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Review	Pdf file on Synergy OPEN omments
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 trans�genesis 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抯 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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