Comparative Proteomic Analysis of Posterior Silk Glands of Wild and

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Comparative proteomic analysis of posterior silk glands of wild and domesticated silkworms reveals functional evolution during domestication Jian-ying Li, Fang Cai, Xiao-gang Ye, Jian-she Liang, Jianke Li, Meiyu Wu, Dan Zhao, Zhen-dong Jiang, Zheng-ying You, and Bo-xiong Zhong J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017 Downloaded from http://pubs.acs.org on June 2, 2017

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Journal of Proteome Research

Comparative proteomic analysis of posterior silk glands of wild and domesticated silkworms reveals functional evolution during domestication ‖

Jian-ying Li†,‡, Fang Cai‡,¶, Xiao-gang Ye‡, Jian-she Liang§, Jian-ke Li , Mei-yu Wu‡, Dan Zhao‡, Zhen-dong Jiang‡, Zheng-ying You‡, Bo-xiong Zhong*‡

†

Institute of Life Sciences, College of Life and Environmental Sciences, Hangzhou Normal University,

Hangzhou 310036, China ‡

College of Animal Sciences, and §College of Environmental & Resource Sciences, Zhejiang University,

Hangzhou 310029, China ‖

¶

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Present Address: Research Center of Buckwheat Industry Technology, Institute of Plant Genetics and

Breeding, School of Life Sciences, Guizhou Normal University

* To whom correspondence should be addressed. Prof. Bo-xiong Zhong, College of Animal Sciences, Zhejiang

University,

Hangzhou

310058,

China.

Tel/Fax:

[email protected].

1

ACS Paragon Plus Environment

+86-571-86971302.

E-mail:


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ABSTRACT

The wild silkworm Bombyx mandarina was domesticated to produce silk in China approximately 5000 years ago. Silk production is greatly improved in the domesticated silkworm B. mori, but the molecular basis of the functional evolution of silk gland remains elusive. We performed shotgun proteomics with label-free quantification analysis and identified 1012 and 822 proteins from the posterior silk glands (PSGs) of wild silkworms on the 3rd and 5th days of the fifth instar, respectively, with 128 of these differentially expressed. Bioinformatics analysis revealed that with the development of the PSG, the up-regulated proteins were mainly involved in the ribosome pathway, similar to what we previously reported for B. mori. Additionally, we screened 50 proteins with differential expression between wild and domesticated silkworms that might be involved in domestication at the two stages. Interestingly, the up-regulated proteins in domesticated compared to wild silkworms were enriched in ribosome pathway, which is closely related to cell size and translation capacity. Together, these results suggest that functional evolution of the PSG during domestication was driven by reinforcing the advantageous pathways to increase the synthesis efficiency of silk proteins in each cell and thereby improve silk yield.

KEYWORDS: Bombyx mandarina; Bombyx mori; posterior silk gland; domestication; functional evolution; proteomics; label-free quantification; KEGG pathway

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INTRODUCTION The wild silkworm (WS), Bombyx mandarina M. is generally accepted as the ancestor of domesticated silkworm (DS), Bombyx mori L..1 The B. mandarina chromosome number varies within the species. It has 28 chromosomes per haploid genome (the same as B. mori) in China and 27 in Japan. B. mori has been domesticated in China for more than 5000 years and can still cross-breed with its wild relative B. mandarina and yield fertile hybrids.2-3 It now has become a model insect for Lepidoptera and has economic importance for silk production. During the domestication process, B. mori experienced intense artificial selection for increased silk production and has become completely dependent on humans for survival, from whom they receive mulberry leaves as feed.1 Artificial selection resulted in genetic variation and has enhanced some economically important traits such as silk production, growth rate and digestion efficiency.1, 4 However, the mechanisms by which the silk gland has enhanced its functionality during evolution by artificial selection remains elusive. The silk gland is the key silk-producing organ of the silkworm. Silk is composed of fibroin and sericin proteins. The gland is physiologically divided into three compartments: the anterior silk gland (ASG), middle silk gland (MSG), and posterior silk gland (PSG). The PSG is a unique sub-organ responsible for the synthesis and secretion of the silk core protein fibroin, which consists of a 350 kDa heavy chain (Fib-H), a 26 kDa light chain (Fib-L), and a 30 kDa P25 (fibrohexamerin) with a molar ratio of 6:6:1.5-8 The insoluble silk core protein fibroin is covered with hydrophilic glue sericin proteins that are synthesized in the MSG. Silk fiber comprises approximately 70-80% fibroin proteins in the inner layer and can be used for many purposes, including textiles, medical and industrial applications.9-10 Due to the economic importance for the silk industry, revealing the mechanism of silk production in B. mori has gained considerable attention, and a wide range of research has been conducted. For instance, 3



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we have characterized the differential expression of silkworm PSG during developmental stages at the transcriptional, translational, and post-translational modification (PTM) levels.11-17 Quantitative proteomic and transcriptomic analysis of PSGs between normal and low-yield silkworm strains revealed that down-regulated genes in the latter are mainly involved in the ribosome pathway.12-13 Lower silk production was ascribed to the decreased capacity for fibroin synthesis and the inefficiency of energy exploitation, along with weakened growth and development of the PSG.12-13 Considering the smaller size and lower silk production in wild silkworm compared to domesticated silkworm, more comparative analyses are needed to determine whether there is a similar variation during silk gland evolution. Comparative transcriptomic analysis of the silk gland between B. mori and B. mandarina showed that some up-regulated genes in the former were involved in the synthesis and secretion of silk proteins.18 Comparative methylomics between B. mori and B. mandarina found increased levels of methylated cytosines in domesticated silkworms and suggested possible epigenetic influences on silk gland enlargement.19 However, there is lack of direct evidence at the proteomic level to explain the phenotypic differentiation between the species. Long-term artificial selection and breeding have resulted in a much higher silk yield in B. mori than in B. mandarina.4 Molecular differences between them can reflect these processes. In this study, we performed shotgun proteomic analysis of the PSGs from Chinese B. mandarina on the 3rd and 5th days of the fifth instar and conducted an in-depth comparison with previously reported data from B. mori.11 The differential expression analysis between different developmental stages and between different species will contribute to the understanding of the mechanisms of silk production and the functional evolution of silk glands during domestication.

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MATERIALS AND METHODS Sample collection and protein extraction. Wild silkworms were collected from the farmland of Tongxiang, Zhejiang Province, China. They have a chromosome number of 2n = 56, which is the same as that of domesticated silkworm strains such as P50.20 PSGs on the 3rd day (I5D3) and 5th day (I5D5) of the fifth instar were dissected from 5 silkworms for each experimental group in cold PBS. The fibroin in the PSG lumen was removed according a previously described method.11, 21 The dissected PSGs at each stage were mixed together for the following analyses. Protein extraction was performed as previously described.22 Briefly, lysis buffer (containing 2.5% SDS, 10% glycerin, 5% β-mercaptoethanol, and 62.5 mM Tris-HCl pH 6.8) was added to the sample for grinding. The homogenate was centrifuged at 4 °C and 20,000 × g for 20 min after sonication. The supernatant was aliquoted and stored at -80 °C. The protein samples were quantified using the 2-D Quant Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. Protein separation and digestion. A total of 200 µg proteins for each sample was separated by 12.5% 1D SDS-PAGE gel under a constant current of 12 mA for 2.5 hours. The gels were stained with Coomassie Brilliant Blue (CBB) after electrophoresis, and the stained gel band was cut into 12 slices. These slices were subjected to in-gel digestion separately as previously described.22 Briefly, the denatured proteins were reduced with 50 mM Tris[2-carboxyethyl] phosphine (TCEP, Sigma-Aldrich, Saint Louis, MO, USA) and alkylated by 100 mM iodoacetamide (IAA, Amersham, Piscataway, NJ, USA). The proteins were subsequently digested with 20 ng/µL porcine trypsin (modified proteomics grade, Sigma-Aldrich) overnight at 37 °C. The resulting tryptic peptides mixture was extracted with 5% trifluoroacetic acid (TFA, Fluka) in 50% acetonitrile (ACN, Fischer Scientific) and dried using a SpeedVac system (SPD111VP2, Thermo Scientific). 5



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Nano-LC_MS/MS analysis. The protocol for LC-MS/MS analysis of wild silkworm PSG proteins was the same as previously reported for domesticated silkworm PSG.11 Briefly, the re-dissolved peptide pellets were applied to an Ettan MDLC nanoflow/capillary LC system (GE Healthcare, Pittsburgh, PA) coupled to a linear ion trap Orbitrap (LTQ-Orbitrap) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The peptides were firstly passed through a C18 trap column (PepMap C18, 300-µm i.d. ×5 mm, 3 µm, 100 Å (P/N 160454), Sunnyvale, CA) for desalting and pre-concentration with buffer A (5% ACN, and 0.1% formic acid in water) for 7 min at a flow rate of 10 µL/min. The peptides were then transferred on an analytical column (PepMap C18, 75-µm i.d. ×15 cm, 3 µm, 100 Å (P/N 160321), Sunnyvale, CA) and eluted with a 65-min gradient of buffer B (84% acetonitrile, 0.1% formic acid in water) from 5% to 60%, and a 10-min gradient from 60% to 95% and 15 min of holding at 95% with a constant 300 nL/min flow rate. The LTQ-Orbitrap, in

nanospray configuration, was operated in

data-dependent acquisition mode with XCalibur software version 2.0 (Thermo Electron, San Jose, CA, USA). Full MS scans (m/z 300‒1600) were acquired in the Orbitrap with the resolution R= 60 000 at m/z 400. Collision-induced dissociation (CID) was conducted with a normalized collision energy of 35% for MS/MS acquisition. The full scan mass spectra were collected in profile mode, and MS/MS data were collected in centroid mode. Charge state screening was enabled, and precursors with an unknown charge state or a charge state of 1 were excluded. The five most intense ions were isolated for CID fragmentation and measured in the linear ion trap with the following dynamic exclusion settings: repeat count 2, repeat duration 30 s, exclusion duration 180 s. Triplicate technical replicates were performed for each sample. Protein identification and quantification. The MS/MS raw data were submitted to an in-house Mascot server for database searches using the Mascot Daemon software (version 2.3, Matrix Science, London, 6


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U.K.). The in-house database consisted of 14 623 predicted proteins from genomic sequences and the reference protein sequences downloaded from NCBI (released on Jan 20, 2010; 1739 entries).11 The protein database was supplemented with a list of common contaminants retrieved from The Global Proteome Machine website (http://www.thegpm.org/crap/index.html). A concatenated target-decoy database search strategy was adopted for protein identification.

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To make comparisons with the

proteomic data from B. mori PSG, the protein identification and quantification processes were the same as previously described.11 Briefly, parent and fragment ion mass tolerances were set at 50 ppm and 0.6 Da, respectively. Trypsin was used to cleave the peptides, and two missing cleavage sites were allowed. A fixed carbamidomethyl alkylation of cysteines and a variable oxidation of methionine were specified. At least 2 identified peptides were required for each protein identification. The database search results of each experimental group with triplicate technical replicates were subjected to Trans-Proteomic Pipeline (TPP)

software

(v4.0

JETSTREAM

rev

2,

http://tools.proteomecenter.org/wiki/index.php?title=Software:TPP) for further peptide and protein validation by PeptideProphet23 and ProteinProphet24 algorithms, with probability thresholds at 0.7 and 0.9, respectively. The final protein identification list was manually validated by excluding the common contaminants and decoy hits and reducing redundant proteins with multiple shared peptides. Stringent parameters and validation processes were adopted to keep the false discovery rate (FDR) at a low level (below 0.5%) in all experiments.11 The relative expression levels of the proteins identified by LC-MS/MS were evaluated with a label-free quantification method, Absolute Protein Expression (APEX).26-28 The APEX score of each protein was computed according to the following formula27:

7



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‫ܺܧܲܣ‬௜ =

݊ ‫݌‬௜ ቀ ௜ ቁ ‫݋‬௜

ே

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×‫ܥ‬

The APEX technique is based on a spectral counting method in which the total observed spectral count for protein i (ni) is normalized by a computationally predicted or expected count (Oi) for one molecule of protein i. The computed values are weighted by the protein identification probability (pi). The relative APEX score of a protein is computed by dividing by the sum of the values for all proteins being quantified and then multiplying the normalization factor (C). The APEX abundance estimates of a protein within a sample are normalized and can be readily compared with those of other samples.26-27 Bioinformatics analysis. Gene Ontology (GO) analyses of the identified proteins were carried out with Blast2GO software (version 3.2),29 using a BLASTp search against the NCBInr database (e-value < 1e-10), followed by mapping and annotation (e-value < 1e-10). Pie graphs of biological process terms were generated by node score level filter 2 or 3. KEGG pathway enrichment analyses were performed by a hypergeometric statistic test with a web server KOBAS 2.0 (http://kobas.cbi.pku.edu.cn/).30 The protein sequences were blasted against the Drosophila melanogaster (fruit fly) database. The Benjamini and Hochberg FDR correction was used to correct the probability values, and only those corrected values with P < 0.05 were considered statistically significantly enriched biological pathways. The chromosome localization of the genes that mapped to the protein identifications were analyzed by

the

Scaffold

Sequence

Search

with

tBlastn

program

in

KAIKObase

(http://sgp.dna.affrc.go.jp/KAIKObase).31 The chromosome locations were obtained from the best matches of the proteins in BLAST results (e-value < e-10). 8


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RESULTS AND DISCUSSION Proteome identification of B. mandarina posterior silk glands. There are obvious differences between B. mandarina and B. mori, including their body size and silk yield. For example, the cocoon shell weight of the wild silkworm used in this study was significantly lower than that of domestic silkworm strain P50 (Fig. 1). Although the proteome of B. mori silk glands has been well studied,11-17, 32 it is difficult for B. mandarina proteomics identification because there is little protein annotation in the available databases. In this study, the MS data were subjected to searches against the previously used database containing the known and predicted protein sequences of B. mori.11 This greatly improved the identification of B. mandarina proteome due to the evolutionary homology between the wild and domesticated silkworm. Ultimately, we identified 1012 and 822 proteins with FDR lower than 0.5% from WS-I5D3 and WS-I5D5, respectively (Fig. 2, Table S1). Gene Ontology analysis for the B. mandarina PSG proteome. The functional categories of the identified proteins were analyzed with Blast2GO software based on their GO annotations. The common proteins in wild silkworm PSGs at I5D3 and I5D5 were classified into several GO categories based on the biological processes with which they are involved (Figs. 2A and 3A). The top three enriched terms were transport, translation, and protein localization. There were 351 and 161 stage-specific proteins that were also classified into different biological process terms with statistical score distribution (Figs. 2A and 3B, C). The specific proteins in PSG at I5D3 were mostly involved in signal-organism cellular processes, gene expression and intracellular transport (Fig. 3B). In contrast, the specific proteins in PSG at I5D5 were overrepresented in the GO terms for oxidation-reduction process, translation, and signal transduction (Fig. 3C). 9



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KEGG pathway analysis for the B. mandarina PSG proteome. To obtain an overview of the function of the wild silkworm PSG proteome, the protein identifications were used to search the KEGG pathway database using KOBAS 2.0 (http://kobas.cbi.pku.edu.cn/). The proteins in PSG at I5D3 were significantly enriched in the pathways for carbon metabolism, proteasome, protein processing in endoplasmic reticulum, citrate cycle (TCA cycle), RNA transport, biosynthesis of amino acids, and 2-Oxocarboxylic acid metabolism (corrected P-value < 0.05; Fig. 4). In contrast, the proteins in PSG at I5D5 were significantly enriched in the pathways of carbon metabolism, proteasome, ribosome, TCA cycle, biosynthesis of amino acids, protein processing in endoplasmic reticulum, protein export, and valine, leucine and isoleucine degradation (corrected P-value < 0.05; Fig. 4). It is likely that the common pathways such as the TCA cycle, whose involved enzymes are primarily covered by the PSG proteins as showed in Fig. 4B, were primary for the normal physiological function of the PSG. The major difference was that RNA transport-related proteins were enriched in the earlier stage, whereas the ribosome-related proteins were enriched in the later stage (Fig. 4). Proteomic differences of PSGs between B. mandarina and B. mori during developmental stages. To get quantitative information about the B. mandarina proteome, differential expression of the shared proteins between I5D3 and I5D5 was analyzed with a label-free quantification method. We found 51 significantly up-regulated and 77 down-regulated proteins expressed at I5D5 compared with I5D3 (fold change >= 2, P < 0.05; Figs. 2A, D, Table S2). The up-regulated proteins were enriched in the ribosome pathway, whereas the down-regulated proteins were enriched in the aminoacyl-tRNA biosynthesis and proteasome pathways. In contrast, we previously identified 57 up-regulated and 154 down-regulated proteins expressed in B. mori PSG at I5D5 compared to I5D3 (fold change >= 2, P < 0.05).11 Similar to the current results, the up-regulated proteins were mostly enriched in pathways of ribosome, protein 10


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export, oxidative phosphorylation, and protein processing in the endoplasmic reticulum (corrected P-value < 0.05). The down-regulated proteins were enriched in RNA transport and proteasome (corrected P-value < 0.05). These data indicate that the PSGs are engaged in transcription at the earlier stage and in translation at the later stage to meet the requirement of fast growing and robust fibroin synthesis from I5D3 to I5D5. To investigate the molecular changes of PSG during silkworm evolution, we compared the PSG proteomes of B. mandarina with that of B. mori at the two developmental stages. We found 532 proteins shared by both B. mandarina and B. mori (Fig. 5A). To determine whether the functional differences were related to differential expression, we did pair-wise proteomic comparisons of WS-I5D3 vs. DS-I5D3, WS-I5D5 vs. DS-I5D5, WS-I5D3 vs. WS-I5D5 (Fig. 2, Table S2-4), and with DS-I5D3 vs. DS-I5D5, as we previously reported 11. We screened a total of 342 proteins with significantly differential expression (fold change >= 2, P < 0.05). The proteins with differential expression in each pair were clustered using gene clustering software and viewed with Java TreeView software. The results showed that B. mandarina and B. mori clustered into two different branches (Fig. 6). There were 762 shared proteins in WS-I5D3 and DS-I5D3, the expression of which included 129 down-regulated and 59 up-regulated proteins in the latter compared with the former (Figs. 2B, E; Table S3). Compared to WS-I5D5, there were 124 down-regulated, and 35 up-regulated proteins expressed in DS-I5D5 (Figs. 2C, F; Table S4). Quantitative analysis revealed that during PSG development from I5D3 to I5D5, there were 19 up-regulated and 26 down-regulated proteins identified both in B. mandarina and B. mori (Fig. 5B). Furthermore, the amplitude of molecular variation during the developmental stages may reflect the different phenotypes of wild and domesticated silkworms. We found 4 up-regulated and 3 11



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down-regulated proteins that had large differences in abundance between B. mandarina and B. mori (differential levels >= 3-fold) (Table S2).11 The up-regulated proteins included ribosomal proteins S9, L18, S24, and ADP-ribosylation factor. For example, in B. mandarina, the ribosomal protein S9, which is required in the early steps of ribosome biogenesis and important for the silk protein synthesis,21, 33 was up-regulated approximately 3 times from I5D3 to I5D5, whereas it up-regulated approximately 24 times in B. mori. Thus, the differential level was up to 8-fold between the species. Furthermore, ribosomal proteins S9, and S24 are also considered important for ribosome biogenesis and are related to sericin yield in MSG.21 In contrast, among the down-regulated proteins, eukaryotic translation initiation factor 3 (BGIBMGA013025-PA) was down-regulated approximately 8 times in B. mandarina, whereas it down-regulated approximately 2 times in B. mori (Table S2).11 The variable amplitudes of translation-related proteins during this process to some extent reflects the different capacity for protein synthesis between the two species. It suggests that the dramatically enhanced protein synthesis capacity and prolonged translational process may in part account for the higher silk yield in B. mori compared to B. mandarina. We also compared the expression of fibroin proteins, including Fib-L and P25, in B. mandarina and B. mori. Label-free quantification analysis revealed that the expression of P25 was down-regulated at I5D5 and was much lower than Fib-L in both B. mandarina and B. mori (Fig. 7). In contrast, the expression of Fib-L was up-regulated both in B. mandarina and B. mori at I5D5 (Table S2).11 Unexpectedly, the expression of L-chain was much higher in B. mandarina than it was in B. mori both at I5D3 and I5D5 (Fig. 7, Table S4). However, we did not identify Fib-H both in B. mandarina and B. mori.11 The absence of Fib-H might have been due to the limitation of gel-based proteomic methods in the separation of high molecular weight proteins and to its simple-repeat-sequence-containing region 12


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that lacks trypsin cleavage sites, thus making it difficult to identify. Fibroins are the major components of silk threads and are composed of Fib-H, Fib-L, and P25, which are organized in a molar ratio of 6:6:1 in domesticated silkworm PSG.8 The protein-coding genes for fibroin did not show significantly differential expression between the wild and domesticated silkworms.18 It was therefore thought that transcriptional regulation or positive feedback of secretion may account for the differences in the silk protein biosynthesis. Furthermore, differential expression of fibroin proteins between the low- and high-yield domesticated silkworm strains was also not obvious, although the protein-coding genes were differentially expressed.12 Together, these data indicate transcriptional and translational modifications during fibroin protein synthesis, and suggest that the relative abundance of fibroin proteins is indirectly related to the silk yield. The higher silk yields in domesticated compared to wild silkworms may primarily due to the larger cell size and stronger protein synthesis capacity of silk glands in the former that leading to a higher total amount of fibroin proteins. Alternatively, considering the diversity among species for the molar ratio of fibroin components and their expression,34-35 it may suggest that the molecular organization of fibroin proteins in wild silkworm PSG may be different from domesticated silkworm. Bioinformatic analysis of the differentially expressed proteins between wild and domesticated silkworms. The differentially expressed proteins between B. mandarina and B. mori at I5D3 and I5D5 were subjected to KEGG pathway analysis. The proteins with differential expression in PSG at I5D3 between wild and domesticated silkworm were significantly enriched in the pathways of aminoacyl-tRNA biosynthesis and ribosome (corrected P-value < 0.05; Fig. 8A). The proteins with higher expression in wild silkworm were enriched in aminoacyl-tRNA biosynthesis, protein processing in the endoplasmic reticulum, and biosynthesis of amino acids (corrected P-value < 0.05). In contrast, 13



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the up-regulated proteins in domesticated silkworm were enriched in the ribosome pathway (corrected P-value < 0.05). The differentially expressed proteins in PSG at I5D5 between wild and domesticated silkworm were significantly enriched in the pathways of ribosome, carbon metabolism, aminocyl-tRNA biosynthesis, and biosynthesis of amino acids (corrected P-value < 0.05; Fig. 8B). The more abundant proteins in wild silkworm were enriched in carbon metabolism, aminoacyl-tRNA biosynthesis, and biosynthesis of amino acids (corrected P-value < 0.05). In contrast, the up-regulated proteins in domesticated silkworm were enriched in the pathways of ribosome, protein processing in endoplasmic reticulum, and protein export (corrected P-value < 0.05). Taken together, the more abundant proteins in wild silkworm at the two stages were enriched in biosynthesis of aminoacyl-tRNA and amino acids, whereas those in domesticated silkworm were primarily enriched in protein synthesis, processing and export. Activation of the ribosome pathway indicates that the enhancement of ribosome biogenesis is an urgent requirement for the fast-growing and robust silk protein synthesis in PSG from I5D3 to I5D5.11 Ma et al. utilized the GAL4/UAS system to overexpress the Ras1CA oncogene specifically in PSG and significantly improved silk yield due to the increased cell size and stimulated ribosome biogenesis for mRNA translation.36 Ribosome biogenesis involves synthesis, processing, and assembly of ribosomal proteins, and controls cell growth and proliferation.37 Ribosomes are the molecular factories for protein synthesis and are essential for every living cell. The reinforced ribosome pathway in B. mori PSG at the very stage that vigorous silk gland growth and silk protein synthesis occurs is the resultant of functional evolution after long-term artificial selection. This advantageous pathway may have contributed to the increase of silk yield in B. mori compared to B. mandarina. Pathways of aminoacyl-tRNA biosynthesis 14


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and biosynthesis of amino acids give rise to aminoacyl-tRNA synthetases (ARSs) and amino acids, respectively. ARSs are key components for protein synthesis because they link speciﬁc amino acids to their cognate tRNAs. Although these two pathways are enriched in B. mandarina, providing abundant translational “material,” the lower efficiency of protein synthesis compared with B. mori may be attributed to the insufficient of “machines”—ribosomes. Screening candidate domestication genes by differential expression analysis. Artificial selection during domestication of B. mori improved important economic traits such as cocoon size and silk yields by altering the expression of some important genes that are considered domestication genes1. Previous studies have identified 354 protein-coding genes as candidate

domestication genes by large-scale

genome sequencing of B. mori and B. mandarina.4 Four genes were found to be enriched in domesticated silkworm silk glands including silk gland factor-1 (Sgf-1), BGIBMGA005127 (homologous to the Drosophila sage gene), BGIBMGA000158 (ecdysone oxidase), and BGIBMGA013304 (a predicated gene without annotation).4,

38

However, these genes were not identified in wild or

domesticated silkworm PSGs.11 This may be due to the different sub-organ location of these genes. Fox example, ecdysone oxidase (BGIBMGA000158-PA) has been identified specifically in the anterior MSG of domesticated silkworm with high-level expression.21 In this study, we compared these candidate domestication genes with PSG proteome identifications in B. mori and B. mandarina, and totally found 11 overlaps. To further identify domestication genes in PSG, we did pair-wise comparison between WS-I5D3 vs. DS-I5D3 and WS-I5D5 vs. DS-I5D5 (Fig. 5B). We identified a total of 50 overlapping proteins with significant differential expression in the two groups that might be related to domestication. Compared with B. mandarina, there were 43 down-regulated and 7 up-regulated proteins expressed in PSG of B. mori both at I5D3 and I5D5 (Fig. 5B, Table 1). 15



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Page 16 of 43

Interestingly, the predicted lysine--tRNA ligase isoform X2 (BGIBMGA002984-PA) and ribosomal protein L7, which were the products previously identified as candidate domestication genes,4 were expressed significantly higher in B. mandarina than in B. mori both at I5D3 and I5D5 (P < 0.05, fold change >= 2). The down-regulated proteins were primarily involved in aminoacyl-tRNA biosynthesis, carbon metabolism, biosynthesis of amino acids, and the pentose phosphate pathway. In contrast, the up-regulated proteins were involved in the ribosome pathway. In addition to the ribosome proteins, there were two up-regulated proteins: DNA supercoiling factor precursor (BGIBMGA001107-PA) and predicted UDP-glucosyltransferase protein 3 isoform X1 (BGIBMGA010289-PA). DNA supercoiling factor (SCF) is a protein capable of generating negative supercoils in DNA in conjunction with topoisomerase II that was first identified from the PSG of B. mori.39 It has been demonstrated that in Drosophila, SCF plays a role in transcriptional activation via the alteration of chromatin structure, which facilitates transcription elongation on chromatin.40 Insect UDP-glucosyltransferases (UGTs) catalyze the conjugation of many small lipophilic compounds with sugars to produce glycosides, playing an important role in the detoxification of xenobiotics in the diets of insects. Here, we identified two UGTs that had been considered to be tissue-specific genes expressed specifically in silk glands.41 UDP-glucosyltransferase protein 3 was expressed significantly higher in B. mori than in B. mandarina at both stages (Table 1). In contrast, another UGT uridine diphosphate glucosyltransferase (BGIBMGA004965-PA, gi|197209932) was expressed significantly more highly in B. mandarina than in B. mori at I5D3 (Table S3). The enhanced expression of UDP-glucosyltransferase protein 3 in B. mori might be attributed to its function in the metabolism and absorption of dietary flavonoids, which are closely associated with the green coloration of cocoons (Fig. 1).42-43 The higher expression of uridine diphosphate glucosyltransferase in B. mandarina might be due to its function in the glucosylation of 16


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flavonoids, which increase the UV-shielding activity of cocoons and thus may increase survival of the insects contained in these cocoons.42 However, surprisingly, the differential expression of these two proteins between B. mandarina and B. mori was similar to that between the low- and high-yield domesticated silkworm strains that were previously identified (Table 1).12-13 These differences reflect the different diets and survival environment between wild and domesticated silkworms and may be related to the silk yield. Our previous studies have performed comparative proteomic analysis of the PSGs between lowand high-yield domesticated silkworm strains at I5D3.12-13 To investigate whether they share similarities with the differentially expressed proteins between B. mandarina and B. mori, we did comparisons and found 26 proteins with differential expression were consistent with that between low- and high-yield silkworm PSGs. Of these proteins, 11 were on the list of our screened candidate domestication genes (Table 1). For instance, the phosphoserine aminotransferase 1, one of the key enzymes for serine biosynthesis that is involved in anti-stress responses of insects,44 was expressed with higher abundance in wild silkworm and low-yield silkworm.12-13, 45 Chromosome localization for the genes encoding differentially expressed proteins. Artificial selection has resulted in massive selective sweep of genetic diversity on chromosome 10 for adaptation during maize domestication.46 To determine whether domestication has predominant effects on the individual chromosome(s), we mapped the differentially expressed proteins between B. mandarina and B.

mori

to

the

chromosomes

with

the

tBlastn

program

in

KAIKObase

(http://sgp.dna.affrc.go.jp/KAIKObase/). There were 87 down-regulated and 210 up-regulated proteins in the PSG of B. mandarina compared with B. mori at the two stages. These proteins were mapped to 82 and 199 genes, respectively. These genes were mapped to 28 chromosomes (Fig. 9A). The results did 17



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Page 18 of 43

not show excessive aggregation of the differential genes on individual chromosomes. The location of screened 50 candidate domestication genes were also distributed in different silkworm chromosomes (Fig. 9B). This suggests that artificial selection did not have an effect on specific chromosome(s) during silkworm domestication.

CONCLUSIONS

The domestication of silkworm, which produces silk material for clothes, has greatly contributed to human civilization. Long-term artificial selection and breeding have impelled silkworms to adapt to new conditions and modify a series of traits towards the phenotypes that meet human needs. Domesticated silkworms differ from their wild ancestors in morphology, physiology, development and behavior, which are controlled by a variety of genes. For the first time, we performed proteomic analysis on wild silkworms. By comparative proteomic analysis of wild and domesticated silkworms, we screened numerous candidate domestication-related proteins that will contribute to better understanding of the evolutionary mechanisms of silk glands in silkworm. Domestication with intense artificial selection significantly improved silk yield by enhancing some advantageous pathways such as the ribosome pathway to increase cell size and translation capacity, rather than by directly increasing the gene expression of silk proteins. This leads to a relatively lower abundance but a higher total production of silk protein in B. mori compared to B. mandarina. Furthermore, the more comfortable living environment decreased the expression of proteins for anti-stress responses, thus saving more energy for silk protein synthesis in domesticated silkworm. In other words, there is a complex network to improve

18


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silk yield by increasing protein synthesis that was the strategy adopted by silkworms during the functional evolution of silk glands.

ACKNOWLEDGEMENTS We thank Dr. Gang Meng, in Ankang University, for his kind help in sample preparation. This work was supported by the grants from the National Basic Research Program of China (No. 2012CB114601), and China Postdoctoral Science Foundation (2016M591989).

ASSOCIATED CONTENT.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

Supplemental Tables Table S1. Proteomic identification of posterior silk glands of wild silkworm at the 3rd and 5th days of the fifth instar.

Table S2. Differentially expressed proteins of the posterior silk glands of wild silkworm during developmental stages.

19



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Page 20 of 43

Table S3. Differentially expressed proteins of the posterior silk glands between wild and domesticated silkworms at the 3rd day of the fifth instar.

Table S4. Differentially expressed proteins of the posterior silk glands between wild and domesticated silkworms at the 5th day of the fifth instar.

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TABLES

Table 1. Proteins with significantly differential expression between B. mandarina and B. mori both at I5D3 and I5D5 APEX Scores ACC

APEX Scores

Changes

DESC WS-I5D3 phosphoribosylformylglycinamidine

BGIBMGA000390

Fold

synthase

DS-I5D3

(WS/DS)

Fold Changes

WS-I5D5

DS-I5D5

(WS/DS)

101.28

38.04

2.66

19.78

6.14

3.22

77.03

10.02

7.69

85.08

12.74

6.68

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, BGIBMGA000897

mitochondrial

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

BGIBMGA001037*

leucine--tRNA ligase, cytoplasmic LOW QUALITY PROTEIN:

BGIBMGA001525

tyrosine--tRNA ligase, cytoplasmic

BGIBMGA001558

isoleucine--tRNA ligase, cytoplasmic

BGIBMGA002508*

pyruvate kinase-like isoform X1 LOW QUALITY PROTEIN:

BGIBMGA002840

translational activator GCN1

BGIBMGA002984

lysine--tRNA ligase isoform X2

BGIBMGA003361*

importin-5 coatomer protein complex subunit

BGIBMGA003892*

beta 2 isoform X2

BGIBMGA003977*

serine--tRNA ligase, cytoplasmic tRNA

BGIBMGA004408

(cytosine(34)-C(5))-methyltransferase bifunctional glutamate/proline--tRNA

BGIBMGA005116

ligase isoform X2 bifunctional glutamate/proline--tRNA

BGIBMGA005117

ligase isoform X2 uncharacterized protein

Page 28 of 43

197.46

20.18

9.78

57.12

12.11

4.72

110.31

19.73

5.59

52.65

16.72

3.15

242.14

12.37

19.57

63.79

11.80

5.41

86.09

23.95

3.59

74.71

21.75

3.44

152.40

23.36

6.52

40.94

20.07

2.04

75.61

18.03

4.19

26.25

6.11

4.29

545.93

116.52

4.69

146.59

63.09

2.32

185.05

74.47

2.48

86.99

23.37

3.72

248.79

17.77

14.00

151.14

39.59

3.82

75.19

14.16

5.31

41.22

14.40

2.86

197.84

14.15

13.98

44.64

13.19

3.38

192.77

60.34

3.19

69.71

21.91

3.18

41.22

11.34

3.63

16.51

2.86

5.77

BGIBMGA005965

C05D11.1-like

BGIBMGA006816

allantoinase isoform X2

97.03

10.75

9.02

24.91

2.49

10.02

BGIBMGA007119

adenylosuccinate lyase

98.06

21.58

4.54

72.01

20.58

3.50

BGIBMGA007637

glycine--tRNA ligase

532.50

80.48

6.62

344.98

68.20

5.06

BGIBMGA007728

cytosolic non-specific dipeptidase

102.11

38.26

2.67

75.96

32.43

2.34

BGIBMGA008551*

hypoxia up-regulated protein 1

257.56

44.40

5.80

96.95

38.57

2.51

BGIBMGA008930

arginine--tRNA ligase, cytoplasmic

88.42

18.07

4.89

54.81

15.32

3.58

BGIBMGA009978

phosphoprotein 1

144.75

16.57

8.74

115.57

6.99

16.52

16.26

5.51

2.95

7.43

2.33

3.18

nucleolar and coiled-body

LOW QUALITY PROTEIN: dynein BGIBMGA010475

heavy chain 2, axonemal-like

28


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


coatomer protein complex subunit BGIBMGA010725

alpha puromycin-sensitive

BGIBMGA011674*

aminopeptidase-like isoform X1

BGIBMGA011695

nucleoprotein TPR

BGIBMGA012659

putative acetyl transferase phosphatidylinositol transfer protein

359.86

93.33

3.86

142.49

47.72

2.99

167.35

46.42

3.60

62.25

31.06

2.00

42.87

11.24

3.81

14.88

2.17

6.87

214.38

67.48

3.18

141.40

70.18

2.01

291.23

51.90

5.61

210.63

21.96

9.59

BGIBMGA012960*

alpha isoform

gi|112982800

ribosomal protein L4

683.24

276.79

2.47

914.45

331.87

2.76

gi|112982844


465.86

117.10

3.98

764.91

278.61

2.75

gi|112983370

transport protein Sec61 alpha subunit

663.03

133.79

4.96

575.43

252.52

2.28

gi|112983926

arginine kinase

264.51

85.69

3.09

229.56

105.90

2.17

gi|112984224

alanyl-tRNA synthetase

467.11

136.50

3.42

182.71

80.77

2.26

gi|112984404


493.56

141.24

3.49

1092.29

516.16

2.12

158.94

56.86

2.80

103.46

38.14

2.71

translation initiation factor 2 gamma gi|112984508

subunit

gi|114051239*

cystathionine gamma-lyase

135.91

12.15

11.18

81.09

7.72

10.50

gi|114051596

adenosine kinase

230.87

45.05

5.13

145.72

41.35

3.52

gi|114052561

cytosolic malate dehydrogenase

303.30

64.51

4.70

263.22

47.21

5.58

111.36

39.83

2.80

80.54

20.63

3.90

phosphoribosyl pyrophosphate gi|114052675

synthetase

gi|114052677*

phosphoserine aminotransferase 1

353.74

74.43

4.75

270.89

92.51

2.93

gi|114052783

serine hydroxymethyltransferase

233.85

106.88

2.19

152.21

45.29

3.36

gi|114053253

6-phosphogluconate dehydrogenase

104.84

23.44

4.47

56.87

9.93

5.73

gi|148298685

fructose 1,6-bisphosphate aldolase

256.78

105.63

2.43

250.71

70.05

3.58

BGIBMGA001107

DNA supercoiling factor precursor

141.30

327.05

0.43

28.64

151.17

0.19

6.28

44.93

0.14

4.84

89.47

0.05

BGIBMGA010289*

UDP-glucosyltransferase protein 3

29



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isoform X1 BGIBMGA013567


318.30

706.70

0.45

37.98

252.85

0.15

gi|112983505

ribosomal protein S10

200.38

585.36

0.34

362.30

966.77

0.37

gi|112983932

ribosomal protein S27

66.25

214.61

0.31

200.26

923.29

0.22

gi|112984158


114.70

492.34

0.23

139.36

389.40

0.36

gi|112984292


122.47

500.67

0.24

394.78

1018.25

0.39

The proteins that have been identified differentially expressed in the PSGs of low- and high-yield silkworm strains are marked with asterisks.12 The products of candidate domestication genes that have been identified are highlighted in bold.4 The differential expression levels of proteins in different groups were judged from the ratios of APEX scores (fold changes ≥ 2 or ≤ 0.5; Z-test, p-value < 0.05).

FIGURE LEGENDS

Figure 1. Comparison of wild and domesticated silkworms.

(A) On the left is the wild silkworm, and on the right is the domesticated silkworm at I5D3. (B) The dissected silk glands of wild (the left) and domesticated (the right) silkworms at I5D3. The white short lines mark the cut-points between the ASG, MSG, and PSG. (C) The cocoons of wild silkworm (the left) and the domesticated silkworm strain P50 (the right). (D) Comparison for the weight of cocoon shell of the wild silkworm (WS) and the domesticated silkworm (DS) strain P50. Ten cocoons were collected for each species, and Student’s t test was performed to analyze their shell weight differentials. **, P < 0.01. Scale bars, 1 cm in A-C. Photograph courtesy of Bo-xiong Zhong. Copyright 2017.

Figure 2. Label-free quantification analysis of wild and domesticated silkworm PSG proteomes. 30


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(A-C) Venn diagram of identified proteins in each set of comparisons. The red area indicates the number of up-regulated proteins, and the green area shows down-regulated proteins. White indicates the proteins without significant differential expression between comparisons. The differential expression in comparison pairs (panels A-C) with APEX score scatter plots are shown in panels D-F, respectively. Red dots and blue blocks represent the proteins with and without significantly differential expression, respectively (P < 0.05). The regression line divides the up- and down-regulated proteins. WS, wild silkworm; DS, domesticated silkworm; I5D3, the 3rd day of the fifth instar; I5D5, the 5th day of the fifth instar.

Figure 3. Gene Ontology analysis of the proteomes of wild silkworm PSGs at I5D3 and I5D5. (A) The biological process category of the shared PSG proteins at I5D3 and I5D5; (B) The biological process category of I5D3-specific proteins compared with I5D5; (C) The biological process category of I5D5-specific proteins compared with I5D3. The pie charts were plotted with Blast2GO software based on the annotation scores of the proteins at GO level 3.

Figure 4. Pathway enrichment analysis of the proteome identifications of the wild silkworm PSG. (A) The top 10 enriched pathways of the identified PSG proteins at I5D3 (red bar) and I5D5 (green bar) are shown. The asterisks mark the significantly enriched pathways of the identified proteins against the reference database (Background, blue bar). Pathway enrichment analysis was performed with the KEGG Orthology-Based Annotation System (KOBAS) by a hypergeometric statistic test. Categories with the corrected p < 0.05 were considered to be significantly enriched pathways. (B) A representative enriched pathway form KOBAS. Green boxes are the matched protein references of fruit fly (D. melanogaster) in 31



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the KEGG PATHWAY database, and the red boxes indicate the protein entries mapped to the significantly enriched pathway.

Figure 5. Venn diagram of the proteome identifications of PSGs in wild and domesticated silkworms during developmental stages. The proteome identifications of PSGs of wild and domesticated silkworms at the I5D3 and I5D5 were pair-wise qualitatively (A) and quantitatively (B) compared. The comparisons in panel A were based on the protein identification lists in Supplemental Table 1 and previous data.11 In panel B, the upper part shows the significantly up-regulated proteins in each pair-wise comparison, and the lower part indicates the down-regulated proteins (P < 0.05). The left panel shows the similarity of differentially expressed proteins from I5D3 to I5D5 between wild and domesticated silkworms. The right panel indicates the similarity of differentially expressed proteins between wild and domesticated silkworms at I5D3 and I5D5. The dark red and dark green areas show the common proteins with up- and down-regulation between the pair-wise comparisons, respectively.

Figure 6. TreeView analysis of the differentially expressed proteins between wild and domesticated silkworm PSGs. The differentially expressed proteins in pair-wise comparisons of wild and domesticated silkworms at I5D3 and I5D5 were clustered with Gene Cluster 3.0 and viewed with Java TreeView software. The heat map on the upper left indicates the differential expression levels. The relative expression of representative proteins evaluated with APEX scores is shown in the right panel.

32


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Figure 7. Quantitative analysis of the expression of fibroin proteins in wild and domesticated silkworm PSGs. The relative expression of fibroin proteins, including Fib-L and P25, in different pair-wise comparisons was evaluated with APEX score. The two asterisks indicate significantly differential expression (P < 0.01). Fib-L, fibroin light chain; P25, fibrohexamerin.

Figure 8. Top 10 enriched KEGG pathways of differentially expressed proteins between the wild and domesticated silkworm PSGs. (A) Enriched pathways of the differentially expressed proteins between wild and domesticated silkworm at I5D3; (B) Enriched pathways of the proteins with differential expression between wild and domesticated silkworm at I5D5. The pathway enrichment analyses were performed with a web server KOBAS 2.0. The asterisk indicates the significantly enriched pathway (corrected P-value less than 0.05) against the reference database (Background). The number of identified proteins (red) is a part of the background (green).

Figure 9. Chromosome localization of the genes encoding differentially expressed proteins between wild and domesticated silkworm PSGs.

(A) The chromosome localization of genes encoding proteins that were differentially expressed in pair-wise comparisons between wild and domesticated silkworms at I5D3 and I5D5. The sequences of down- and up-regulated proteins in wild compared with domesticated silkworms at the two stages were subjected to KAIKObase and analyzed with tBlastn to find the best matches (e-value < e-10). (B) The

33



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chromosome localization of the screened domestication candidate genes. WS, wild silkworm; DS, domesticated silkworm.

for TOC only

34


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Figure 1. Comparison of wild and domesticated silkworms. (A) On the left is the wild silkworm, and on the right is the domesticated silkworm at I5D3. (B) The dissected silk glands of wild (the left) and domesticated (the right) silkworms at I5D3. The white short lines mark the cut-points between the ASG, MSG, and PSG. (C) The cocoons of wild silkworm (the left) and the domesticated silkworm strain P50 (the right). (D) Comparison for the weight of cocoon shell of the wild silkworm (WS) and the domesticated silkworm (DS) strain P50. Ten cocoons were collected for each species, and Student’s t test was performed to analyze their shell weight differentials. **, P < 0.01. Scale bars, 1 cm in A-C. Photograph courtesy of Bo-xiong Zhong. Copyright 2017. 82x56mm (300 x 300 DPI)



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Figure 2. Label-free quantification analysis of wild and domesticated silkworm PSG proteomes. (A-C) Venn diagram of identified proteins in each set of comparisons. The red area indicates the number of up-regulated proteins, and the green area shows down-regulated proteins. White indicates the proteins without significant differential expression between comparisons. The differential expression in comparison pairs (panels A-C) with APEX score scatter plots are shown in panels D-F, respectively. Red dots and blue blocks represent the proteins with and without significantly differential expression, respectively (P < 0.05). The regression line divides the up- and down-regulated proteins. WS, wild silkworm; DS, domesticated silkworm; I5D3, the 3rd day of the fifth instar; I5D5, the 5th day of the fifth instar. 170x90mm (300 x 300 DPI)


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Figure 3. Gene Ontology analysis of the proteomes of wild silkworm PSGs at I5D3 and I5D5. (A) The biological process category of the shared PSG proteins at I5D3 and I5D5; (B) The biological process category of I5D3-specific proteins compared with I5D5; (C) The biological process category of I5D5-specific proteins compared with I5D3. The pie charts were plotted with Blast2GO software based on the annotation scores of the proteins at GO level 3. 175x92mm (300 x 300 DPI)



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Figure 4. Pathway enrichment analysis of the proteome identifications of the wild silkworm PSG. (A) The top 10 enriched pathways of the identified PSG proteins at I5D3 (red bar) and I5D5 (green bar) are shown. The asterisks mark the significantly enriched pathways of the identified proteins against the reference database (Background, blue bar). Pathway enrichment analysis was performed with the KEGG Orthology-Based Annotation System (KOBAS) by a hypergeometric statistic test. Categories with the corrected p < 0.05 were considered to be significantly enriched pathways. (B) A representative enriched pathway form KOBAS. Green boxes are the matched protein references of fruit fly (D. melanogaster) in the KEGG PATHWAY database, and the red boxes indicate the protein entries mapped to the significantly enriched pathway. 124x180mm (300 x 300 DPI)


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Figure 5. Venn diagram of the proteome identifications of PSGs in wild and domesticated silkworms during developmental stages. The proteome identifications of PSGs of wild and domesticated silkworms at the I5D3 and I5D5 were pairwise qualitatively (A) and quantitatively (B) compared. The comparisons in panel A were based on the protein identification lists in Supplemental Table 1 and previous data.11 In panel B, the upper part shows the significantly up-regulated proteins in each pair-wise comparison, and the lower part indicates the downregulated proteins (P < 0.05). The left panel shows the similarity of differentially expressed proteins from I5D3 to I5D5 between wild and domesticated silkworms. The right panel indicates the similarity of differentially expressed proteins between wild and domesticated silkworms at I5D3 and I5D5. The dark red and dark green areas show the common proteins with up- and down-regulation between the pair-wise comparisons, respectively. 82x112mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 6. TreeView analysis of the differentially expressed proteins between wild and domesticated silkworm PSGs. The differentially expressed proteins in pair-wise comparisons of wild and domesticated silkworms at I5D3 and I5D5 were clustered with Gene Cluster 3.0 and viewed with Java TreeView software. The heat map on the upper left indicates the differential expression levels. The relative expression of representative proteins evaluated with APEX scores is shown in the right panel. 140x140mm (300 x 300 DPI)



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Figure 7. Quantitative analysis of the expression of fibroin proteins in wild and domesticated silkworm PSGs. The relative expression of fibroin proteins, including Fib-L and P25, in different pair-wise comparisons was evaluated with APEX score. The two asterisks indicate significantly differential expression (P < 0.01). Fib-L, fibroin light chain; P25, fibrohexamerin. 82x50mm (300 x 300 DPI)


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Figure 8. Top 10 enriched KEGG pathways of differentially expressed proteins between the wild and domesticated silkworm PSGs. (A) Enriched pathways of the differentially expressed proteins between wild and domesticated silkworm at I5D3; (B) Enriched pathways of the proteins with differential expression between wild and domesticated silkworm at I5D5. The pathway enrichment analyses were performed with a web server KOBAS 2.0. The asterisk indicates the significantly enriched pathway (corrected P-value less than 0.05) against the reference database (Background). The number of identified proteins (red) is a part of the background (green). 119x95mm (300 x 300 DPI)



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Figure 9. Chromosome localization of the genes encoding differentially expressed proteins between wild and domesticated silkworm PSGs. (A) The chromosome localization of genes encoding proteins that were differentially expressed in pair-wise comparisons between wild and domesticated silkworms at I5D3 and I5D5. The sequences of down- and upregulated proteins in wild compared with domesticated silkworms at the two stages were subjected to KAIKObase and analyzed with tBlastn to find the best matches (e-value < e-10). (B) The chromosome localization of the screened domestication candidate genes. WS, wild silkworm; DS, domesticated silkworm. 119x131mm (300 x 300 DPI)


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Comparative Proteomic Analysis of Posterior Silk Glands of Wild and

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