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Up-regulation of lysozyme gene expression during metamorphosis and immune challenge of the cotton bollworm Helicoverpa armigera.

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A r t i c l e
UP-REGULATION OF LYSOZYME
GENE EXPRESSION DURING
METAMORPHOSIS AND IMMUNE
CHALLENGE OF THE COTTON
BOLLWORM, Helicoverpa
armigera
Yong Zhang, Jianhua Huang, and Bo Zhou
Institute of Plant Physiology and Ecology, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s
Republic of China
Graduate School of the Chinese American Academy of Sciences, Beijing,
People’s Republic of China
Chunlin Zhang
Department of Biology, Guiyang Medical University, Guiyang, People’s
Republic of China
Wenbin Liu
Institute of Plant Physiology and Ecology, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s
Republic of China
Graduate School of the Chinese American Academy of Sciences, Beijing,
People’s Republic of China
Xuexia Miao and Yongping Huang
Institute of Plant Physiology and Ecology, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s
Republic of China
Grant sponsor: National Natural Sciences of China 30771445; Grant sponsor: National Basic Research Program
of China 2005BC121000; Grant sponsor: National High-Tech R&D Program 2006AA10A119.
Correspondence to: Yongping Huang, Institute of Plant Physiology and Ecology, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences, 300 Feng Lin Road, Shanghai, 200032, People’s Republic
of China. E-mail: yphuang@sibs.ac.cn
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 70, No. 1, 18–29 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2008 Wiley Periodicals, Inc. DOI: 10.1002/arch.20258
Up-regulation of Lysozyme Gene Expression
19
Lysozymes act as crucial bacteriolytic enzymes in insect immune system by
hydrolyzing the b (1-4) bonds between N-acetylglucosamine and Nacetylmuramic acid in the peptidoglycan of prokaryotic cell walls. We have
isolated and characterized a Helicoverpa armigera cDNA encoding an
insect lysozyme named HaLyz. We amplified a fragment by PCR, using
degenerate primers derived from the conservative amino acid sequences for
performing 50 and 30 RACE. The full-length cDNA was 661 base pairs.
The theoretical pI and molecular weight of the protein were computed to be
9.08 and 15.6 kDa, respectively. Prokaryotic expression of the HaLyz ORF
by Escherichia coli confirmed the calculated molecular weight of the protein.
The deduced 135 amino acids showed high homology with known lysozymes
from other insects, ranging from 47% to 89% by BLASTp search in
NCBI. Analyses revealed that this protein has a typical lysozyme C
signature among amino acids 93–111, CNVTCAEMLLDDITKASTC.
An interesting relation between immunity and larva to pupa metamorphosis
in insects was discovered. Real time-PCR showed that HaLyz gene
expression was transiently enhanced at the onset of metamorphosis of the
cotton bollworm, Helicoverpa armigera. The gene expression was upregulated after the injection of E. coli or entomopathogenic fungi,
C 2008
Beauveria bassiana, but showed different expression patterns. Wiley Periodicals, Inc.
Keywords: Helicoverpa armigera; lysozyme; lysozyme gene; insect immunity; insect development
INTRODUCTION
Insects are the most widely distributed and successful animal class in the world. Rapid
propagation, complicated metamorphosis, and a robust immune system are the pivotal
reasons for this phenomenon. Insects defend themselves against many kinds of
microbial organisms, both prokaryotic and eukaryotic. Among these, various bacteria
and entomopathogenic fungi compose the main pathogenic threats to insects. Besides
the physical barriers, defense systems include both cellular and humoral immune
response elements (Lemaitre and Hoffmann, 2007). Humoral immune factors,
antimicrobial proteins, for example, cecropin, lebocin, and lysozyme, play an effective
role in insect response to microbial infection (Lemaitre and Hoffmann, 2007).
Lysozyme (EC 3.2.1.17) is a crucial bacteriolytic enzyme in animal innate immune
systems. This enzyme hydrolyzes the b-1,4 glycosidic bond between N-acetylmuramic
acid and N-acetylglucosamine in the peptidoglycan layer of bacterial cell walls, and
cause bacterial lyses. Lysozymes can be classified into at least five different classes: C
(chicken type), G (goose type), phage-type (T4), fungi (Chalaropsis), and bacterial
(Bacillus subtilis), but there are few similarities in the sequences of the different types of
lysozymes (Kamei et al., 1985; Nitta et al., 1988).
So far in many kinds of insects, such as tobacco hornworm, silkworm, soft tick, and
mosquito, lysozymes have been cloned and have shown significant similarity with
chicken type lysozyme. Their important role in innate immune system has been shown
in insect immune responses to pathogens (Mulnix and Dunn, 1994; Lee and Paul,
1995; Grunclová et al., 2003; Li et al., 2005; Kollien et al., 2003; Fujimoto et al., 2001;
Bedoya et al., 2005). Spatial expression of lysozyme showed that the higher lysozyme
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
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Archives of Insect Biochemistry and Physiology, January 2009
activity was present in the midgut and hemolymph than other tissues. In the wax
moth, Galleria mellonella, lysozyme expression is enhanced significantly after lava-topupa metamorphosis (Altincicek and Vilcinskas, 2006).
The cotton bollworm H. armigera (Hübner) is one of the most devastating pests in
Asia, Australia, and Africa. This polyphagous pest attacks a variety of crops, including
cotton, corn, tobacco, hot pepper, tomato, as well as kidney beans. However, there are
few reports about the immune related genes and their mode of action. Immunity is a
very important aspect of insect biology and understanding insect immunity is crucial to
developing improved biological control systems. In the present study, we cloned a
cDNA encoding a H. armigera lysozyme named HaLyz. By investigating its expression
level among the developmental stages, we found a link between the immunity and the
larva to pupa metamorphosis from the transiently enhanced expression level at the
onset of metamorphosis of the cotton bollworm, H. armigera. After treatment with
injection of Escherichia coli or entomopathogenic fungi, Beauveria bassiana, the gene
expression was up-regulated, but showed remarkably different patterns.
MATERIALS AND METHODS
Insects
H. armigera larvae were reared on an artificial diet as described by Wu and Gong (1997)
at 2770.51C and 490% R.H. with a light/dark photoperiod of 14:10.
cDNA Isolation and Sequencing
Total RNA was isolated from the whole larvae using Trizol (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions. In this study, 1 mg total RNA was used to
synthesize first-strand cDNA, using the Rever Tra Ace-a-TM kit (cat. no. FSK-100; Toyobo,
Osaka, Japan). Based on the two highly conserved amino acid sequences: WVCLIEN and
DDITKAS, a pair of degenerated primers was designed as follows: the forward primer 50 TGG GTN TGY CTN ATH GAR AAT-30 , and the reverse primer 50 -GA NGC YTT NGT
DAT RTC RTC-30 . PCR amplification of the target cDNA was performed in the reaction
mixtures containing 1 ml of the cDNA. PCR was performed using 35 cycles as follows:
941C, 30 s; 541C, 30 s, and 721C, 1 min. A 1.3-kb fragment was cloned into the T vector
(Takara, Tokyo, Japan) and then sequenced by ABI 3730 sequencer.
50 and 30 RACE
Rapid amplification of the 50 and 30 end of the full-length HaLyz was performed using
the GeneRacerTM Kit (cat. no. L1500-01; Invitrogen) according to the manufacturer’s
instructions. About 200 ng of the cotton bollworm mRNA was treated with calf
intestinal phosphatase (CIP) at 501C for 1 h to remove the 50 -phosphates. Tobacco acid
pyrophosphatase (TAP) was added to the reaction to remove the 50 -cap structure from
intact, full-length mRNA at 371C for 1 h. RNA oligo was ligated to the decapped
mRNA with T4 RNA ligase at 371C for 1 h. RNA oligo provides a known priming site
for the 50 RACE, with the sequence 50 -CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-30 . The cDNA was synthesized with Cloned AMV RT
reverse transcriptase in this kit through oligo (dT) primers: 50 -GCTGTCAACGATACGCTACGTAACGGCATGACAGTG (T)18-30 . The adaptor-ligated cDNAs were
subjected to PCR under the following conditions: 951C for 5 min, followed by five
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
Up-regulation of Lysozyme Gene Expression
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cycles at 941C for 30 s and 721C for 3 min, five cycles at 941C for 30 s, and 701C for
3 min, and 25 cycles at 941C for 30 s, 621C for 30 s, and 681C for 3 min. The reactions
were maintained at 681C for 10 min after the last cycle. The forward universal primer
was 50 -CGACTGGAGCACGAGGACACTGA-30 , homologous to the RNA oligo and the
reverse primer was 50 -CTCATTTTCTATCAGGCATACCCA-30 , designed on the
sequence of the fragment amplified by the degenerated primers as described above.
The 30 end was amplified using the forward primer 50 -AAGAACGGCTCCCGAGACTAC-30 , and the universal reverse primer was 50 -CGCTACGTAACGGCATGACAGTG30 . These PCR products were then ligated to the T vector (Takara, Tokyo, Japan) and
then sequenced in ABI 3730 sequencer.
Computer Analyses of cDNA
The amino acid sequence of lysozyme from H. armigera was deduced and analyzed.
Deduced protein sequences were analyzed using tools available at http://us.expasy.org/,
including translation, molecular weight, and the PI calculator program. Sequence
identities were verified using the BLASTp program. Clustalx 1.8 was used to align the
HaLyz (GenBank accession no. DQ493869) with known amino acid sequences in
GenBank: Trichoplusia ni (2211308A), Pseudoplusia includens (AAS48094), Heliothis
virescens (AAD00078), Spodoptera exigua (AAP03061), Hyphantria cunea (AAA84747),
Manduca sexta (AAB31190), Antheraea pernyi (ABC73705), Galleria mellonella (P82174),
Antheraea mylitta (Q7SID7), Bombyx mori (AAB40947), Aedes aegypti (AAU09087), Aedes
albopictus (AAM11885), Anopheles gambiae (AAC47326), Anopheles stephensi (BAC82382),
Anopheles darlingi (AAB61345), Musca domestica (2119303A), Drosophila melanogaster
(CAA80229), Bos taurus (U19466), Trichosurus vulpecula (U40664), Allenopithecus
nigroviridis (P79687), Mus musculus (BC054463), and Homo sapiens (U25677).
Phylogenetic analysis was performed using the neighbor-joining method. Bootstrap
confidence limit probabilities were estimated from 1000 replications.
Expression of HaLyz in E. coli and Western blotting
The HaLyz open reading frame (ORF) was amplified by PCR, using following primers:
50 -ATGC GGATCC G ATGCAGAAGTTAACTTTGTTCGTGG-30 (sense primer; italic
nucleotides indicate the location of a BamH I site); and 50 -ATCG CTCGAG
TAATACAAGTCTAGCAGTTGCTAAGG-30 (antisense primer; italic nucleotides indicate the location of an XhoI site). The PCR product was digested by BamH I/XhoI
(PROMEGE) and inserted into vector pET-41b (Novagen, Darmstadt, Germany)
with an N-terminal GST-tag. The expression vector was first transformed into E. coli
Top10 for molecular verification, and then transformed into E. coli BL21 for
expression. After sequencing and ORF confirmation of the inserted sequence, the
positive clone was grown in 30 ng/ml kanamycin-supplemented LB media to an OD600
of 0.6–0.8 at 371C and 250 rpm, and then induced with 0.4 mM IPTG for 4 h.
Expression of the HaLyz protein was measured by SDS-PAGE in Coomassie Blue G250
staining. Following SDS-PAGE, we transferred the membrane and blocked it for 3 h
after washing with TBST buffer, then incubated with a mouse monoclonal anti-GST
Tag antibody (Sigma, Salt Lake City, UT) 41C overnight, then treated with Goat antimouse IgG HRP (Sigma) for 45 min. Western blots were visualized by enhanced
chemiluminescence (Millipore, Billerica, MA).
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
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Archives of Insect Biochemistry and Physiology, January 2009
Quantitative RT-PCR Analysis of HaLyz Expression at Different Development Stages
Quantitative analysis of HaLyz expression was done by real-time PCR (RT-PCR). To
compare expression during the developing period, total RNA was isolated from 1st
instar, 2nd instar, 3rd instar, 4th instar, 5th instar, and 6th instar and early, middle, and
later pupae stages, as well as adults. After denaturing electrophoresis using 1.2%
agarose gel, total RNAs without degradations were used to synthesize the first-strand
cDNA by an oligo (dT)20 primer. RT-PCR was performed using the SYBR Premix EX
TaqTM kit (cat. no. DRR041S; Takara, Tokyo, Japan). First-strand cDNA synthesis was
primed with oligo (dT)20, using the ReverTra Ace-aTM kit (cat. no. FSK-100; Toyobo,
Osaka, Japan) and served as the template in 25 ml PCR assays. Specific primers for
HaLyz were designed to do the RT-PCR experiments. The forward primer was 50 AGATGAGGGATTGGGTATGCC-30 and the reverse primer was 50 -TGTCGTCTAGCAGCATTTCAGC-30 . H. armigera actA3a gene was used as an internal control for data
analysis and the primers are: 50 -TGCCCATCTACGAGGGTTACG-30 (forward) and 50 GCCGTGGTGGTGAACGAGTA-30 (reverse). PCR amplification and fluorescence
detection were performed using the Rotor-Gene Real-Time Analysis system under
the following thermal cycle conditions: 951C for 10 s, followed by 40 cycles of 951C for
5 s and 601C for 20 s. To reach reproducibility, each sample was performed three times.
The HaLyz expression level was calculated on the basis of the difference in the Ct value
in relation the actA3a transcripts, according to the instructions of the Rotor-Gene
software, version 6.0.19. Each time point was replicated three times using
independently collected samples from different larvae with an error bar 5 1 SD.
HaLyz Expression Analysis With the E. coli and B. bassiana Treatment by RT-PCR
Routine culturing of E. coli was carried out with Luria-Bertani (LB) medium in a 371C
shaking incubator (200 rpm). Before use in experimental challenge assays, overnight
cultures of E. coli were diluted 1:100 in distilled water, and numbers adjusted to 1.0 106 cells/ml.
B. bassiana strain Bb13 was kindly provided by the Research Center of
Entomopathogenic Fungi (Anhui Agricultural University, Hefei, China). This strain
was propagated on a 9-cm diameter Sabouraud dextrose agar yeast (SDAY) plate
covered with cellophane paper at 25 1; 2 ml of the conidial suspension of the
selected fungal species, adjusted to 1 106 conidia ml1.
In this study, 2 ml of E. coli or B. bassiana or distilled water (for control) was injected
into the hemocoel of newly ecdysed 5th-instar H. armigera larvae with a 10-ml syringe,
respectively. All treatments were injected at the dorsal side of the larvae abdomen. We
sampled the whole body of insects at various intervals of 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 h
after microbe injection. Since most of the treated larvae died over 72 h after injection, we
sampled until 72 h. Then we stored the samples immediately in liquid nitrogen for RNA
extraction. Total RNA was extracted using Trizol reagent (cat. no. 15596-018; Invitrogen)
according to the manufacturer’s protocol. RT-PCR was performed as described above.
Statistical Analysis
A multiple comparison test was conducted to compare the significant differences
between E. coli injection group and fungi group using Graph-Pad Prism, version 2.0
(GraphPad Software). A significance level of Po0.01 was chosen. Statistical analysis
was performed using analysis of one-way analysis of variance (ANOVA).
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
Up-regulation of Lysozyme Gene Expression
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RESULTS
Cloning and Characterization of HaLyz
To obtain the HaLyz cDNA, we designed one pair of degenerate primers from the
highly conserved amino acid sequences: WVCLIEN and DDITKAS. The primers were
used for RT-PCR, in which mRNA from whole larval body was the template. The
expected PCR product was cloned and sequenced. Specific primers derived from the
fragment were used for RACE to obtain both the 50 and 30 end sequences of the cDNA.
The novel nucleotide sequence reported here (HaLyz full-length cDNA, total 661 bp),
was constructed by assembly of these overlapping sequences, deposited in the
GenBank (NCBI sequence data bank) under accession number DQ493869. Figure 1
shows the nucleotide sequence and deduced amino acid sequence. This cDNA
contained an ORF of 408 bp between nucleotides 44 and 451, encoding 136 amino acid
residues. A predicted polyadenylation signal (AATAAA) was located 15 bp upstream of
the poly A tail. The molecular mass of the deduced HaLyz protein was predicted to be
15.6 kDa, and the calculated isoelectric point (pI) was 9.08.
Sequence Comparisons
Sequence analysis with the NCBI BLASTp program showed that the deduced
HaLyz protein of the cotton bollworm was highly similar to other insect lysozymes.
High sequence identity was found with these lysozymes: H. virescens (89%), S. exigua
(84%), T. ni (82%), and P. includens (82%). We also found significant similarity
with the lysozymes of some other insects, including H. cecropia (78%), H. cunea (77%),
A. mylitta (77%), M. sexta (77%), A. convolvuli (75%), A. pernyi (70%), B. mori (63%), and
A. aegypti (48%).
From the alignments of selected known lysozymes, it was seen that four highly
conversed cysteine residues (marked with asterisks) are involved in two of these
disulfide bonds. Identical amino acid residues were marked with dark background
(Fig. 2). Figure 3 shows the dendogram constructed by the neighbor-joining method
using amino acid sequences of known lysozymes. The statistical robustness of nodes in
the tree was verified by performing a bootstrap analysis of 1,000 resampled data sets.
The dendogram showed that three groups were clearly clustered as lepidopteran,
dipteran, and mammalian, respectively.
Expression of HaLyz in E. coli and Western Blotting
Induction of the correct recombinant clone with 0.4 m spells out M IPTG resulted in
accumulation of the protein with the molecular mass of 40 kDa (Fig. 4). There was an
N-terminal GST-tag in the pET-41b vector, and the size of the GST protein was
26 kDa. Thus, the weight of HaLyz protein corresponded to the calculated molecular
of 15.6 kDa. Western blot analysis with anti-GST antibodies also revealed the presence
of the protein at the expected size.
HaLyz Expression Level During Metamorphosis
Quantitative RT-PCR was performed to estimate the expression level of HaLyz at
different development stages as described in Materials and Methods. Dramatic
changes of HaLyz levels were detected in the six larval instars, different pupal stages,
and adults of the cotton bollworm (Fig. 5). The H. armigera actA3a was used as an
internal control. Comparative analysis results revealed that in the larval and adult
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
Figure 1. Nucleotide and deduced amino acid sequences of H. armigera lysozyme. Possible polyadenylation signal (AATAAA) and poly (A)1 tail are underlined. The
sequence was deposited in the GenBank with accession number DQ493869.
24
Archives of Insect Biochemistry and Physiology, January 2009
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
Figure 2. Alignments of conserved sequences of some lysozymes. Four highly conversed cysteines residues involved in two of these disulfide bonds are marked with
asterisks. Aa: Aedes aegypti; Ad: Anopheles darlingi; Ag: Anopheles gambiae; Al: Aedes albopictus; Am: Antheraea mylitta; An: Allenopithecus nigroviridis; As: Anopheles stephensi;
Bm: Bombyx mori; Bt: Bos taurus; Dm: Drosophila melanogaster; Gm: Galleria mellonella; Ha: Helicoverpa armigera; Hc: Hyphantria cunea; Hs: Homo sapiens; Hv: Heliothis
virescens; Md: Musca domestica; Mm: Mus musculus; Ms: Manduca sexta; Pi: Pseudoplusia includens; Se: Spodoptera exigua; Tn: Trichoplusia ni; Tv: Trichosurus vulpecula.
Up-regulation of Lysozyme Gene Expression
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
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Archives of Insect Biochemistry and Physiology, January 2009
Figure 3. Neighbor-joining dendogram showing the relationship among some known lyszymes. Analysis
was performed with multiple alignments from amino acid sequences using Clustalx 1.8. Aa: Aedes aegypti; Ad:
Anopheles darlingi; Ag: Anopheles gambiae; Al: Aedes albopictus; Am: Antheraea mylitta; An: Allenopithecus
nigroviridis; As: Anopheles stephensi; Bm: Bombyx mori; Bt: Bos taurus; Dm: Drosophila melanogaster; Gm: Galleria
mellonella; Ha: Helicoverpa armigera; Hc: Hyphantria cunea; Hs: Homo sapiens; Hv: Heliothis virescens; Md: Musca
domestica; Mm: Mus musculus; Ms: Manduca sexta; Pi: Pseudoplusia includens; Se: Spodoptera exigua; Tn:
Trichoplusia ni; Tv: Trichosurus vulpecula.
stages HaLyz expressed at a relatively low level, while in the early pupal stage the
expression increased more than four times and reached the peak at the middle pupal
stage, and then decreased in the later pupal stage and adult stages.
HaLyz Expression After E. coli and B. bassiana Treatment by RT-PCR
We carried out the gram-positive bacteria and fungi challenge assays by injection of E.
coli and B. bassiana into H. armigera larvae. We sampled at various intervals of 0.5, 1, 2,
4, 6, 12, 24, 48, and 72 h after microbe infection. HaLyz expression level was analyzed
by RT-PCR. The influence of bacterial and fungal challenge on relative lysozyme
expression is displayed in Figure 6. The HaLyz transcripts appear in expression
patterns differently (with a significance level Po0.01). The HaLyz transcripts increase
in response to the B. bassiana challenge was weaker and much slower than the
expression response to E. coli. After 12 h, the gene expression of HaLyz reached to its
highest level when treated with E. coli compared with that of control which increased at
a certain low level. However, the gene expression profiles of B. bassiana treatment
reached to the highest level on 24 h.
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
Up-regulation of Lysozyme Gene Expression
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Figure 4. Expression of H. armigera lysozyme and Western blotting analysis. Lane 1, Coomassie staining of
the induced protein without IPTG ; lane 2, Coomassie staining of the induced protein with IPTG; lane 3,
staining of the induced protein without IPTG the protein detected with anti-GST antibodies; lane 4, the
protein detected with anti-GST antibodies.
Figure 5. Relative quantification of lysozyme transcripts in different developmental stages of the cotton
bollworm, Helicoverpa armigera. L1–L6: 1st, 2nd, 3rd, 4th, 5th, and 6th instar larval stages; Pe: early pupal
stage, Pm: middle pupal stage, Pl: later pupal stage, A: adults stage. Each time point was replicated three
times using independently collected samples from different larvae. Error bar 5 1 SD.
DISCUSSION
We cloned the lysozyme full-length cDNA of the cotton bollworm H. armigera, by 50 and
30 Race. After investigating its expression pattern at the different developmental stages
by RT-PCR, we found that lysozyme has constitutively low expression in the six larval
instars. However, in the very beginning of pupal stage, the expression increases sharply
with about four times more expression than seen in the sixth instar. Expression reaches
a peak at the middle pupal stage and decreased in the later pupal stage and adult stages.
Lysozyme may protect Manduca sexta against bacteria that could be released into the
hemolymph during metamorphosis. The M. sexta lysozyme gene may be developmenArchives of Insect Biochemistry and Physiology DOI: 10.1002/arch
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Archives of Insect Biochemistry and Physiology, January 2009
Figure 6. Helicoverpa armigera expression was detected using RT-PCR analysis after E. coli and fungi B. bassiana
treatment with H2O injection as the control. They were sampled at various intervals of 0.5, 1, 2, 4, 6, 12, 24, 48,
and 72 h. Each time point was replicated three times using independently collected samples. Error bar 5 1 SD.
tally regulated as seen here (Russell and Dunn, 1991). A peak of lysozyme expression
occurs in late larvae/early pupae transition in many insects. In diptera, the Drosophila
LysX gene is expressed only in the midgut of late larvae and early pupae (Daffre et al.,
1994). The expression of lysozyme in hemimetabolous T. infestans is also up-regulated
immediately after the molt (Kollien et al., 2003). The immune response of antimicrobial
genes including lysozyme, gallerimycin, and the insect metalloproteinase inhibitor were
stimulated by metamorphosis in the wax moth, Galleria mellonella (Altincicek and
Vilcinskas, 2006). We found a link between the lysozyme and larval/pupal ecdysis
because the transiently enhanced expression level appears at the onset of metamorphosis of Helicoverpa armigera. However, whether it is due to the occurrence of bacteria
challenge in the larval gut tissue during metamorphosis or some other reasons is still not
clear. Although we speculate the transient increased in lysozyme expression is controlled
by a developmental mechanism, the point needs to be elucidated by further studies.
Lepidopteran c-type lysozyme is supposed to have two important roles in insect
defense mechanism. In M. sexta and B. mori, respectively, lysozyme can liberate
peptidoglycan layer of bacterial cell walls, and act as an elicitor for antibacterial protein
synthesis and secretion (Dunn et al., 1985; Iketani, 1993). Second, lysozyme also acts to
remove the bacterial cell, which is left after the action of cecropins and attacins (Boman
et al., 1991).
Lysozymes and some antimicrobial peptides involved in defense responses can be
induced by the microbial challenge. In this study, we confirmed the immune-inducible
nature of HaLyz. B. bassiana is a cosmopolitan entomopathogenic fungi with a diverse
host spectrum (Inglis et al., 2001), and has been widely used to manage pests. In the
two challenge assays HaLyz transcripts exhibited different expression patterns. The
HaLyz transcripts induce a weaker and slower response to the B. bassiana than that to
E. coli. There may be a couple of reasons: first, compared with E. coli, the fungi
propagate much slower; and second, lysozymes recognize and hydrolyze the b-1,4
glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in the
peptidoglycan layer of bacterial cell walls. As fungi have different chemistry at the out
cell walls, we may expect stronger reactions to E. coli than to fungi.
ACKNOWLEDGMENTS
The authors thank Prof. Xiangxiong Zhu for his suggestions on experiments
design.
Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
Up-regulation of Lysozyme Gene Expression
29
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Archives of Insect Biochemistry and Physiology DOI: 10.1002/arch
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expressions, metamorphosis, challenge, immune, lysozyme, bollworm, genes, armigera, regulation, cotton, helicoverpa
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