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Acta Biochim Biophys
Sin 2008, 40: 601-611
doi:10.1111/j.1745-7270.2008.00432.x
From genome to proteome: great progress in
the domesticated silkworm (Bombyx mori L.)
Zhonghua Zhou, Huijuan Yang, and Boxiong Zhong*
College of Animal Sciences, Zhejiang
University, Hangzhou 310029, China
Received: April 25,
2008
Accepted: May 5,
2008
This work was
supported by the grants from the National Basic Research Program of China (No.
2005CB121003), the National Hi-Tech Research and Development Program of China
(No. 2006AA10A118), the National Postdoctoral Fund of China (No. 20070411197)
and the Doctoral Fund of the Ministry of Education of China (No. 20070335148)
*Corresponding
author: Tel/Fax, 86-571-86971302; E-mail, [email protected]
As the only truly domesticated insect, the
silkworm not only has great economic value, but it also has value as a model
for genetics and molecular biology research. Genomics and proteomics have
recently shown vast potential to be essential tools in domesticated silkworm
research, especially after the completion of the Bombyx mori genome
sequence. This paper reviews the progress of the domesticated silkworm genome,
particularly focusing on its genetic map, physical map and functional genome.
This review also presents proteomics, the proteomic technique and its
application in silkworm research.
Keywords genomics; proteomics; Bombyx mori
The mulberry silkworm, Bombyx mori, has been bred to produce
silk for more than 5000 years. There are millions of farms raising silkworms in
many countries, such as China, India and Thailand [1,2], as they have
commercial value and are an effective form of pest control [3]. Additionally, B.
mori has been used as an important bioreactor for the production of
recombinant proteins [4,5]. The economic and scientific significance of the
silkworm has made it the subject of intensive genetic studies since the 20th
century and the most important genetic model insect after Drosophila
melanogaster. Furthermore, the fields of genomics and proteomics have
developed, with particular progress having been made after the completion of B.
mori genome sequence. This review will concentrate on recent progresses in
silkworm genomics and proteomics.
Genomic Studies
Genetic maps
Genetic and molecular linkage maps provide a means of cloning genes,
tracking inheritance of traits of interest, finding transgene landing sites and
uncovering patterns of chromosome evolution. The first genetic map for the
silkworm was constructed in the early decades of the 20th century and used
genes as markers [1]. The classical linkage map for B. mori consisted
of approximately 240 visible and biochemical markers on 28 linkage groups with
an approximately 900 cM recombination length [6]. Although genes and biochemical markers are useful, they are not
ideal, given their limited numbers. Therefore, molecular markers have been
employed in constructing linkage maps, which were initially made using random
amplified polymorphic DNA (RAPD) or restriction fragment length polymorphic
(RFLP) markers [79]. However, these maps were of low-to-medium density and they
only contain few markers. Then a high-density linkage map was constructed for
the silkworm B. mori with an approximately 200 cM recombination length,
which contained 1018 RAPD markers on all 27 autosomes and the Z chromosome,
and an approximately 2 cM average interval [10]. More complete maps followed,
including a map constructed of 356 amplified fragment length polymorphism
markers [11], a map of 407 amplified fragment length polymorphism markers [12],
a map of RAPD and selectively amplified DNA fragments with 544 markers [13], a
map of RFLP markers with expressed sequence tags (ESTs) comprising over 200
markers [14], and a map of 518 simple sequence repeat (SSR) markers [15].
Further RAPD, SSR and fluorescent inter SSR (FISSR) markers were integrated
into the map construction of the Z chromosome [16]. Moreover, four markers from
the classical linkage map, og, w-1, Lp and Pfl,
were assigned
to the molecular linkage maps using sequence tagged sites (STSs), which attempted to fill the gap between molecular and classical linkage
maps [17]. To enable the sharing of reference markers and genetic resources, a
pair of inbred strains, C108 and p50 (also called Daizo in Japan or Dazao in
China), were used in many of these studies.
Physical map
Due to their low accuracy and resolution, genetic maps are rarely
sufficient for directing the sequencing phase of a genome project in most
eukaryotes; they must be checked and supplemented by alternative mapping
procedures. Lots of physical mapping techniques, such as restriction mapping,
fluorescent in situ hybridization (FISH) and STS mapping, have been
developed to address these problems.Manning and Gage reported a physical map of the DNA containing the
gene for silk fibroin, which was developed from direct hybridization analysis
of restriction endonuclease digests of total B. mori DNA using fibroin 125I-messenger RNA (mRNA) [18]. In addition, based on single
nucleotide polymorphisms (SNPs) between strains C108T and p50T initially
found on regions corresponding to the end sequences of bacterial
artificial chromosome (BAC) clones, Yamamoto et al constructed a
physical map composed of 534 SNP markers spanning 1305 cM distributed over
28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used
to construct the linkage map, 89 were associated with 107 different ESTs
[19]. Further, Yasukochi et al reported a physical map focused on Bombyx
sequences appearing in public nucleotide databases and BAC contigs. A
total of 874 BAC contigs containing 5067 clones (or 22% of the library) were
constructed by polymerase chain reaction-based screening with sequence
tagged sites derived from whole-genome shotgun (WGS) sequences. A total of 523
BAC contigs including 342 independent genes registered in public databases
and 85 ESTs were placed onto the linkage map. Yasukochi et al also
found significant synteny as well as conserved gene order between B.
mori and Heliconius melpomene in four linkage groups, and
proposed that B. mori could be used as a reference for comparative
genomics in Lepidotera [20]. Yamamoto et al mapped 1755 SNP markers from BAC end sequences
onto 28 linkage groups using a recombining male backcross population with
an average inter-SNP distance of 0.81 cM (or approximately 270 kb). The
integrated map contained approximately 10% of predicted silkworm genes and
had an estimated 76% genome coverage by BACs, which can provide a new
resource for improved assembly of WGS data, gene annotation and positional
cloning. This map will serve as a platform for comparative genomics and
gene discovery in Lepidoptera and other insects [21]. Song et al reported the chromosomal locations of two
single-copy genes, Ser-1 and CI-13, in B. mori by FISH.
The results showed that Ser-1 was located near the distal end of the
11th linkage group with a relative position of 12.51.4 in pachytene, while
CI-13 was mapped near the distal end of the second linkage group with
a relative position of 8.21.2 in pachytene [22].
Genome sequencing
The haploid genome size of B. mori, originally estimated at
530 Mb by Cot analysis, is approximately 2.5-fold the size of the D.
melanogaster genome (175 Mb) and 1.6-fold the size of the Anopheles
gambiae genome (280 Mb) [23]. Facilitated by both recent
advances in sequencing facilities and genome informatics applied to the Human
Genome Project, WGS sequence analyses have been completed in some key insects,
such as D. melanogaster [24] and Anopheles gambiae [25]. As
such, it was natural to adopt the WGS strategy for the B. mori genome
project. In 2004, Japanese and Chinese groups independently accomplished
the WGS sequencing in B. mori of 3 and 5.9?coverage, respectively [26, 27].In the Japanese WGS, 2,843,020 single-pass sequences were
constructed and then assembled into 49,345 scaffolds averaging 10 kb in length.
Based on the estimated genome size of 530 Mb, almost 97% of the genome, of
which 75% was sequenced, was organized into scaffolds. Furthermore, the
validity of the sequence was evaluated by carrying out a Basic Local Alignment
Search Tool (BLAST) search for 50 characteristic Bombyx genes and 11,202
non-redundant ESTs in a Bombyx EST database against the WGS sequence
data. Analysis of the WGS data revealed that the silkworm genome
contained many repetitive sequences with an average length of less than
500 bp. These repetitive sequences appeared to have been derived from
truncated transposons and were interspersed at approximately 2.5–3.0 kb intervals
throughout the genome, which suggested that the silkworm may have an
active mechanism that promotes removal of transposons from the genome. In
addition, the WGS data found that genome DNA fragments were homologous to
mitochondrial DNA at nine sites, which approved the incorporation of exogenous
DNA into the silkworm genome. Moreover, the search for Bombyx orthologs
to Drosophila genes controlling sex determination in the WGS
data revealed 11 Bombyx genes and suggested that the
sex-determining systems differ profoundly between the two species [25].While in the Chinese WGS, 4,903,289 single-pass sequences were
determined and assembled into 23155 scaffolds averaging 26.9 kb in length. The
WGS proved that, at 428.7 Mb, the genome size of B. mori was smaller
than the previously estimated size of 530 Mb. Almost 92.8% of the
genome was organized in scaffolds, of which approximately 85.2% has been
sequenced. In addition, the WGS found that the final corrected gene count for
the silkworm was 18,510 genes, far exceeding the official fruit fly gene count
of 13,379. However, the WGS data found that only 14.9% of predicted genes were
confirmed by ESTs, 63.1% were validated by GenBank non-redundant proteins and
60.4% were similar to fruit fly genes. In addition to the silkworm having more
genes than the fruit fly, it also has larger genes, which was discovered as a
result of the insertion of transposable elements in introns. The fact that
the silkworm has bigger and more genes than the fruit fly explains 86% of the
factors involved in the silkworm’s larger genome size. The rest of the factors
relate to the silkworm’s genes having slightly more exons than the fruit fly,
with a mean exons per gene ratio of 1:15 (and a median ratio of 1:12).
Comparative analyses between the domesticated silkworm and the fruit fly,
mosquito, spider and butterfly all revealed both similarities and differences
at genome level [27].
Functional genome
Despite the completion of the nucleotide sequence of the entire
silkworm genome and the achievement of many of the Silkworm Genome Project’s
declared aims, these successes are only the first step towards a functional
understanding of the silkworm’s genome. Functional genomics attempts, through
computer analysis and experimentation, to better understand the genome’s
contents, locate specific genes and determine their functions. Some of the
approaches involved in exploring the silkworm genome include as follows.
RNA interference (RNAi) RNAi was first reported in fungi as a phenomenon of
post-transcriptional gene silencing [28], which had been developed as a
powerful tool for gene-specific knockdown in many species, including silkworms
(B. mori). Combined with transgenic technology of virus infection [29], piggyBac
transposon plasmid or direct RNA injection [4,30], RNAi has been applied to
verify the functional role of specific genes, such as a transcription factor, BR-C
[29]; a ribonuclease inhibitor, BmRLI [31]; a argonaute2 homolog gene, BmAGO2
[32]; a lysosomal aspartic proteinase, BmCatD [33]; a baculoviral
immediate early-1 gene, ie-1 [34]; and an endogenous eclosion hormone
gene EH [35], in silkworms.Transgenesis Transgenesis
technology allows for the functional analysis of newly identified genes, but it
can also be used to produce specialized silks or value-added products, such as
recombinant proteins for pharmacological activity, or to improve productivity
and pathogen resistance in silkworms. piggyBac, a transposon discovered
in the lepidopteran Trichoplusia ni [36], has been confirmed as a valid
method to achieve silkworm transgenesis and has been employed to analyze
silkworm gene function over the past several years [4,36–40]. As the piggyBac
promoter used in early B. mori research, BmA3 cytoplasmic actin
drove the expression of a reporter gene, EGFP, as well as the piggyBac
transposase gene. However, this system had major drawbacks in that
transformation efficiencies, which ranged from 0.7% to 3.9%, was inefficient
and the expression of the fluorescent transgene was low relative to the high
background from vitellophages [4]. Then, the artificial promoter 3XP3, the Drosophila
heat shock 70 promoter Fib-L, and EGFP or other spectral
derivatives as reporters were introduced to overcome these shortcomings. So
these systems work well for egg injections and can be applied for many
functional studies, such as conditional knockouts and knockdowns via antisense
or double-stranded short interfering RNA constructs [37,38]. Furthermore, the GAL4/UAS system, a more effective tool for studying
gene and promoter function in vivo, was adopted in the silkworm research
[41,42]. The system relies on the generation of two transgenic lines that carry
an activator and effector, respectively. The activator expresses the GAL4 yeast
transcription factor under the control of promoter, whereas the effector
contains the GAL4-binding sequence linked to the gene of interest [43]. The
system’s transformation efficiencies ranged up to 17.7%, which makes it a
candidate for a wide range of functional genomics applications in the silkworm.
Additional methods, such as viral vectors [44–46], gun bombardment [47],
electroporation [48,49], and minos transposon [50,51], have been
developed and applied to the transgenesis in recent years.
EST EST has been proven as an effective
tool for discovering new genes?annotating unknown genes, generating gene expression profiles and performing
comparative genomics. To date, more than 180,000 ESTs from independent projects
are available in public databases (http://www.ncbi.nlm.nih.gov/Genbank).
The two largest EST projects were constructed by Mita et al. at the National
Institute for AgrobiologicalSciences in Tsukuba, Japan
(http://morus.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi/)
[52], and by Cheng et al at Southwest Agricultural University in
Chongqing, China (http://www.ncbi.nlm.nih.gov/UniGene/lbrowse2.cgi?TAXID=7091&CUTOFF=1000)
[53]. In addition, Zhong et al have developed the posterior silk gland
library at Zhejiang University in Hangzhou, China (http://www.ncbi.nlm.nih.gov/UniGene/library.cgi?ORG=Bmo&LID=15568)
[54]. Currently, almost all silkworm tissues, including Malpighian
tubules, Verson? glands, antennae, blood, brains, embryonic tissues, epidermises,
eyes, fat body, imaginal disks, maxillae, midguts, ovaries, pheromone glands,
prothoracic glands, silk glands and testes, have been involved in EST projects.
Moreover, EST projects have involved nearly all developmental stages of
silkworms, including the egg, embryo, larval, spinning, molting, pupa, newly
closed and adult stages. All EST projects have a policy to distribute
complementary DNA (cDNA)
clonesfor free, upon request, for any non-commercial use.
Serial analysis of gene expression (SAGE) SAGE is one of the more
versatile methods for functional genomics studies, as it has the ability
to detect and quantify the expression of large numbers of known and unknown
transcripts [55]. The SAGE technique works by isolating short fragments of
genetic information from the expressed genes, connecting these unique sequence
tags serially into long DNA molecules for sequencing, collecting
information from genes expressed in the tissue of interest, identifying
each gene expressed in the cell and the levels at which each gene is
expressed, and analyzing the differences in gene expression between cells
[56]. The technology has been used to study gene expression in a wide
range of organisms, including yeast, Arabidopsis thalianae, rice,
mice and humans [57,58]. SAGE has also been used to derive profiles of
expressed genes during the developmental life cycle [59], examine the profile
of expressed genes during embryonic development [60], identify genes involved
in cystoblast differentiation [61], and monitor the global gene expression
profile during larval development as well as larva-pupa metamorphosis [62] in
the silkworm.
DNA microarray Although
several technologies have been widely applied in functional genomics, DNA
microarray is still an excellent and high-throughput method for
large-scale expression measurements in silkworm due to its
cost efficiency, accessibility and standardized protocol [63]. DNA
microarrays rely on the ability of single strand nucleic acid
fragments to hybridize with high specificity to a second complementary
single strand and generate a double-stranded DNA molecule [65]. The
sample or target (ie, DNA, RNA or cDNA) is labeled
using either radioactive or fluorescent dyes that are hybridized to
the array surface [65]. This technology allows the simultaneous, quick and
efficient analysis of thousands of variables in a single sample and in a
simple hybridization experiment. Therefore, it has been applied
extensively to establish gene expression patterns of different organisms, such
as yeast, fruit flies and humans [66–68]. This approach was first used to isolate
an ecdysone up-regulated cuticle protein gene from wing discs of B. mori
in 2003 [69]. Subsequent investigators monitored the gene expression in
silkworm wing discs during metamorphosis using a cDNA microarray constructed
from over 5000 ESTs [70,71]. The microarray has also been used to identify
functional characterizations of BmADAMTS-1, BmADAMTS-like and
carboxypeptidase A in B. mori [72,73]. Earlier researchers used a microarray constructed with 2445 ESTs to
screen gene expression profiles during germ-band formation at six specific time
points in the early embryonic stage [74]. More recently, the technology was
applied in a similar way to determine the secondary structure of RNA [75,76],
explore the expression pattern of the chemosensory protein gene family [77],
identify Toll-related genes [78], verify elicitor efficacy of
lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression
[79], and investigate global gene expression profile during larval development
and larva-pupa metamorphosis [62]. Moreover, researchers designed and
constructed a genome-wide microarray with 22,987 70-mer oligonucleotides covering
the presently known and predicted genes in the silkworm genome and surveyed the
gene expression in multiple silkworm tissues on 3 d of the fifth instar [80].
Proteomic Studies
Proteomic technique
DNA acts like a blueprint of cell, while proteins are the dynamic
components. DNA or mRNA sequences cannot sufficiently describe the structure,
function and cellular location of proteins. Moreover, some important
functional, post-translational modifications, such as glycosylation and
phosphorylation, may not even be seen at the genome level. The term
“proteome” denoted as the entirety of proteins expressed by the
genome [81], was first introduced in the early 1990s, and since then, the field
of proteomics has attracted international attention. The technical achievements
of the past decade have driven proteomic analyses and have enabled
quantitative analysis of protein expression inside cells. Some useful proteomic
technologies will be reviewed as follows.
Two-dimensional gel electrophoresis (2-DE) In proteome research, 2-DE is a common separation technique to
examine the proteome of cells, cell lines, organs and tissues. The method
couples isoelectric focusing in the first dimension with sodium
dodecylsulfate-polyacrylamide gel electrophoresis in the second dimension
and enables the separation of complex mixtures of proteins according to
pI, Mr, solubility and relative abundance. Since the
2-DE technique was first implemented by O’Farrel [82] and Klose [83]
in 1975, it has had numerous developments, such as the invention of
IPG strips for different pH ranges [84–86], that have improved reproducibility and
have allowed for major breakthroughs in proteome research. Depending
on the gel size and pH gradient used, 2-DE can resolve several
thousand proteins simultaneously and detect a protein spot smaller than 1
ng. In addition, compared to LC-mass spectrometry(MS)/MS based methods, another
protein separation approach, 2-DE delivers a map of intact proteins with
no loss of molecular mass and pI information, and that can analyze proteins
that have undergone some form of post-translational modifications or
limited proteolysis. 2-DE also permits proteins to be isolated for further
structural analyses by matrix-assisted laser desorption/ionization (MALDI)-TOF/MS,
electrospray ionization (ESI)-MS or Edman micro-sequencing. Difference gel electrophoresis (DIGE) An important
improvement in the application of 2-DE was the introduction of DIGE by Unl et
al in 1997 [87]. DIGE avoided some basic problems encountered with 2-DE,
such as gel-to-gel variations and limited accuracy. In
DIGE-based proteomics, the experimental and control samples are
labeled with different fluorophores (Cy2, Cy3, or Cy5) and run in the same
gel, which can reduce spot pattern variability and the number of gels in an
experiment, shortening the time involved in this laborious procedure. Moreover,
DIGE covers a dynamic detection range of 3–5 orders of magnitude while
conventional 2-DE can only detect 30-fold changes [87–89]. However, one
significant shortcoming of DIGE is that proteins with a low percentage of
lysine residue may not be labeled as efficiently as than proteins with a high
percentage. Another potential drawback of the approach is that the
fluorophores, equipment and software are currently proprietary to GE
Healthcare, which may make its application cost prohibitive for some academic
labs. Biological MS Rapid advances
in biological MS have made proteomics a key technology in molecular cell
biology and biomedical research. MALDI and ESI represent the two predominant
ionization techniques in MS-based proteomics. MALDI is mainly used to volatize
and ionize simple polypeptide samples for MS analysis at high speeds [90],
while ESI-MS is usually used to analyze more complex peptide mixtures [91].
Therefore, MS-based proteome has primarily two analysis strategies: MS analysis
of substantially purified proteins and MS analysis of complex peptide mixtures
[92]. 2-DE is the classic proteomic approach to analyzing substantially
purified proteins. Although 2-DE provides unprecedented separation power for
proteins, this approach suffers several limitations, especially when compared
to the ability of MS to identify proteins in gel spots, including difficulties
in resolving proteins with extreme size, pI or hydrophobicity and in relation
to automation and reproducibility. The analysis of complex peptide mixture, or shotgun proteomics,
involves digested protein samples, the resulting peptides from which are
separated and subject to tandem MS analysis, and the proteins are
then identified by databases searching. The shotgun approach is advantageous
due to its conceptual and experimental simplicity, high-throughput,
increased proteomic coverage and more accurate quantification relative to
the 2-DE method. However, the shotgun method suffers from limited
dynamic range, informatics challenges related to inferring peptide and
protein sequence identities from the large number of acquired
mass spectra, a high redundancy and the enormous complexity of the
generated peptide samples [92,93].
Protein biochips Benefiting
from DNA microarray technologies and its application in genomics, protein
biochips have emerged as a possible protein-screening tool. In recent years, different
formats of protein biochips have been developed, including protein, peptide,
antibody/antigen, tissue, living cell, carbohydrate and small molecule arrays
[9496]. As a crucial tool for large-scale, high-throughput biology, protein
biochips technology has shown great potential for basic research, diagnostics
and drug discovery. It has been applied to analyze antibody-antigen,
protein-protein, protein-nucleic-acid, protein-lipid and protein-small-molecule
interactions as well as enzyme-substrate interactions. However, protein
biochips have several drawbacks, including the relatively large sample size
required, the unpredictable rate of protein degradation, and false positives
caused by nonspecific or multi-specificity binding [96].
Progress of silkworm proteomics
All the techniques mentioned so far have revolutionized
the ability to characterize the proteome in some model organisms,
especially in humans. However, silkworm proteomics are still in the developing
stages with research, primarily form China and Japan, focusing on a variety of
fields.Sample preparation Sample
preparation is the first important step towards successful 2-DE and
identification in proteomics study. Zhong et al established a sequential
extraction technique to prepare protein samples from the body wall of the fifth
instar larvae of the silkworm; the results have indicated that most species of
proteins could be obtained by this method [97]. Long et al reported a
robust approach in which the extract enriched in ESP and 30 KP was fractioned
and mixed with the re-extract of a residual pellet in an optimal proportion.
This new method improved the 2-DE pattern by increasing enhancement in spots by
one-third relative to the one-step method [98].Sample loading Rehydration
loading and cup loading are the most common methods for sample loading. In the
former method, the sample is mixed directly with rehydration buffer and loaded
during rehydration of the strip, whereas in the latter method, the sample is
applied after the strip rehydration step by face-up loading via a sample cup.
Long et al reported a novel procedure called droplet-tap mode, which was
devised for sample application in 2-DE expression profiles. The results showed
that the method resulted in significantly improved resolution, compared with
cup loading, when high concentrations of proteins were present [99]. In-gel digestion Although wet
gels are usually used for in-gel digestion after 2-DE analysis, dried gels are
easier to handle, less fragile and more suitable for long-term storage to avoid
contamination. Zhang et al compared the use of wet and dry 2-DE gels for
in-gel tryptic digestion and subsequent analysis by MS, and the results
confirmed that dry gels were also suitable for proteomic analysis [100].
Protein database Zhong
constructed a silkworm protein databank to facilitate better understanding of
gene expression and post-translational modifications. A total of 40 proteins
and their homology from silkworm body wall, fat body and middle
intestines were separated by 2-DE and determined by the N-terminal amino acid
sequencing method. The N-terminal sequences of 27 proteins were first found in
silkworms, and all these data were registered in Swiss-Prot through the
Internet [101]. Having benefited from vital techniques such as N-terminal
amino acid sequencing, MS-sequencing, WGS applied in B. mori and the
development of functional genomics, more proteins have been identified in the
domesticated silkworm. Although there is still no protein database specifically
for silkworms, more than 2400 proteins have been registered to date in protein
databases, such as NCBI (http://www.ncbi.nlm.nih.gov/),
Swiss-Prot (http://ca.expasy.org/sprot/)
and the Protein Information Resource (http://pir.georgetown.edu/).
Protein expression profile analysis
Fat body is the principal organ responsible for metabolic processing
of digestive products following absorption and for the storage and synthesis of
carbohydrates, proteins and lipids. Hou et al constructed a protein
expression profile for fat body from fifth instar of the silkworm with high
resolution 2-DE, in which a total of 722 spots were obtained, most of which
were distributed in the area from 15 kDa to 90 kDa with pI 4–8 [102]. Midgut is the chief locus of digestion, absorption and secretion of
digestive enzymes in the silkworm. The protein expression profile of midgut
from the fifth instar of the silkworm was constructed by 2-DE and showed that
over 600 spots were obtained, most of which were distributed in the area from
15 kDa to 80 kDa with pI 3.0–8.5 [103]. Hemolymph plays a very important role in transporting nutrients to
other tissues, eliminating metabolic wastes and protecting against harmful
microorganisms. Li et al utilized the proteomic approach to investigate
the proteome of the fifth instar hemolymph during growth and development. The
results showed that 241 protein spots were expressed at the beginning of the
fifth instar while 298 protein spots were expressed on 7 d of the fifth instar
[104]. The silk gland, an important organ that produces liquid silk for
cocoon fiber, is broadly divided into the anterior, middle and posterior parts.
Yan et al analyzed changes in protein expression patterns of the
posterior silk gland of the fifth instar from the p50 silkworm strain. The
study found that individual silkworms expressed proteins consistently
regardless of the part of the posterior silk gland used [105]. In addition,
some research has shown that protein expressions differ between the posterior
silk gland on 1 d and 4 d of the fifth instar, but that this difference is far
less conspicuous than that in EST expressions [106]. Likewise, some reports
have also confirmed that the proteins expression patterns of different parts of
the middle silk gland at different times were significantly varied [107,108]. Additionally, protein expression profiles of other tissues, such as
the colleterial gland [109], and at other stages such as embryonic stage were
also analyzed by 2-DE [110–112].
Functional proteomic analysis
Wang et al used 2-DE and MS to examine the effects of
lipopolysaccharide injections on changes in polypeptides in the hemolymph, fat
body and three portions of the midgut. The results showed that no polypeptides
were significantly induced in the midgut. In contrast, FB1 and H1-4
polypeptides, thought to be antitrypsin, serpin-2 protease inhibitors, novel
polypeptides and attacin antibacterial polypeptide, were significantly induced
in fat body and hemolymph. In addition, the results showed that all the
presence of induced polypeptides decreased at 48 h after the injection [113]. Zhong et al investigated the relationship between the 30K
protein family and the embryonic development of a temperature-sensitive,
sex-linked mutant strain of silkworm by 2-DE and MALDI-TOF/MS. The results
suggested that 30K proteins must have reasonable metabolism for an embryo to
develop normally [114].Zhang et al separated eight p25 isoforms of whole silk gland
protein by 2-DE and identified them by peptide mass fingerprinting. The results
indicated that the diversity of p25 isoforms depended on phosphorylation
modification in addition to glycosylation [115]. Zhang et al identified 93 silk gland proteins by 2-DE and
protein mss fingerprinting. These proteins were categorized into groups
involved in silk protein secretion, transport, lipid metabolism, defense
etc. The carotenoid-binding protein was confirmed by Western blot analysis
using its antibody, and multiple isoforms of L-chain and p25, some of
which contained varying amounts of phosphate residue as determined by
on-probe dephosphorylation, were found [116]. Li et al investigated the hemolymph proteome of the fifth
instar of the silkworm during its growth and development, identified some
proteins of interest, and discussed the relationship between these
proteins and the growth and development of silkworm [104]. Using high-resolution 2-DE and computer-assisted analysis, Jin et
al screened the secretory region of colleterial gland for protein
patterns during development to find the quantitative and qualitative
differences in protein expression during the pupae and moth stages. More
than 700 protein spots were observed in different developmental stages,
and three proteins were found to be expressed only in the later pupae stage and
moth stage. Furthermore, these proteins, especially actin, were not expressed
in the no glue mutant. The results indicated that actins participated in or
regulated the exocytosis of colleterial gland, while other differentially
expressed proteins might be related to colleterial gland development or the
secretion of a glue-like substance [117]. Hou et al used high-resolution 2-DE and computer-assisted
analysis to investigate quantitative and qualitative differences between the
middle and posterior silk glands. The results showed that there were
significant differences in spot distribution and expression between the glands;
some proteins identified from the posterior silk gland were related to heat
shock proteins, chaperones, redox system proteins, DNA replication proteins and
serpin proteins. In addition, two novel serpin proteins were identified in the
middle silk gland, which were presumed to be involved in regulating proteolytic
activity and preventing silk proteins from degradation [118]. Zhang et al produced the initial profile of the
intersegmental muscle proteins of the silkworm during larval-pupal metamorphosis.
In total, 258 protein spots were observed by 2-DE. Fifty-seven larval proteins
were identified; three of these were detected exclusively in larval samples.
Fifty-four other proteins were common in pupal samples; 12 of these belonged to
the contractile apparatus and their metabolism, regulation and signal
transduction were altered during metamorphosis from larvae to pupae. Three
pupa-defective proteins were identified as isoforms of troponin I and validated
by immunoblotting [119]. On the basis of morphological changes of silkworm during pupal
metamorphosis and the occurrence of a DNA ladder, Jia et al conducted a
comparative proteomic analysis to identify the proteins involved in the
programmed cell death (PCD) process. Among the approximately 1000 detected
reproducible protein spots on each gel, 43 were down-regulated and 34 were
up-regulated in the PCD process. MS identified 17 differentially expressed
proteins including some well-studied proteins as well as some novel PCD-related
proteins, such as caspases, proteasome subunit, elongation factor, heat shock
protein and hypothetical proteins. The results suggested that these proteins
may participate in the silk gland PCD process of B. mori and provided
new insights into this mechanism [120]. Chen et al identified some specific protein spots of silkworm
eggs at critical development II with proteomic approach. Of all 287 newly
expressed protein spots identified, five spots from four stages, juvenile
hormone-binding protein in the shortening stage, epidermal growth factor
receptor in the head thorax differentiation stage, larval cuticle protein and
accessory gland-specific peptide in the tubercle appearance stage, and amylase
respectively in the body pigmentation stage, were identified. The results
indicated that the larvae-relevant genes have been expressed logically in the
embryonic stages, which may be a preparation for larval life activity [121].
Conclusion
The accomplishment of the B. mori genome sequence has moved
the focus of biological research towards the functional analysis of the
genome and has catalyzed the emergence of proteomics, a new branch of
biological science focusing on proteins immediately relevant to
biological function. The technological achievements, such as transgenesis,
RNAi, DNA microarray, 2-DE and MS, have only emerged in the last decade. These
recent developments have enabled the quantitative analysis of the
DNA sequence, mRNA and protein expression inside cells, and have driven the
development of genomic and proteomic studies of the silkworm. New technologies
developed in proteomics or genomics, such as the shotgun approach, as well
as new researching strategies, such as system biology-base approach, have
provided new insight into the complex cellular processes in B. mori
and will continue to promote greater development of genomics and proteomics.
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