Genomics 87 (2006) 356 365

www.elsevier.com/locate/ygeno

Structures, regulatory regions, and inductive expression patterns of

antimicrobial peptide genes in the silkworm Bombyx mori
Tingcai Cheng, Ping Zhao, Chun Liu, Pingzhen Xu, Zhihong Gao, Qingyou Xia , Zhonghuai Xiang
The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Chongqing 400716, China
Received 8 July 2005; accepted 25 November 2005
Available online 9 January 2006

Abstract
Antimicrobial peptides (AMPs) are a group of immune proteins that protect the host from infection. In Drosophila, seven groups of inducible
AMPs have been identified, with activities against fungi and gram-positive and gram-negative bacteria. On the basis of the silkworm genome
sequence and expressed sequence tags, we identified 35 AMP genes, mostly belonging to the cecropin, moricin, and gloverin gene families. We
predicted the core promoters required for gene transcription and the cis-regulatory elements for NF-B/Rel and GATA transcription factors. The
expression profiles of these genes after an immune challenge with lipopolysaccharide were examined by reverse transcription PCR. Members of
the cecropin B and gloverin A subfamilies were intensely expressed in the fat body after induction. In contrast, those of the moricin B subfamily
were not expressed under the same conditions. Such results suggest that these regulatory elements and their positions in the upstream regions play
an important role in regulating the transcription of these defense genes.
2005 Elsevier Inc. All rights reserved.
Keywords: Antimicrobial peptide; Regulatory element; Gene family expansion; Insect immunity

Innate immunity in multicellular organisms is the first line of

defense against invading microbes such as bacteria, fungi, and
viruses. On the other hand, adaptive immunity endowed with
memory is restricted to most vertebrates, where it coexists with
innate immunity. Invertebrates lack an adaptive immune
system, yet their innate immunity effectively kills invading
microbes. They fight against foreign organisms by humoral and
cellular reactions such as antimicrobial protein (AMP) synthesis, nodule formation, and melanization [1]. In insects, AMPs
such as cecropins, defensins, proline/glycine-rich antibacterial
peptides, and antifungal proteins play an important role in
eliminating invaders. These peptides/proteins are synthesized
mainly in the fat body (equivalent of liver in mammals) and
secreted into the hemolymph (blood) to combat microorganisms
[2].
Since Bomans group purified P9A and P9B first from the
hemolymph of immunized pupae of Hyalophora cecropia [3],
a large number of AMPs have been identified from insect and
mammalian species. Typically, these molecules have low
Corresponding author. Fax: +86 23 68251128.
E-mail address: xiaqy@swau.cq.cn (Q. Xia).
0888-7543/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygeno.2005.11.018

molecular weight, high heat stability, good water solubility,

and broad-spectrum antimicrobial activity. In Drosophila,
seven distinct groups of AMPs (cecropins, drosocin, attacins,
diptericins, defensin, drosomycins, and metchnikowin) have
been identified and characterized by whole-genome microarray analysis [4,5]. Cecropins are active against grampositive and gram-negative bacteria as well as fungi, whereas
defensin kills gram-positive bacteria only [6]. In the
domesticated silkworm, Bombyx mori, cecropins are classified
into three subtypes, A, B, and D. Cecropins contain two
helices, and they act on gram-negative bacteria most
effectively [79]. Proline-rich lebocin, similar to abaecin
isolated from the honeybee, has weak antibacterial activity
under physiological conditions. When lebocin is glycosylated
or exists together with cecropin D, its antibacterial activity
becomes much higher [10]. Both attacin and moricin are
active against gram-negative bacteria. Moricin also kills
gram-positive bacteria. Attacin inhibits the synthesis of the
outer membrane protein of bacteria, whereas moricin
increases their membrane permeability [11,12]. Several other
antibacterial factors have been reported from B. mori,
including lysozyme and two lectins [13].

T. Cheng et al. / Genomics 87 (2006) 356365

B. mori is the only lepidopteran insect whose genome

sequence is available [14]. With 18,510 predicted genes, about
400 mutant lines, and a relatively large body size, B. mori serves
as a good model for genetic and biochemical study of insect
immune responses [15,16]. In this paper, we report the structure
and transcription regulation of AMP genes in the silkworm
genome and give a comparative analysis of their upstream
regions, which has revealed the possible relationships between
their regulatory elements and expression profiles.
Results
Families of AMP genes in the silkworm genome
A local BLASTP search of the silkworm genome database
identified 35 amino acid sequences similar to those of other
insect AMPs (Table 1). They include 12 moricins, 11 cecropins,
7 gloverins, 1 lebocin, 2 enbocins, and 2 attacins. Five of the
cecropin genes belong to the B subfamily, while 6 belong to the
A, C, D, or E subfamily. A cluster of 8 moricin genes was
identified by searching for its conserved sequence. Seven
gloverins fall into two classes and they are highly similar in
amino acid sequence to a lepidopteran antibacterial peptide
from Helicoverpa armigera (P81048). Apparently, AMP gene
ancestors underwent several rounds of duplication, giving rise
to these gene families in the genome. As a result, AMP genes
representing species-specific expansion may be crucial to innate
immunity, permitting finely tuned responses to multiple
pathogens.

Table 1
Antimicrobial peptide genes in silkworm
Name

Subfamily

Cecropins

EST

Tissue
(amount of ESTs)

Reference

[7]

Fat body (7),

testis (3)
Fat body (63),
testis (17),
hemocyte (9)

1
2
1
4

Fat body (2)

[9]

Moricins

C
D
E
A

Fat body (5),

testis (1),
hemocyte (1)

[12]

Gloverins

B
A

8
6

B
Attacins

1
2

+
+

Enbocins
Lebocin

2
1

+
+

Total

Number

35

Fat body (109),

testis (3),
silk gland (3),
midgut (3),
ovary (1)
Fat body (11)
Fat body (109),
testis (2),
hemocyte (1)
Fat body (9)
Fat body (13),
hemocyte (2)

[8]

[11]

[17]
[10]

357

Expressed sequence tags (ESTs) for AMPs

To test whether these AMPs are conceptual translates of
inactive genes, we searched the silkworm EST database
using the assembled nucleotide sequences as queries. With
the exception of BmcecC, BmcecE, and the moricin B
subfamily, all the AMP genes were expressed (Table 1).
The amounts of ESTs were distinctly different among the
gene families. Gloverin A families and attacins matched
well in more than 100 ESTs. AMP genes had a
characteristic tissue-specific expression, as was shown by
the analysis of ESTs, and most of them were shown to be
from the fat body and a few from the hemocyte, testis, and
other tissues.
New subfamilies or members of AMP genes
Cecropins
Among all the AMPs, cecropins are well understood from
insects to vertebrates. Initially discovered in Hy. cecropia
[18], cecropin is an alkaline peptide resisting gram-negative
and gram-positive bacteria and fungi. Later, five and three
cecropin genes were discovered in succession in Drosophila
melanogaster and Anopheles gambiae, respectively, and
categorized into four types, A, B, C, and D. In the
silkworm genome, six (BmcecA1 and A2, BmcecB1 and B2,
and BmcecD1 and D2) have been reported [9,13]. Another
five (BmcecB3, B4, and B5; BmcecC and BmcecE) are
newly discovered (Fig. 1). BmcecC is 70% similar in amino
acid sequence to Heliothis virescens cecropin C. BmcecE
has nucleotide and amino acid sequence identities of 62 and
66%, respectively, with BmcecA1. Three additional cecropin
genes are tandemly located on the same scaffold. The
clustering of cecropin genes has also been reported in D.
melanogaster and A. gambiae [19]. All these genes consist
of two exons and are conserved in the splicing sites.
Phylogenetic tree analysis indicates that the majority of
cecropins represent species-specific expansion of the gene
family (Fig. 1A).
Moricins
Moricin, a highly alkaline AMP, was initially isolated from
B. mori [12]. Twelve moricin genes have been identified, 3 of
which are similar to moricin1 (BmmorA1) and are thus
designated BmmorA2A4. The other 8 genes, located in the
genome tandemly, constitute a new subfamily named moricin B.
BmmorB1B8 are less than 20% similar in sequence to
BmmorA1 [12] (Fig. 2A). However, IntPro analysis has
shown that they are of the same type. Moricins of subfamily
B contain a high level of glycine residues (16.9%) (Fig. 2B).
Their nucleotide sequences in the coding region are highly
identical (N89.9%) (Fig. 2C). BmmorB1 and B2 reside in one
scaffold, whereas BmmorB3B8 reside in another. Comparison
of the coding regions further indicates that recent gene
duplications have given rise to BmmorB3, BmmorB4, and
BmmorB5 (Fig. 2D) and this gene cluster probably originated
from BmmorB1. These evolutionary relationships are further

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T. Cheng et al. / Genomics 87 (2006) 356365

Fig. 1. (A) Phylogenetic tree and (B) sequence alignment of insect cecropins. Amino acid sequences of cecropins from Manduca sexta, MscecB5 (D32021) and
Mscec6 (AAO74638); D. melanogaster, DmcecA1 (CAA34843), DmcecA2 (CAA34844), DmcecB (CAA34845), DmcecC (P29561), and Dmcec2 (NP_788767);
and A. gambiae, AgcecA (AAM82610), AgcecB (AAM82611), and AgcecC (AAM82612) are presented. The arrowhead indicates the possible signal peptidase
processing site, mostly between Ala and Ala.

supported by our analysis of the intron sequences (data not

shown).
So far, moricins have been found in only Lepidoptera. The
silkworm moricins contain 65 or 66 residues, the first 22 or 23
of which form the predicted secretion peptide. Mature
BmmorB1, 42 residues long, attacks gram-negative and grampositive bacteria by inserting its amino-terminal helix into the
bacterial plasma membrane to increase its permeability [20].
Gloverins
Gloverin, an inducible antibacterial protein, was first isolated
from the pupae of the giant silk moth Hy. cecropia. It is a basic,
heat-stable protein containing a large number of glycine
residues (18.5%) but no cysteine [21]. Gloverins interact with
lipopolysaccharide (LPS) and specifically inhibit the formation
of the outer membrane, which leads to increased permeability of
the membrane.

In the silkworm genome, there are at least seven gloverin

genes, of which six belong to subfamily A and one to
subfamily B (Fig. 3A). BmglvB is orthologous to gloverins
from other lepidopteran insects, while subfamily A has
probably resulted from species-specific gene expansion in
the process of adapting to various environments. As family
members of the glycine-rich antibacterial peptides, these
silkworm gloverins contain up to 15% glycine. Positions of
these glycine residues are highly conserved (Fig. 3B). The
signal peptidase and endopeptidase procession sites have
been verified in Trichoplusia ni [22].
Other AMPs
Two attacin genes are next to each other in the same scaffold,
similar to the attacin genes in the D. melanogaster genome.
There are at least two enbocin genes in the silkworm genome.
We have identified one lebocin gene, and other members of the

T. Cheng et al. / Genomics 87 (2006) 356365

359

Fig. 2. Phylogenetic tree and sequence alignment of moricins. (A) Phylogenetic tree showing expansion of the moricin gene family. Msmor (AAO74637), M. sexta
moricin. (B) Amino acid sequence alignment. The intron and signal peptidase procession sites are indicated by a diamond and an arrowhead, respectively. (C)
Nucleotide sequence alignment of moricin B subfamily. (D) Proposed order of expansion of moricin B subfamily. The orientations of the genes on two scaffolds are
indicated. Closest paralogs are connected with brackets.

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T. Cheng et al. / Genomics 87 (2006) 356365

Fig. 3. Phylogenetic tree and sequence alignment of gloverins from lepidopteran insects. (A) Evolutionary relationships. Haglv, He. armigera gloverin (P81048);
Msglv, M. sexta gloverin (AAO74639); Tnglv, T. ni gloverin (AAG44367). (B) Amino acid sequence alignment. Gloverins are glycine-rich antibacterial proteins. The
conserved glycine sites are indicated by short horizontal bars. The signal peptidase and endopeptidase procession sites are indicated by and , respectively.

gene family are probably located in the genome sequence gaps

[10].
Regulatory elements
The upstream regions of the AMP genes contain
sequence motifs similar to the binding sites of NF-B/
Rel and GATA transcription factors in mammals. Yamakawa and Tanaka [13] reviewed these two elements in
seven antibacterial peptide genes (BmcecA1, BmcecA2,
BmcecB1, BmcecB2, Bmatt1, Bmleb3, and Bmleb4). In
addition, CATTW is known as a common LPS-responsive
element in insects and mammals [23]. The adjacent B and
GATA boxes work cooperatively in the transcriptional
activation of the Drosophila antibacterial peptide gene
cecropin A1 [24].
Using GGGRAYYYYY, WGATAR, KKGNNCTTTY, and
CAATW as queries, we searched the upstream sequences of

29 AMP genes in the silkworm genome and identified

clusters of B-like, GATA, R1, and CATT motifs, whereas
the other 6 genes were not searched due to the fact that they
had no intact upstream regulatory sequences. Most of these
genes were found to contain the putative regulatory
elements. In the moricin B2 and B3 genes, GATA box
and B-like element were farther apart than those in other
AMP genes. Furthermore, we found that the consensus
sequence of GATA-like elements in moricin B genes was
NGATAM, instead of the WGATAR in the cecropin and
gloverin families (Fig. 4A). In addition to BmcecC, at least
one B-like element was present in the upstream region.
The B-like consensus sequences of the moricin and
cecropin families had markedly different 3 end sequences,
GGKWNNRRNR and GGRRANYMMC, respectively (Fig.
4B). We found that more than one of the CAATW boxes
matched perfectly in the upstream region of each AMP
gene, which probably mediates the start frequency of gene

T. Cheng et al. / Genomics 87 (2006) 356365

361

Expression patterns of AMP genes

In general, AMP genes are not expressed at high levels in
insect larvae under normal conditions, but are actively
transcribed upon bacterial infection. To compare the relative
abundances of AMP transcripts, we performed the semiquantitative reverse transcriptase polymerase chain reaction (RTPCR) to analyze total RNA samples of the fat body at different
times after LPS injection. In most cases, the band intensities
increased gradually with time, reached a maximum level 4
h after the immune challenge, and persisted up to 36 h or longer
(Fig. 6). In the control larvae injected with saline (lane 1), weak
signals were detected. Compared with LPS-injected treatment,
the transcripts of AMP genes were not rapidly and abundantly
produced after saline injection (data not shown).
As had been expected, some differences were present in the
expression profiles. For instance, the transcription of gloverin A
lasted much longer (from 1 to 4272 h) than that of gloverin B
(116 h). The expression of cecropin B was more intense than
that of cecropin A. BmcecE had a delayed and low level of
expression, compared with the cecropin A and B subfamilies.
BmmorA1 and Bmleb3 were expressed at a high level. On the
other hand, induced expression of moricin B was not detected
(data not shown). Gene regulation for moricins A and B
appeared to be different. Although expression of the attacin
gene and two enbocin genes was induced, their mRNA levels
were low.
Discussion

Fig. 4. AMP regulatory element logos. (A) Sequence logo of GATA elements
found upstream of the AMP genes. (B) Sequence logo of the B-like sequences
found upstream of the AMP genes. The height of the letters indicates the relative
frequency of the letter at that position and the overall height of the stack
indicates the sequence conservation in terms of information content in bits.
Logos were created using the WebLogo program available at http://weblogo.
berkeley.edu/ [26].

transcription [25]. Based on the R1 consensus sequence

(KKGNNCTTTY), similar regions were also found in the
upstream sequences of 14 AMP genes.
The variations in nucleotide and distance between the
motifs may lead to the change in gene expression pattern.
Therefore, the mismatches with consensus sequences were
tested (Fig. 5). Taking the B-like elements as examples, the
mismatches in moricin families were comparatively obvious.
The means of nucleotide variation in the three families,
cecropin, gloverin, and moricin, were 2.1/10, 2.3/10, and
3.7/10, respectively.

AMPs are the crucial component of insect innate immunity,

which combats invading microbes in the hemolymph. In this
work, we analyzed their sequences retrieved from the silkworm
genome database. In the silkworm genome, at least 35 AMP
genes exist, compared with 21 in D. melanogaster [6]. In
previous research, six types of AMPs (cecropin A, cecropin B,
cecropin D, lebocin, enbocin, and moricin) were identified as
purified proteins or cDNAs by differential display [13]. AMP
genes identified from the genome sequence can be divided into
six gene families. The structures and functions of cecropins are
best understood. We have confirmed 5 cecropin B genes in the
haploid genome, one gene more than was estimated by Taniai et
al. [8]. Two genes named BmcecA1 and BmcecA2 were
identified from the genome. A new gene named BmcecD2
was found, which, together with BmcecD1 isolated and
identified by differential display, constitutes the cecropin D
subfamily [9]. Another two genes, BmcecC and BmcecE, were
found for the first time.
The results from the sequence analysis of the cecropin and
moricin gene families are consistent with a model of sequential
gene duplications, potentially enabling diversified pathogen
defense. Moreover, adaptation to specific microbial environments may be reflected by expansion of certain AMP families.
For example, the moricin and gloverin gene families are
apparently unique of Lepidoptera. While such sequence
similarities allow identification of closely related genes through
comparative analysis, the same approach is not useful in the

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T. Cheng et al. / Genomics 87 (2006) 356365

Fig. 5. The B-like, GATA, and R1 elements in the upstream regions of 29 B. mori AMP genes. 500 nucleotides before and 100 nucleotides after the predicted
transcription start site (+1) are drawn to scale. Swallow-tailed arrows mark the translation start sites and numbers indicate the distance to the transcription start site.
Orientation of the elements is indicated. The number of mismatches is shown at the bottom of each arrow.

discovery of AMPs with low or no sequence resemblance. In

our search for AMP genes, we did not find any special
antifungal peptides.
Some AMP genes of B. mori, similar to those of D.
melanogaster and A. gambiae [27], have experienced major
family expansions. Many adjacent genes are very similar in
their nucleotide sequences. The expansion of gene families is
essentially produced by gene duplication. Unequal crossing
over may be an important mechanism for generating these AMP
gene clusters. Due to a high probability of misalignment, the

presence of these genes may have greatly increased the

opportunity of unequal crossing over and thus yielded larger
gene clusters during evolutionary processes [28]. While
sequence divergence following gene duplication may give rise
to new functions, concerted evolution could be crucial for
maintaining a basic biological role in defense, possibly
permitting finely tuned responses to multiple pathogens. Taking
the moricin family as an example, the family of moricin genes
may have sequentially duplicated. On the basis of sequence
changes, the last duplication may have occurred among

T. Cheng et al. / Genomics 87 (2006) 356365

Fig. 6. Time courses of AMP gene expression after LPS injection. Total RNA
samples were isolated from fat bodies at the indicated times (h) after LPS
injection. The control (C) was fat body RNA isolated 4 h after saline injection
from the larvae. RT-PCR products were separated by 1.2% agarose gel
electrophoresis. Actin transcripts were analyzed as internal control under the
same experimental conditions.

BmmorB3, BmmorB4, and BmmorB5, while the initial duplication may have occurred between BmmorB1 and BmmorB2. The
big cluster is likely to have derived from BmmorB1. If we adopt
the D. melanogaster neutral rate of 1.56 108 substitutions per
year [29], the mean sequence divergence 6.06% will imply that
the initial gene may date back to 1.9 MYA, thus suggesting that
the gene family is very young. Detailed study of the
evolutionary history by more sophisticated methods and
investigation of AMPs in the genomes of related species may
provide useful clues on how the silkworm genome has evolved.
An increase in mRNA level after immune challenge is
considered to be evidence that the corresponding gene is
involved in defense responses. The experimental evidence
indicates that transcription of the AMP genes is up-regulated.
For instance, the transcripts of gloverins and cecropins are
elevated after LPS injection. The fine differences in expression
profiles are probably caused by the upstream regulatory
sequences that bind transcription factors differently. Therefore,
it will be essential to identify experimentally the regulatory
elements of these genes. It is also important to identify
transcription factors involved in immunity. The transcription
factor BCFI of B. mori has a highly conserved zinc finger-type
DNA binding motif, which recognizes WGATA [30,31], thus
suggesting that the factor could be a member of the GATA

363

family of DNA-binding proteins. In comparison, factors

corresponding to vertebrate NF-B are less known in the
silkworm.
Based on the regulatory elements and gene expression
profiles, we attempted to explore the relationships between
them. Although the B-like and GATA sequences were found in
the upstream regions of moricin B genes, the distances between
B-like and GATA sequences were greater and the density was
lower than those in the other genes, which could have resulted
in the failure of transcription of the subfamily [24]. The
expression of AMP genes is regulated by a complex system
similar to the innate immune system in mammals. The process
from infection to elimination of pathogens needs the participation of multiple factors involved in recognition, signal
transduction, protein synthesis, and secretion [15,27]. Therefore, we should not only focus on the upstream regulatory
elements and their binding proteins, but also pay more attention
to the pattern recognition receptors and signal transduction
factors in the immune responses on the basis of the draft
sequence of the silkworm genome.
The roles of AMPs during immune responses need to be
more closely investigated. We have explored the AMPs and
compared their upstream regions involved in the gene
regulation of immune responses of B. mori. In this study, we
examined only the gene expression profiles in the fat body after
induction by LPS. We will be investigating the expression
profiles of these genes when they are induced by different
microorganisms, such as gram-positive and -negative bacteria,
virus, fungi, and Nosema parasites. Moreover, the first wholegenome oligonucleotide microarray, which will soon be
completed, will greatly facilitate the systematic comparative
analysis of the differences in expression profiles under
induction by different microbes and the identification of the
genes involved in immune response. It will help us to
understand the mechanism(s) of AMP gene expression
regulation. The research on silkworm antibacterial peptides
can contribute to medicine by leading to the development of
new antibiotics, to agriculture by establishing transgenic
antimicrobial strains of B. mori, and even to biocontrol of
pests in general in the near future.
Materials and methods
Identification of antibacterial peptide genes in the silkworm genome
The silkworm genome was sequenced by the whole-genome shotgun
method at the Beijing Institute of Genomics [14]. Genes were predicted using
BGF software [32]. To find AMP genes, a local BLASTP search was performed
using sequences from the Antimicrobial Peptide Database (http://aps.unmc.edu/
AP/main.php) as queries [33]. Since some AMPs are less than 20 residues long,
an E-value threshold was set at 102 for the searches. The resulting similar
sequences were examined manually.

ESTs of the silkworm AMPs

To determine the availability of ESTs for individual genes, a local
BLASTN search was performed against the silkworm EST database
downloaded from GenBank (http://www.ncbi.nlm.nih.gov/). The EST
sequences originated mainly from the tissues of silk gland, fat body,

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T. Cheng et al. / Genomics 87 (2006) 356365

hemocyte, testis, ovary, embryo, and midgut. The assembled exon sequences
of the AMP genes were used as queries for individual searches. The
availability of an EST was determined based on sequence similarity with
the query. A 90% or greater identity was considered to be an EST
corresponding to a specific gene.

Alignment and domain analysis

Sequences were aligned using Clustal X [34]. Based on the amino acid
sequences, phylogenetic trees were constructed by the neighbor-joining method
with options of bootstrap test. Bootstrapping was performed with 1000 trials.
The alignments were shaded using Boxshade (http://www.ch.embnet.org/
software/BOX_form.html) in such a way that identical residues were shaded
black and conserved ones were shaded gray [35]. The threshold fraction was set
at 0.5.

Search for promoters and regulatory elements

The core promoter regions of AMP genes were predicted by Neural
Network Promoter Prediction, a promoter-finding program designed on the
basis of a time-delay neural network (http://www.fruitfly.org/seq_tools/
promoter.html). A promoter score N0.8 was required for each gene [36].
Regulatory elements in the 500-bp upstream regions were predicted using
Find Pattern of the BioEdit package [37]. The searched sequences included
B-like (GGGRAYYYYY), GATA (WGATAR), and R1 (KKGNNCTTTY)
elements. Nucleotide positions were counted from the predicted transcription
start site (+1).

Maintenance of silkworms
The silkworm (Dazao) was reared on an artificial diet (Nihonnosanko) at
25C. The fifth-instar day 3 larvae were injected with 10 l of LPS (1 mg/ml in
150 mM NaCl). Controls were injected with saline solution. Fat bodies were
collected at different time intervals after the immune challenge and kept at
80C.

RT-PCR
For expression profiling, total RNA was extracted from the fat body
using TriPure Reagent (Roche Molecular; 1 ml/100 mg tissue). The RNA
samples were treated with RNase-free DNase I. RT-PCR was carried out
following the manufacturers instructions. All primers were designed based
on the genomic and EST sequences of silkworm AMPs. Primers Fa3 (5AACACCCCGTCCTGCTCACTG-3) and Ra3 (5-GGGCGAGACGTGTGATTTCCT-3) were used for amplifying silkworm actin-3 cDNA as an
internal control. The volume of each cDNA pool was adjusted to give the
same amount of the actin cDNA product in the exponential phase. PCR was
carried out in a 25-l reaction system containing normalized cDNA, 1.5
mM MgCl2, 50 mM KCl, 10 mM TrisHCl (pH 8.3), 0.25 mM each dNTP,
and 2.5 units of Taq DNA polymerase. After an initial denaturation at 94C
for 2 min, the cDNA fragments were amplified for 25 cycles at 94C for
30 s, 53C for 30 s, and 72C for 40 s, followed by a final extension at
72C for 10 min. PCR products were separated by gel electrophoresis,
recovered from the gel, and sequenced directly using an ABI 3100
automated sequencer.

Acknowledgments
We are grateful to Dr. Haobo Jiang (Oklahoma State
University, Stillwater, OK, USA) for his critical comments on
the manuscript. This work was supported by the National Basic
Research Program of China under Grant 2005CB121000 and by
the National Natural Science Foundation under Grants
30470991 and 30330460.

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