statistically significant differences (one-way ANOVA, LSD-test).
significantly greater than the three other tissues. The differences in relative expression
abundance of SePBAN in ventral nerve cord, ovary, and pheromone gland were not
statistically significant.
The expression abundance of SePBANR mRNA in four tissues. SePBANR mRNA in the
various tissues was also quantitatively different (Fig. 3). In contrast to the SePBAN
mRNA expression, the maximal relative expression abundance of SePBANR mRNA
was found in the sex pheromone gland, followed by the ventral nerve cord, brainsubesophageal ganglion complex, and ovary in decreasing order. Relative expression
abundance of SePBANR mRNA in the sex pheromone gland was significantly greater
than that in the three other tissues and there was no significant difference in the latter
three tissues.
Relative Expression Abundance of SePBAN and SePBANR in a Diurnal Rhythm
The expression abundance of SePBANR in a diurnal rhythm. The maximal relative
expression abundance of SePBAN mRNA in 3-day-old females occurred at 23:30,
followed by 19:30, 11:30, 15:30, 03:30, and 07:30 in decreasing order (Fig. 4).
Although there were no significant differences among six of the time points for the
expression abundance of SePBAN mRNA, the expression abundance during
the scotophase in average was greater than expression during the photophase. The
expression abundance in the scotophase fluctuated greatly while it was rather stable
during the photophase, except at the 7:30 time point.
The expression abundance of SePBANR in a diurnal rhythm. The relative expression
abundance of SePBANR mRNA was synchronized with that of PBAN mRNA in 3-dayold female moths (Fig. 4). The maximal relative expression abundance of SePBANR
Archives of Insect Biochemistry and Physiology
PBAN and PBANR Expression in S. exigua
21
2.1
PBAN
PBANR
A
Relative expression level
1.8
1.5
A
A
a
A
1.2
a
a
0.9
a
a
a
A
A
07:30
11:30
15:30
19:30
23:30
03:30
Time
Figure 4. Relative expression abundance of PBAN mRNA in brain-subesophageal ganglion complex and
PBANR mRNA in pheromone glands of S. exigua at six time points during a photoperiod. For PBAN and
PBANR, the gene expression at 03:30 was set as the calibrator. Line graph represents mean7SE. Different
letters indicate statistically significant difference (one-way ANOVA, LSD-test).
mRNA occurred at 23:30, followed by 03:30, 07:30, 11:30, 15:30, and 19:30. Although
these values were not significantly different, those obtained in the scotophase were
much greater than those obtained in the photophase. Again, the relative expression
abundance of SePBANR mRNA in the scotophase was rather variable while that in the
photophase remained low and stable.
Relative Expression Abundance of SePBAN and SePBANR From Day 1 to Day 5
in Female Moths
The expression abundance of SePBAN from day 1 to day 5 in female moths. The relative
expression abundance of SePBAN mRNA in females increased generally from day1 to
day5 (Fig. 5). The maximal value for the relative expression abundance of SePBAN
mRNA was in the 5-day-old female, which was significantly greater than that in the
1- and 2-day-old, but not significantly different from that in the 3- and 4-day-old. The
relative abundance of PBAN mRNA in female moths from day 1 to day 4 was not
significantly different (Fig. 5).
The expression abundance of SePBANR from day 1 to day 5 in female moths. The relative
expression abundance of SePBANR mRNA during the same adult period was
generally similar to that of the SePBAN mRNA (Fig. 5). However, the maximal
expression abundance of SePBANR mRNA occurred on day 4 rather than on day 5 of
the adult life. Regression analysis demonstrated that relative expression abundance of
both PBAN and SePBANR were positively correlated to the age from the 1st day to the
5th day of the adult female S. exigua life (Fig. 5). The linear equation for the expression
of PBAN and SePBANR was Y 5 0.217510.5999X, (R 5 0.7790, Po0.05), and
Y 5 0.847910.2655X (R 5 0.6470, Po0.05), respectively (Fig. 5). In comparison with
the expression abundance of SePBANR, expression of SePBAN increased more rapidly
Archives of Insect Biochemistry and Physiology
22
Archives of Insect Biochemistry and Physiology, September 2010
The relative expression level
5
PBAN
PBANR
4
3
2
1
0
1
2
3
4
Days after emergence
5
Figure 5. Linear regression analysis of relative expression abundance of PBAN and PBANR mRNA in 1- to
5-day-old S. exigua female adults. The linear equation for PBAN and PBANR is Y 5 0.847910.2655X,
R 5 0.6470; and Y 5 0.217510.5999X, R 5 0.7790, respectively. The correlation equation indicates that the
relative expression abundance of both PBAN and PBANR mRNA is positively correlated to the age of the
female moth during the sampling period (N 5 15, Po0.05).
as the female moth aged, as demonstrated by their respective linear regression
equation and R value.
DISCUSSION
Identification of PBANR in Female Moth of S. exigua
PBANR is one member of GPCRs, and plays an important role in regulating
pheromone biosynthesis together with its ligand PBAN. Several points support our
identification of SePBANR. First, the structural analysis of the deduced amino acid
sequence indicates that 7 transmembrane domains are present, which is characteristic
of GPCRs (Palczewski et al., 2000; Zhang et al., 2005). Second, there are two possible
N-linked glycosylation sites at Asn19 and Asn22, which are necessary for the efficient
expression of GPCRs on the cell surface (Kaushal et al., 1994; Ding et al., 1995;
Rodrı́guez et al., 1995), although the glycosylation may not be necessary for signal
transduction (Choi et al., 2003). Third, an ERY motif (AA137–139) is highly conserved
for GPCRs downstream the TM3. For one thing, it is responsible for G-protein
coupling and the normal production of cAMP (Yamano et al., 2004); for another, in
mammalian cells the residue Arg forms a salt bridge with the neighboring Glu and/or
another charged residue Glu on TM6 (review: Rovati et al., 2007) to stabilize the intramolecular interaction. The ERY motif, therefore, may play a key role in maintaining a
stable receptor conformation in the cell membrane and coupling with intracellular
G-proteins. Last, according to the BLAST analysis the deduced amino acid sequence of
SePBANR is highly homologous (98%) to PBANRs of S. littoralis, which has been
functionally identified (Zheng et al., 2007). The homology of SePBANR to other
species varies depending on the phylogenetic relationship with the S. exigua. The
Archives of Insect Biochemistry and Physiology
PBAN and PBANR Expression in S. exigua
23
deduced amino acid sequence of the SePBANR has an exact identity with PBANRs of
other species: 96% (H. zea), 95% (H. armigera), 95% (H. virescens), 86% (B. mori), and
74% (P. xylostella). The high homology with other PBANRs and the deduced amino acid
sequence functional motifs suggest that SePBANR most probably has the same
function on target tissues.
SePBAN and SePBANR Expression in Different Tissues
SePBAN mRNA abundance in brain-subesophageal ganglian complex is significantly
greater than that in the three other tissues. We infer that SePBAN is produced mainly
in the brain-subesophageal ganglian complex of adult females. This is consistent with
reports on H. zea (Raina and Klun, 1984; Raina et al., 1989, 1991), and many others
(Davis et al., 1992; Matsumoto et al., 1992; Sato et al., 1993; Masler et al., 1994; Choi
et al., 1998; Duportets et al., 1998; Jacquin-Joly et al., 1998; Iglesias et al., 2002). We
infer from the different distribution of SePBAN mRNA in four tissues that SePBAN
may play different roles. Indeed, SePBAN not only acts as a pheromone biosynthesis
regulator but also plays diverse roles in several insect species. For example, PBAN may
stimulate the contraction of hindgut muscles in the cockroach, induce embryonic
diapause, terminate pupal diapause, melanize moth larvae, and accelerate fly
puparium formation (see reviews: Rafaeli, 2002; Rafaeli and Jurenka, 2003).
The expression of SePBANR mRNA has also been verified in four adult tissues.
However, SePBANR mRNA was differentially expressed relative to SePBAN expression. SePBANR expression in sex pheromone gland was 2-fold greater than in the
other tissues. We surmise that PBANR is mainly produced in sex pheromone glands.
SePBANR mRNA also appeared in ventral nerve cord, brain-subesophageal ganglion
complex, and ovary of female adults. These results are similar to those found in B. mori
(Hull et al., 2004) and H. armigera females (Rafaeli et al., 2007). The existence of
SePBAN and SePBANR in different tissues indicates to us that they are involved in
multi-functional roles in cell activity, including sex pheromone production. We infer
from the different spatial distribution patterns of both SePBAN and SePBANR that
they have a ligand-receptor relationship.
The Diurnal Expression Rhythm of SePBAN and SePBANR
The relative expression abundance of SePBAN and SePBANR occurs in a diurnal
rhythm in 3-day-old females. Expression is low and stable in photophase but rises
greatly during scotophase and peaks at 23:30. This is consistent to the report on
2–4-day-old virgin females by Northern blot analysis (Xu et al., 2006). The sex
pheromone titer in the pheromone gland is greater in scotophase than photophase
and the expression peak is at 6.5 h (03:30) into scotophase in 3-day-old virgin females
(Dong and Du, 2001). It is reasonable that the expression of SePBAN and SePBANR
mRNA peaks earlier than the initiation of sex pheromone production, since as an
upstream regulator of sex pheromone biosynthesis, the biosynthesis of the SePBANR
and a complicated signal transduction from the cell membrane on will occur before the
production of sex pheromone. So SePBAN/SePBANR is probably a key rhythmic
messenger in regulating the production of sex pheromone. We infer from our results
that the diurnal expressions of both SePBAN and SePBANR are tightly associated with
sex pheromone production and release.
Archives of Insect Biochemistry and Physiology
24
Archives of Insect Biochemistry and Physiology, September 2010
Expression of SePBAN and SePBANR in Relation to Adult Age
The expression abundance of both SePBAN and SePBANR is correlated with adult age
from day 1 to day 5. We infer that the increasing abundance of SePBAN and SePBANR
mRNA is necessary to maintain enough sex pheromone biosynthesis and to support
subsequent calling behavior. This is supported by the observation that the onset and
duration of calling of 1- and 2-day-old adults are significantly later and shorter than
that at 3- to 7-day-old adults, respectively (Dong and Du, 2001). It is similar to the
pattern recorded for other moths, including H. armigera (Hou and Sheng, 2000),
Condylorrhiza vestigialis (Ambrogi et al., 2009), Agrotis ipsilon (Swier et al., 1977),
Hydraecia micacea (West et al., 1984), Chilo suppressalis (Kanno, 1979), Platynota stultana
(Webster and Cardé, 1982), and H. zea (Raina et al., 1986). This data in this report
support our hypothesis that the expression pattern of SePBAN and SePBANR is
related to the release of sex pheromone in S. exigua, and SePBAN and SePBANR
probably positively regulate the biosynthesis of sex pheromone.
ACKNOWLEDGMENTS
We thank Dr. Yi Hu for her help in rearing the insects. We also thank Dr. Russell
Jurenka of the Department of Entomology, Iowa State University, for his great help in
writing this article. This study was supported by Programs of Industrial Science and
Technology of Ministry of Agriculture of China (200803007), Research Projects of
State Key Laboratory for Plant Diseases and Insect Pests (SKL2007SR09,
SKL2009SR02) and the Natural Science Foundation of Beijing (6072023).
LITERATURE CITED
Adams MD, Celniker SE, Holt RA, Evan CA, Gocayne JD, Amanatides PG, et al. 2000. The
genome sequence of Drosophila melanogaster. Science 287:2185–2195.
Ambrogi BG, Fonseca MG, Coracini MDA, Zarbin PHG. 2009. Calling behaviour and male
response towards sex pheromone of poplar moth Condylorrhiza vestigialis (Lepidoptera:
Crambidae). J Pest Sci 82:55–60.
Choi MY, Tanaka M, Kataoka H, Boo KS, Tatsuki S. 1998. Isolation and identification of the
cDNA encoding the pheromone biosynthesis activating neuropeptide and additional
neuropeptides in the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera:
Noctuidae). Insect Biochem Mol Biol 28:759–766.
Choi MY, Fuerst EJ, Rafaeli A, Jurenka R. 2003. Identification of a G protein-coupled receptor
for pheromone biosynthesis activating neuropeptide from pheromone glands of the moth
Helicoverpa zea. Proc Natl Acad Sci USA 100:9721–9726.
Chu Y-I, Wu H-T. 1992. Studies on emergence, copulation and oviposition of adult beet
armyworm (Spodoptera exigua Hübner). Chin J Entomol 12:91–99.
Davis MT, Vakharia VN, Henry J, Kempe TG, Raina AK. 1992. Molecular cloning of the
pheromone biosynthesis-activating neuropeptide in Helicoverpa zea. Proc Natl Acad Sci USA
89:142–146.
Ding DX-H, Vera JC, Heaney ML, Golde DW. 1995. N-glycosylation of the human granulocytemacrophage colony-stimulating factor receptor alpha subunit is essential for ligand binding
and signal transduction. J Biol Chem 270:24580–24584.
Dong S-L, Du J-W. 2001. Diel rhythms of calling behavior and sex pheromone production of
beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). Entomol Sin 8:89–96.
Archives of Insect Biochemistry and Physiology
PBAN and PBANR Expression in S. exigua
25
Dong S-L, Xu S-F, Du J-W, Shen J-H. 2001. Behavioral responses of the males to components
and blends found in the female sex pheromone gland of beet armyworm, Spodoptera exigua
(Lepidoptera: Noctuidae) in China. Insect Sci 8:93–100.
Duportets L, Gadenne C, Dufour MC, Couillaud F. 1998. The pheromone biosynthesis
activating neuropeptide (PBAN) of the black cutworm moth, Agrotis ipsilon: immunohistochemistry molecular characterization and bioassay of its peptide sequence. Insect
Biochem Mol Biol 28:591–599.
Groot AT, Fan Y, Browne C, Jurenka RA, Gould F, Schal C. 2005. Effect of PBAN on pheromone
production by mated Heliothis virescens and Heliothis subflexa females. J Chem Ecol 31:14–28.
Hou ML, Sheng CF. 2000. Calling behaviour of adult female Helicoverpa armigera (Hübner)
(Lep., Noctuidae) of overwintering generation and effects of mating. J Appl Entomol
124:71–75.
Hull JJ, Ohnishi A, Moto Ki, Kawasaki Y, Kurata R, Suzuki MG, Matsumoto S. 2004. Cloning
and characterization of the pheromone biosynthesis activating neuropeptide receptor from
the silkmoth, Bombyx mori: significance of the carboxyl terminus in receptor internalization.
J Biol Chem 279:51500–51507.
Iglesias F, Marco P, Francois MC, Camps F, Fabrias G, Jacquin-Joly E. 2002. A new member of
the PBAN family in Spodoptera littoralis: molecular cloning and immunovisualisation in
scotophase hemolymph. Insect Biochem Mol Biol 32:901–908.
Jacquin-Joly E, Burnet M, Francois MC, Ammar D, Nagnan-Le Meillour P, Descoins C. 1998.
cDNA cloning and sequence determination of the pheromone biosynthesis activating
neuropeptide of Mamestra brassicae: a new member of the PBAN family. Insect Biochem Mol
Biol 28:251–258.
Jiang X-F, Luo L-Z, Hu Y. 1999. Influence of larval diets on development, fecundity and flight
capacity of the beet armyworm, Spodoptera exigua. Acta Entomol Sin 42:270–272.
Jurenka RA, Jacquin E, Roelofs WL. 1991. Control of the pheromone biosynthetic pathway in
Helicoverpa zea by the pheromone biosynthesis activating neuropeptide. Arch Insect
Biochem Physiol 17:81–91.
Kanno H. 1979. Effects of age on calling behaviour of the rice stem borer, Chilo suppressalis
(Walker) (Lepidoptera: Pyralidae). Bull Entomol Res 69:331–335.
Kaushal S, Ridge KD, Khorana HG. 1994. Structure and function in rhodopsin: the role of
asparagine-linked glycosylation. Proc Natl Acad Sci USA 91:4024–4028.
Lee DW, Boo KS. 2005. Molecular characterization of pheromone biosynthesis activating
neuropeptide from the diamondback moth, Plutella xylostella (L.). Peptides 26:2404–2411.
Luo L-Z, Cao W-J, Qian K, Hu Y. 2003. Mating behavior and capacity of the beet armyworm,
Spodoptera exigua (Lepidoptera: Noctuidae). Acta Entomol Sin 46:494–499.
Masler EP, Raina AK, Wagner RM, Kochansky JP. 1994. Isolationand identification of a
pheromonotropic neuropeptide from the brain-suboesophageal ganglion complex of
Lymantria dispar: a new member of the PBAN family. Insect Biochem Mol Biol 24:829–836.
Matsumoto S, Fonagy A, Kurihara M, Uchiumi K, Nagamine T, Chijimatsu M, Mitsui T. 1992.
Isolation and primary structure of a novel pheromonotropic neuropeptide structurally
related to leucopyrokinin from the armyworm larvae, Pseudaletia separata. Biochem Biophys
Res Commun 182:534–539.
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Trong IL, Teller DC,
Okada T, Stenkamp RE, Yamamoto M, Miyano M. 2000. Crystal structure of rhodopsin: A G
protein-coupled receptor. Science 289:739–745.
Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT–PCR.
Nucleic Acids Res 29:2002–2007.
Rafaeli A. 2002. Neuroendocrine control of pheromone biosynthesis in moths. Int Rev Cytol
213:49–91.
Archives of Insect Biochemistry and Physiology
26
Archives of Insect Biochemistry and Physiology, September 2010
Rafaeli A. 2005. Mechanisms involved in the control of pheromone production in female moths:
recent developments. Entomol Exp Appl 115:7–15.
Rafaeli A, Jurenka R. 2003. PBAN regulation of pheromone biosynthesis in female moths. Insect
Pherom Biochem Mol Biol. New York: Academic Press. p 107–136.
Rafaeli A, Zakharova T, Lapsker Z, Jurenka RA. 2003. The identification of an age-and femalespecific putative PBAN membrane-receptor protein in pheromone glands of Helicoverpa
armigera: possible up-regulation by juvenile hormone. Insect Biochem Mol Biol 33:371–380.
Rafaeli A, Bober R, Becker L, Choi MY, Fuers EJ, Jurenka R. 2007. Spatial distribution
and differential expression of the PBAN receptor in tissues of adult Helicoverpa spp.
(Lepidoptera: Noctuidae). Insect Mol Biol 16:287–293.
Raina AK, Kempe TG. 1990. A pentapeptide of the C-terminal sequence of PBAN with
pheromonotropic activity. Insect Biochem 20:849–851.
Raina AK, Kempe TG. 1992. Structure activity studies of PBAN of Helicoverpa zea (Lepidoptera,
Noctuidae). Insect Biochem Mol Biol 22:221–225.
Raina AK, Klun JA. 1984. Brain factor control of sex pheromone production in the female corn
earworm moth. Science 225:531–533.
Raina AK, Klun JA, Stadelbacher EA. 1986. Diel periodicity and effect of age and mating on
female sex pheromone titer in Heliothis zea (Lepidoptera: Noctuidae). Ann Entomol Soci Am
79:128–131.
Raina AK, Jaffe H, Kempe TG, Keim P, Blacher RW, Fales HM, Riley CT, Klun JA, Ridgway RL,
Hayes DK. 1989. Identification of a neuropeptide hormone that regulates sex pheromone
production in female moths. Science 244:796–798.
Raina AK, Kempe TG, Jaffe H. 1991. Pheromone biosynthesis-activating neuropeptide:
regulation of pheromone production in moths. ACS Symp Ser Am Chem Soc 453:100–109.
Rasmussen R. 2001. Quantification on the light cycler. In: Meuer S, Wittwer C, Nakagawara K,
editors. Rapid cycle real-time PCR, methods and applications. Heidelberg: Springer Press.
p 21–34.
Rodrı́guez CG, Cundell DR, Tuomanen EI, Kolakowski Jr LF, Gerard C, Gerard NP. 1995. The
role of N-glycosylation for functional expression of the human platelet- activating factor
receptor. J Biol Chem 270:25178–25184.
Rovati GE, Capra V, Neubig RR. 2007. The highly conserved dry motif of class A G proteincoupled receptors: beyond the ground state. Mol Pharmacol 71:959–964.
Sato Y, Oguchi M, Menjo N, Imai K, Komiya T, Saito H, Ikeda M, Isobe M, Yamashita O. 1993.
Precursor polyprotein for multiple neuropeptides secreted from the suboesophageal
ganglion of the silkworm Bombyx mori: characterisation of the cDNA encoding diapause
hormone presursor and identification of additional peptides. Proc Natl Acad Sci USA
90:3251–3255.
Swier SR, Rings RW, Musick GJ. 1977. Age-related calling behavior of the black cutworm, Agrotis
ipsilon. Ann Entomol Soci Am 70:919–924.
Tang JD, Charlton RE, Jurenka RA, Wolf WA, Phelan PL, Sreng L, Roelofs WL. 1989.
Regulation of pheromone biosynthesis by a brain hormone in two moth species. Proc Natl
Acad Sci USA 86:1806–1810.
Torfs P, Nieto J, Cerstiaens A, Boon D, Baggerman G, Poulos C, Waelkens E, Derua R,
Calderón J, De Loof A, Schoofs L. 2001. Pyrokinin neuropeptides in a crustacean: isolation
and identification in the white shrimp Penaeus vannamei. Eur J Biochem 268:149–154.
Webster RP, Cardé RT. 1982. Relationships among pheromone titre, calling and age in the
omnivorous leafroller moth (Platynota stultana). J Insect Physiol 28:925–933.
West RJ, Teal PEA, Laing JE, Grant GM. 1984. Calling behavior of the potato stem borer,
Hydraecia micacea (Esper) (Lepidoptera: Noctuidae), in the laboratory and the field. Environ
Entomol 13:1399–1404.
Archives of Insect Biochemistry and Physiology
PBAN and PBANR Expression in S. exigua
27
Xu G-Q, Cong B, Luo L-Z. 2006. Expression of PBAN gene in Spodoptera exigua female adults in
different ages. Chin Bull Entomol 43:38–41.
Yamano Y, Kamon R, Yoshimizu T, Toda Y, Oshida Y, Chaki S, Yoshioka M, Morishima I. 2004.
The role of the DRY motif of human MC4R for receptor activation. Biosci Biotechnol
Biochem 68:1369–1371.
Zhang T-Y, Sun J-S, Liu W-Y, Kang L, Shen J-L, Xu W-H. 2005. Structural characterization and
transcriptional regulation of the gene encoding diapause hormone and pheromone
biosynthesis activating neuropeptide in the cotton bollworm, Helicoverpa armigera. Biochim
Biophys Acta 1728:44–52.
Zheng L, Lytle C, Njauw C-N, Altstein M, Martins-Green M. 2007. Cloning and characterization
of the pheromone biosynthesis activating neuropeptide receptor gene in Spodoptera littoralis
larvae. Gene 393:20–30.
Archives of Insect Biochemistry and Physiology
Документ
Категория
Без категории
Просмотров
0
Размер файла
857 Кб
Теги
expressions, activation, spodoptera, mrna, adults, neuropeptide, exigua, pbanr, lepidopteranoctuidae, female, receptov, biosynthesis, pheromones
1/--страниц
Пожаловаться на содержимое документа