Comparative Proteomic Analysis of Posterior Silk Glands of Wild and Domesticated Silkworms Reveals Functional Evolution during Domestication.

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 third and fifth 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 the 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.

Proteome identification of the silkworm middle silk gland.

To investigate the functional differentiation among the anterior (A), middle (M), and posterior (P) regions of silkworm middle silk gland (MSG), their proteomes were characterized by shotgun LC-MS/MS analysis with a LTQ-Orbitrap mass spectrometer. To get better proteome identification and quantification, triplicate replicates of mass spectrometry analysis were performed for each sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014) [1] via the PRIDE partner repository (Vizcaino, 2013) [2] with the dataset identifier PXD003371. The peptide identifications that were further processed by PeptideProphet program in Trans-Proteomic Pipeline (TPP) after database search with Mascot software were also available in .XML format files. Data presented here are related to a research article published in Journal of Proteomics by Li et al. (2015) [3].

Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production.

The silkworm middle silk gland (MSG) is the sericin synthesis and secretion unique sub-organ. The molecular mechanisms of regulating MSG protein synthesis are largely unknown. Here, we performed shotgun proteomic analysis on the three MSG subsections: the anterior (MSG-A), middle (MSG-M), and posterior (MSG-P) regions. The results showed that more strongly expressed proteins in the MSG-A were involved in multiple processes, such as silk gland development and silk protein protection. The proteins that were highly expressed in the MSG-M were enriched in the ribosome pathway. MSG-P proteins with stronger expression were mainly involved in the oxidative phosphorylation and citrate cycle pathways. These results suggest that the MSG-M is the most active region in the sericin synthesis. Furthermore, comparing the proteome of the MSG with the posterior silk gland (PSG) revealed that the specific and highly expressed proteins in the MSG were primarily involved in the ribosome and aminoacyl-tRNA biosynthesis pathways. These results indicate that silk protein synthesis is much more active as a result of the enhancement of translation-related pathways in the MSG. These results also suggest that enhancing ribosome biogenesis is important to the efficient synthesis of silk proteins.

Quantitative proteomic and transcriptomic analyses of molecular mechanisms associated with low silk production in silkworm Bombyx mori.

To investigate the molecular mechanisms underlying the low fibroin production of the ZB silkworm strain, we used both SDS-PAGE-based and gel-free-based proteomic techniques and transcriptomic sequencing technique. Combining the data from two different proteomic techniques was preferable in the characterization of the differences between the ZB silkworm strain and the original Lan10 silkworm strain. The correlation analysis showed that the individual protein and transcript were not corresponded well, however, the differentially changed proteins and transcripts showed similar regulated direction in function at the pathway level. In the ZB strain, numerous ribosomal proteins and transcripts were down-regulated, along with the transcripts of translational related elongation factors and genes of important components of fibroin. The proteasome pathway was significantly enhanced in the ZB strain, indicating that protein degradation began on the third day of fifth instar when fibroin would have been produced in the Lan10 strain normally and plentifully. From proteome and transcriptome levels of the ZB strain, the energy-metabolism-related pathways, oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle were enhanced, suggesting that the energy metabolism was vigorous in the ZB strain, while the silk production was low. This may due to the inefficient energy employment in fibroin synthesis in the ZB strain. These results suggest that the reason for the decreasing of the silk production might be related to the decreased ability of fibroin synthesis, the degradation of proteins, and the inefficiency of the energy exploiting.

Proteome analysis of silkworm, Bombyx mori, larval gonads: characterization of proteins involved in sexual dimorphism and gametogenesis.

Sexual dimorphism is initialed by the components of the sex determination pathway and is most evident in gonads and germ cells. Although striking dimorphic expressions have been detected at the transcriptional level between the silkworm larval testis and the ovary, the sex-dimorphic expressions at the protein level have not yet been well characterized. The proteome of silkworm larval gonads was investigated using a shotgun-based identification. A total of 286 and 205 nonredundant proteins were identified from the silkworm testis and ovary, respectively, with a false discovery rate (FDR) lower than 1%. Only 40 and 16 proteins were previously identified, and 246 and 189 proteins were newly identified in the silkworm testis and the ovary, respectively. The gametogenesis mechanism of silkworm was demonstrated using the protein expression profile and bioinformatics analysis. Cellular retinoic acid binding protein (CRABP) showed to be highly abundant in testis, while tubulins were abundant in ovary. Several homologies of Drosophila essential proteins for gametogenesis were identified in silkworm, such as male meiotic arrest gene product ALY and VISMAY in testis, and maternal mRNA localization protein exuperantia and SQUID in ovary. The gene ontology (GO) annotation and pathway analysis provide system-level insights into the sexual dimorphism and gametogenesis.

Comparative analysis on the expression of inducible HSPs in the silkworm, Bombyx mori.

Heat shock proteins (HSPs), well-known in respond to various kinds of stress situations, have been widely studied in Drosophila. However, a few reports related to silkworm bombyx mori. Genetic and non-genetic factors affecting on the expression of some HSPs in heat-treated silkworm were studied at the present paper. The mRNA levels of HSPs were quantified by real-time quantitative RT-PCR method and compared with their expression in the proteome profiles. The results showed up-regulation of two small heat shock proteins (sHSPs), HSP19.9 and HSP20.4 and down-regulation of HSP70 in the fat body, testis and ovary of heat exposed larvae. Higher variation of the sHSPs than HSP70 was observed in the different conditions such as heat exposures and genetic backgrounds. Significant difference in the HSP19.9 expression between two breeds was observed which implied the importance of this gene in the genetic differences. There was significant difference between responses of severe and mild heat shocks after 4 h heat recovery. The HSPs expression in male was significantly higher than that in female silkworm larva for all transcript measurements (P < 0.001). Comparison of two methods of quantification showed a fair similarity between HSPs expression in the transcriptome and proteome levels. Nistari breed as a naturally thermo-tolerant breed was expressed lower HSPs than a thermo-sensitive breed.

[Study on the hemostatic effects in raw and charred Mongolian drug Agi and its mechanism].

OBJECTIVE: To observe the effects of raw and charred Agi on hemostasis and its mechanism. METHODS: The rabbit bleeding time was measured by traumatic hemorrhage test, and the clotting time was measured by tube test. The rabbit prothrombin time (PT), activated partial thormboplastia time (APTT), thrombin time (TT), fibrinogen (FIB), plasma recalcification time (PRT), euglobulin lysis time (ELT), max platelet aggregation rate (MPAR) were measured by solidification method, turbidimetry and tube test to analyze the effects of raw and charred Agi on rabbit coagulation-fibrinolysis system and platelet function. RESULTS: The medium doses and high doses of raw Agi groups, all of the groups of charred Agi decreased rabbit BT obviously (P<0.01 or P<0.05); all of the groups of raw and charred Agi declined rabbits CT (P<0.01 or P<0.05). All of the doses groups of raw and charred Agi had no apparent influences on PT (P>0.05); the high dose of raw Agi group and all of the groups of charred Agi decreased APTT apparently (P<0.01 or P<0.05) and prolonged ELT (P<0.01 or P<0.05); the high doses groups of raw and charred Agi decreased TT apparently (P<0.01 or P<0.05); the medium and high doses groups of charred Agi increased FIB obviously (P<0.01 or P<0.05); the high doses group of charred Agi showed the decreased PRT significantly (P<0.05) and increased MPAR obviously (P<0.01). CONCLUSIONS: Raw Agi can play its role in hemostasis and coagulation by affecting the intrinsic pathway of coagulation and fibrinolytic system. These effects are inhanced after processing drugs; moreover, the charred Agi could increase FIB and MPAR with promoting more in blood coagulation.

Expression profiling and regulation of genes related to silkworm posterior silk gland development and fibroin synthesis.

The posterior silk gland (PSG) is the most important suborgan responsible for the synthesis and secretion of silk core fibroin proteins in silkworm. Here, we performed genome-scale expression profiling analysis of silkworm PSG at the fourth molting (M4) and at day 1 (V1), day 3 (V3), day 5 (V5), and wandering stage (W) of the fifth instar by microarray analysis with 22 987 probes. We found that the five genes of silk proteins secreted from PSG including fibroin heavy (H) and light (L) chains, P25, seroin 1, and seroin 2 basically showed obvious up-regulation at V3 which lasted to V5, while slight down-regulation at W. The expression of translation-related genes including ribosomal proteins and translation initiation factors generally remained stable from M4 to V5, whereas it showed clear down-regulation at W. Clustering analysis of the 643 significantly differentially expressed transcripts revealed that 43 of the important genes including seroin 1 and sugar transporter protein had co-expression patterns which were consistent with the rate changes of fibroin synthesis and PSG growth. Pathway analysis disclosed that the genes in different clusters might have co-regulations and direct interactions. These genes were supposed to be involved in the fibroin synthesis and secretion. The differential expression of several hormone-related genes also suggested their functions on the regulation of PSG development and fibroin synthesis. 2D gel-based proteomics and phosphoproteomics profiling revealed that the phosphorylated proteins accounted for no more than one-sixth of the total proteins at each stage, which was much lower than the level in normal eukaryotic cells. Changes in the phosphorylation status and levels of several proteins such as actin-depolymerizing factor 1 and enolase might be deeply involved in fibroin secretion and tissue development. Shotgun proteomic profiling combined with label-free quantification analysis on the PSG at V3, V5, and W revealed that many small heat shock proteins (sHSP) were specially expressed at W, which was substantially consistent with the results from 2-DE analysis, and implied the close correlations of sHSP with the physiological states of PSG at W. A majority of significantly up-regulated proteins at V5 were related to ribosome pathway, which was different from the microarray results, implying that the translation-level regulation of ribosomal proteins might be critical for fibroin synthesis. In contrast, the ubiquitin-proteasome pathway related proteins appeared obviously up-regulated at W, suggesting that the programmed cell death process of PSG cells might be started before cocooning.

Shotgun proteomic analysis on the embryos of silkworm Bombyx mori at the end of organogenesis.

Embryonic development of silkworm, Bombyx mori is a process of systematical expression of genes and proteins which is dominated by complex regulatory networks. To gain comprehensive insight into the molecular basis of embryonic development and its regulation mechanisms, the proteome profile of the B. mori embryos at the end of organogenesis (tubercle appearance stage, TA) was characterized using LTQ-Orbitrap mass spectrometer. Totally 963 proteins were identified with a false discovery rate (FDR) of 0.12%. They were involved in embryonic development, chemoreception, and stimuli response and so forth. The proteins with the largest number of identified unique peptides, implying their possibly higher abundance, were involved in heat shock response, lipid transport and metabolism, and apoptosis. It was consistent with the physiological status of embryo at the end of organogenesis. Many functionally important proteins were identified for the first time in B. mori embryo such as the progesterone receptor membrane component 2, antennal binding protein, sericotropin, and molting fluid carboxypeptidase A (MF-CPA). 253 (26.27%) specific proteins in TA versus labrum appearance stage (LA, four days before TA) embryos were identified, which were mainly associated with musculature, nervous system, and chemoreception system. They disclosed the differential temporal and spatial expression of proteins in the process of organogenesis. The relative mRNA levels of fifteen identified proteins in the two experimented stages were also compared using quantitative reverse transcription PCR (qRT-PCR) and showed some inconsistencies with protein expression. Gene Ontology (GO) annotation of the identified proteins showed that the most proteome representations were in the categories of "binding" and "catalytic" in molecular function, and "cellular process" and "metabolic process" in biological process.

Proteomic and bioinformatic analysis on endocrine organs of domesticated silkworm, Bombyx mori L. for a comprehensive understanding of their roles and relations.

Three organs of silkworm larva endocrine system, including brain (Br), subesophageal ganglion (SG) and prothoracic glands (PG), were studied employing shotgun LC-MS/MS combined with bioinformatic analysis to comprehensively understand their roles and relations. Totally, 3430, 2683, and 3395 proteins were identified including 1885 common and 652, 253, and 790 organ-specific ones in Br, SG, and PG, respectively. Identified common-expressed proteins indicated the existence of intrinsic complex interactions among these parts of endocrine system. Most of the reputed organs-specific proteins were identified by this approach. KEGG pathway analysis showed 162 same pathways among the 169, 164, and 171 relating Br, SG, and PG. This analysis revealed functional similarities with exceptional resemblance in their metabolism and signaling pathways of the three organs. On the other hand, 70, 57, and 114 organ-specific enzymes related pathways were detected for Br, SG, and PG confirming their functional differences. These results reveal a cooperative mechanism among the three endocrine organs in regulating various physiological and developmental events, and also suggest that the organ-specific proteins might be the fundamental factors responsible for the functional differentiation of these organs.

Proteomic identification of the silkworm (Bombyx mori L) prothoracic glands during the fifth instar stage.

Although the ecdysteroid of the silkworm had been studied for decades, the proteome of the prothoracic gland, the primary source of ecdysteroid hormones, has not been studied previously. In the present paper, we utilized a proteomic approach to investigate the fifth instar prothoracic gland during the growth and development of the silkworm, Bombyx mori L. The two-dimensional electrophoresis results showed that the majority of proteins were acidic proteins, especially concentrated in the area of 25-65 kDa, with pI values of between 4 and 7, and the difference was not distinct. When compared with Qiufeng (Japanese strain), the interspecific distinction was larger than the intraspecific distinction, and 19 particular spots, excized from the third, fifth and ninth days of p50 (Chinese strain) and Qiufeng were subjected to MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) analysis. We sorted them into seven catagories: energetics and/or metabolism, storage proteins, protection, lipid metabolism, signal transduction, cell function and unknown function proteins. Of these proteins, arginine methyltransferase is discussed as playing an important role in regulating the activation of ecdysteroidogenesis via transcription or translation.

Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet.

To gain an insight into the effects of different diets on growth and development of the domesticated silkworm at protein level, we employed comparative proteomic approach to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet. Seventy-six differentially expressed proteins were identified by MALDI TOF/TOF MS, and among them, 41 proteins were up-regulated, and 35 proteins were downregulated. Database searches, combined with GO analysis and KEGG pathway analysis revealed that some hemolymph proteins such as Nuecin, Gloverin-like proteins, PGRP, P50 and beta/-N-acetylglucosamidase were related to innate immunity of the silkworm, and some proteins identified in silkworm midgut including Myosin 1 light chain, Tropomyosin 1, Profilin, Serpin-2 and GSH-Px were involved in digestion and nutrition absorption. Moreover, two up-regulated enzymes in fat body of larvae reared on artificial diet were identified as V-ATPase subunit B and Arginine kinase which participate in energy metabolism. Furthermore, 6 down-regulated proteins identified in posterior silk gland of silkworm larvae reared on artificial diet including Ribosomal protein SA, EF-2, EF-1gamma, AspAT, ERp57 and PHB were related to silk synthesis. Our results suggested that the different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis poor nutrition and absorption of nutrition in silkworm. The results also confirmed that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for low cocoons yield, inferior filament quality, low survival rate of young larvae and insufficient resistance against specific pathogens in the silkworms fed on artificial diet.

Microsatellite analysis of royal jelly producing traits of Italian honeybee (Apis mellifera Liguatica).

Genetic variations at 10 microsatellite loci were surveyed to determine the evolutionary relationships and molecular characteristics of three different honeybee (Apis mellifera L.) populations from Italy and China, i. e., native Italian (Ee), Chinese-Italian (Eb) and selected high royal jelly producing bees (Ea). A total of 96 alleles,an average of 9.6 alleles per locus,were scored in Ee,Eb and Ea bees at 10 loci. Out of which 48 (5%) were different. This indicated a high degree of polymorphism and ever, some genetic differentiation among the three populations due to artificial selection and geographical isolation. The polymorphic information contents (PIC) and heterozyosity of the three populations at 10 loci were 0.57, 0.50, 0.57, and 0.60, 0.57, 0.61, for Ee, Eb, Ea populations respectively, neither of which were different. This indicated same gene diversity within the three populations. The genetic distance was shorter between Ee and Eb bees as well as between Eb and Ea bees. Whereas that between Ee and Eb bees was longer. Further analysis indicated that the allele frequency of seven alleles at six loci (159 bp at A29,100 bp and 104 bp at A24; 110 bp at A7; 126 bp at A43, 221 bp at A14 and 221 bp at A113) increased going from Ee to Eb to Ea bees. Paired tests showed significant higher allele frequency between Ea and Eb bees,as well as between Ea and Eb bees. This indicates that these seven alleles are likely molecular markers of the high royal jelly producing bees. In addition,the allele frequency of four alleles at four loci (106 bp at A24,140 bp at A43;215 bp at A113 and 219 bp at A14) decreased going from Ea bees to Eb to Ee. Paired tests indicated significant lower allele frequency between Ea and Ee bees,as well as between Ea and Eb bees. Those four alleles may be the genetic markers for low royal jelly production.

Possible effect of 30K proteins in embryonic development of silkworm Bombyx mori.

The silkworm Bombyx mori possesses a 30K protein family of 3x10(4) Da, the biological functions of which have not been fully identified. The relationship between the 30K protein family and the embryonic development of temperature sensitive sex-linked mutant strain of silkworm was investigated by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results show that protein spots 1-5 of the 30K protein family, mainly existing in normal strain, are possibly related to embryonic development. The early consumption of a 30K protein named 6G1-30K-1 and the accumulation of 30K proteins named 6G1-30K-3 and 6G1-30K-4 are likely caused by the destruction of physiological balance in normal embryonic development, which may lead to lower hatchability of the temperature sensitive strain. The results suggest that reasonable metabolism of 30K proteins is a prerequisite for the embryo's normal development.

RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly.

The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonas fragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method, respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonas fragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.

Accumulation of pathogenesis-related type-5 like proteins in phytoplasma-infected garland chrysanthemum Chrysanthemum coronarium.

Soluble proteins extracted from leaves, apical shoots, axillary shoots, and stems of garland chrysanthemum plants infected by onion yellows phytoplasma were analyzed by two-dimensional gel electrophoresis. Computerized matching analysis revealed that at least six soluble proteins were accumulated specifically in phytoplasma-infected garland chrysanthemum. N-terminal amino acids sequences of these soluble proteins, determined by Edman degradation, shared high sequence similarities with those of pathogenesis-related type-5 (PR-5) proteins such as tobacco thaumatin-like protein. Accumulation of these six proteins was also found in garland chrysanthemum plants infected by other phytoplasmas. These results demonstrate that phytoplasmal infection induces the accumulation of PR-5 like proteins in garland chrysanthemum plants.

[Cloning and sequence analysis of mrjp1 cDNA from Apis cerana cerana].

A cDNA library was constructed from eight-day-old worker heads of Apis cerana cerana. A probe derived from part of Apis cerana genomic mrjp3 segment was used to screen this library. A total of 120 positive clones of mrjps were screened out from the cDNA library with DIG-probe. The positive clones were amplified and sequenced with T3/T7 primer. 12 sequences were similar to Apis cerana india and Apis mellifera mrjp1 gene by BLAST analysis. The cDNA sequence analysis indicated that the homology was 93.78% between Apis mellifera mrjp1 and Apis cerana mrjp1 while the homology was 99.36% between Apis cerana cerana and Apis cerana india. This result confirmed in molecular level that Apis cerana cerana and Apis cerana india had a common ancestor since the relationship between Apis cerana and Apis mellifera was more far.

[Genetic analysis for developmental behavior of honeybee colony's royal jelly production traits in western honeybees].

Analyses of genotypic effects for colony's royal jelly (RJ) yields and RJ in each queen cell as well as acceptances of queen cell cups of three lines of Western honeybees (Apis melliffera Lingistica) were conducted by using a genotypic model for analyzing the genetic and non-genetic effects. Analytis approaches of conventional and conditional variance and correlation were employed to evaluate the developmental behavior of honeybee colony's RJ producing ability. The results indicated that significant variance due to genotypic effect was detected for colony's RJ yields and RJ in each queen cell cup at all stages, while the acceptance of queen cell cups was found variance significantly at 10 stages of its total 11. It meant that the three traits were dominated by genotypic effect. Significant conditional variances were found at some stages when no unconditional one being detected. Correlation analysis between same trait at different stages indicated that the significant coefficients always existed due to genotypic effects for RJ yields and RJ in each queen cell cup. Although the significant coefficients of the acceptance of queen cell cup at different stages were found at most of the stages, the coefficients were not found in come of the stages. These results indicated that the genotypic effects dominated in early stages of colony's RJ yield and RJ in each queen cell cup influencing its late stages in the same way, but the acceptance of queen cells cup was not. The analysis of correlation between different pair of traits showed that the coefficients between colony's RJ yields and RJ in each queen cell cup were significant at all stages, and while the coefficients between colony's RJ yields and the acceptance of queen cell cups were not always significant at all stages. It was because the genotypic effects dominated the former pair of traits had harmonious effects, while the later pair of traits were not.