Protein Of Bombyx Mori Research Articles

De-novo long-read genome assembly and annotation of the luna moth (Actias luna) fully resolves repeat-rich silk genes.

We present the first long-read de-novo -assembly and annotation of the luna moth (Actias luna) and provide the full characterization of heavy chain fibroin (h-fibroin)--, a long and highly repetitive gene (>20 Kbp) essential in silk fiber production. There are more than 160,000 described species of moths and butterflies (Lepidoptera), but only within the last five years have we begun to recover high-quality annotated whole genomes across the order which capture h-fibroin. Using PacBio HiFi reads, we produce the first high-quality long-read reference genome for this species. The assembled genome has a length of 532 Mbp, a contig N50 of 16.8 Mbp, an L50 of 14 contigs, and 99.4% completeness (BUSCO). Our annotation using Bombyx mori protein and A.luna RNAseq evidence captured a total of 20,866 genes at 98.9% completeness with 10,267 functionally annotated proteins and a full-length h-fibroin annotation of 2,679 amino acid residues.

Identification and Functions of JHE 6 Specifically Expressed in Bombyx mori Silk Gland.

Juvenile hormone esterase (JHE) is the specific enzyme that degrades juvenile hormone (JH) and regulates the JH titer in insects. JH also regulates the development of the silk gland and the synthesis and secretion of silk proteins in Bombyx mori. Here, we identified nine possible JHE family members, Bmjhe1-9. Notably, Bmjhe6 is specifically expressed in the silk gland. Using semi-quantitative, quantitative real-time RT-PCR and Western blot, it was confirmed that Bmjhe6 was specifically expressed in the middle silk gland (MSG) with high levels in the anterior region of the MSG (A-MSG). The immunofluorescence localization analysis revealed that Bmjhe6 is produced within cells, secreted into the gland lumen, and co-transported with silk proteins into the anterior silk gland (ASG). In vitro hormone induction experiments demonstrated that Bmjhe6 responds to a JH analog, increasing its expression after 12-24 h, whereas 20-hydroxyecdysone inhibited it. In addition, Bmjhe6 knockdown using dsBmjhe6 injections accelerated larval development, resulting in increased larval body and silk gland weight. This induced disordered sericin genes (Ser2, Ser3) expression, and key genes in the JH synthesis pathway (BmKr-h1 and BmMet1) were significantly upregulated along with the transcription factors (SGF-1 and Sage). These results indicate that Bmjhe6 plays an important role in silk gland growth and silk protein synthesis by modulating JH signal.

Biologically Active Feeds Based on Local Raw Materials

An efficient technology for protein extraction from silk production waste has been developed. The dynamics of the degree of precipitation of alkaline hydrolysates by acetic acid has been studied. The amino acid composition of the isolated protein was determined, which contains 9 essential acids. IRspectroscopy and elemental analysis showed the purity of the isolated protein. Approbation of protein in the composition of feed in fish farming showed high fish productivity and efficiency in terms of feed ratio. The use of Bombyx mori protein for poultry in the amount of 0.5% 1% increases the gain in poultry meat and egg production by 48.25%.

Nutrition regulates the expression of storage proteins in Bombyx mori via insulin-like/FoxO signaling pathway

Insect serum proteins, also termed storage proteins (SPs), are hexamer proteins that form amino acid reservoirs important for the development of pupae and embryos in most insects. In this study, we investigated the SP genes expression and regulation pathways in silkworms (Bombyx mori). We observed that B. mori SPs (BmSPs) in the fat body of larvae were strongly decreased by starvation, suggesting they respond to nutrition deprivation. Further, we examined the relationship between BmSP expression and the insulin-like signaling pathway (ILS) to study the regulation of BmSPs expression. The results showed that insulin up-regulated the expression of BmSPs, but an inhibitor of the ILS pathway protein PI3K downregulated the expression of BmSPs in B. mori larvae. Similar results were observed in cultured fat body in vitro and BmE cells. We then over-expressed FoxO, an ILS transcriptional factor, in BmE cells and B. mori larvae to further verify the regulatory role of ILS on expression of BmSPs and found BmFoxO negatively regulates the expression of BmSPs in both BmE cells and larvae. Moreover, BmFoxO was dephosphorylated and translocated from the cytoplasm to the nucleus under starvation treatment. Finally, an element on −2627–2644 bp upstream of the transcription start site of BmSP1 was identified as the binding site of BmFoxO by electrophoretic mobility shift assay and verified by chromatin immunoprecipitation. In summary, our results indicate that nutrient uptake triggers the expression of BmSPs via the ILS/FoxO signaling pathway. This study provides a reference for further study on the expression and regulation of insect SP genes.

Polymolecular Complexes of Chitosan with the Bombyx Mori Protein

The interaction of chitosan (ChS) and the Bombyx mori protein on different pH ranges was studied, and the fundamental possibility of obtaining complexes of ChS with the Bombyx mori protein was revealed. The for-mation of a polymolecular complex of protein with ChS in aqueous solutions was confirmed by the results of physico-chemical methods. It is shown that the ChS structure is characterized by a certain rigidity and ionogenicity. The results indicate the complexation of the pupae protein with ChS in 2% acetic acid in the range of pH = 4.8–6.7. The detected changes and shifts of the absorption bands in the IR spectra confirm the occurrence of the complex formation reaction between the molecules of ChS and protein at pH = 4.8–6.7, which is characterized by absorption bands in the IR spectra at 1641 cm–1, 1538 cm–1 and 1068 cm–1. Quan-tum-chemical DFT study of ChS complexes with amino acids (AAs) was carried out. The stability of com-plexes of ChS with AAs (ChS-AA) was shown except for the complex formed with histidine in the gas phase. The calculation results indicate the presence of a strong thermodynamic driving force in the complexation of ChS with AAs.

Role of Toll-receptors in the inhibition of BmNPV proliferation and their interplay with antimicrobial seroin proteins in Bombyx mori

The domesticated silkworm, Bombyx mori, is an economically important insect that majorly contributes in financially viable sericulture industry. The baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV) is a deadly virus that infects B. mori and inflicts heavy loss to silk production by causing high mortality of silkworms. A number of studies have been done to identify antiviral genes and pathways against BmNPV infection in B. mori . Toll-pathway is a well-known immune pathway responsible for combating bacterial and fungal infections in silkworm. However, the role of Toll-receptors in inhibiting BmNPV has not been explored. In the present study, we have attempted to examine the role of Toll-receptors in inhibiting BmNPV proliferation in B. mori larvae. The effect of Toll-receptors on viral proliferation was determined by quantifying viral DNA and counting viral occlusion bodies upon RNA interference-mediated knockdown of Toll-receptors in B. mori larvae. Knockdown of BmToll, BmToll-6, BmToll-7, BmToll-8 and BmToll-9 led to higher accumulation of BmNPV. Further, interplay between Toll-receptors and the antimicrobial proteins of B. mori, called seroins were studied using RNA interference. As a result, we found a downregulation of Seroin1 transcript level upon knockdown of BmToll , BmToll-7 , BmToll-8 , BmToll-10 , BmToll-11 and BmToL K2, whereas Seroin2 expression was found to decrease only upon BmToll-7 knockdown. Moreover, knockdown of seroins did not result in clearly interpretable expression patterns of Toll-receptors. These results suggest the involvement of B. mori Toll-pathway in defense against BmNPV infection and provides a new insights into the antiviral defense mechanism of B. mori. • BmNPV proliferation increases upon knockdown of B. mori Toll-receptors in larvae. • Knockdown of B. mori Toll-7 significantly enhances viral load. • Seroins are likely to function downstream of Toll-7-mediated signaling pathway in B. mori.

Bombyx mori protein BmREEPa and BmPtchd could form a complex with BmNPV envelope protein GP64

Our previous studies have indicated that Bombyx mori receptor expression enhancing protein a (BmREEPa) could participate in BV invasion in vivo and in vitro, however, the mechanism is still unclear. In this study, we screened BmREEPa interacting protein through co-immunoprecipitation and finally identified a membrane protein, Bombyx mori patched domain containing protein (BmPtchd, KR338939), which contains receptor activity. Further studies showed that BmPtchd, BmREEPa and Glycoprotein 64 could form a protein complex and the expression level of BmREEPa and BmPtchd could be affected by cellular cholesterol level. These findings may provide an important basis for explaining the invasion mechanism of Bombyx mori Nucleopolyhedrovirus budded virus.

Transcriptome Analysis in Cotton Boll Weevil (Anthonomus grandis) and RNA Interference in Insect Pests

Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families’ data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.

Effect of a Bombyx mori Protein Disulfide Isomerase on Production of Recombinant Antibacterial Peptides

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.

Characterization and expression of attacin, an antibacterial protein-encoding gene, from the beet armyworm, Spodoptera exigua (Hübner) (Insecta: Lepidoptera: Noctuidae)

To isolate antimicrobial-related genes from the beet armyworm, Spodoptera exigua, we performed GeneFishing, a polymerase chain reaction (PCR)-based differential display technique. An attacin-like complementary DNA (cDNA) including a 3'-untranslated region was identified from among 18 over-expressed genes in microbial-infected larvae. The full-length attacin cDNA from S. exigua cDNA (Seattacin) was cloned using rapid amplification of cDNA ends PCR. The attacin-like cDNA transcript was 765 nucleotides in length, and the predicted polypeptide was 254 amino acids in length with a calculated molecular mass of 27.6kDa and an isoelectric point of 6.44. The protein sequence of the attacin-like cDNA showed high identity to that of Trichoplusia ni (61.2%). The amino acid sequence identity of Seattacin to the orthologous proteins in Bombyx mori, Manduca sexta, Heliothis virescens, Hlicoverpa armigera, Hyphantria cunea, Hyalophora cecropia, and Drosophila melanogaster was 61.2, 46.1, 44.5, 42.2, 39.5, 45.1, and 24.0%, respectively. To examine possible immune functions of the attacin-like cDNA, its expression was investigated by reverse transcriptase PCR analysis after challenging S. exigua with microorganisms. The attacin-like cDNA was expressed at high levels 12h post-infection, and its expression was slightly induced 4-8h post-infection compared to control larvae inoculated with sterile water. Furthermore, induced Seattacin showed biological activity against several bacteria including Escherichia coli DH5α, Pseudomonas cichorii, Bacillus subtilis, and Listeria monocytogenes. These results suggest that the attacin-like cDNA of S. exigua codes for antimicrobial peptides.

Small GTPases of the Rab family in the brain of Bombyx mori

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.

Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane

P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604-4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence-emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324-1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP-Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP-Cry1Ab was reduced from an LD₅₀ of 1.62 to 5.05 μg, but Bm252RFP-Cry1Aa and Bm252RFP-Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP-Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP-Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab α-helix was significantly decreased in Bm252RFP-Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of α-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP-Cry1Ab.

Bombyx mori protein disulfide isomerase enhances the production of nuecin, an antibacterial protein

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpressions of foreign proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.

Secretion of the Antibacterial Recombinant Protein Enbocin

The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant enbocin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins.

Mapping Analysis of Carotenoid-binding Protein of Bombyx mori by Restriction Fragment Length Polymorphism

Mapping Analysis of Carotenoid-binding Protein of Bombyx mori by Restriction Fragment Length Polymorphism

Structural Basis of Ligand Binding and Release in Insect Pheromone-binding Proteins: NMR Structure of Antheraea polyphemus PBP1 at pH 4.5

The NMR structure of the Antheraea polyphemus pheromone-binding protein 1 at pH 4.5, ApolPBP1 A, was determined at 20 °C. The structure consists of six α-helices, which are arranged in a globular fold that encapsulates a central helix α7 formed by the C-terminal polypeptide segment 131–142. The 3D arrangement of these helices is anchored by the three disulfide bonds 19–54, 50–108 and 97–117, which were identified by NMR. Superposition of the ApolPBP1 A structure with the structure of the homologous pheromone-binding protein of Bombyx mori at pH 4.5, BmorPBP A, yielded an rmsd of 1.7 Å calculated for the backbone heavy-atoms N, C α and C′ of residues 10–142. In contrast, the present ApolPBP1 A structure is different from a recently proposed molecular model for a low-pH form of ApolPBP1 that does not contain the central helix α7. ApolPBP1 exhibits a pH-dependent transition between two different globular conformations in slow exchange on the NMR chemical shift timescale similar to BmorPBP, suggesting that the two proteins use the same mechanism of ligand binding and ejection. The extensive sequence homology observed for pheromone-binding proteins from moth species further implies that the previously proposed mechanism of ligand ejection involving the insertion of a C-terminal helix into the pheromone-binding site is a general feature of pheromone signaling in moths.

Changes in cellular secretory processing during baculovirus infection

In the baculovirus expression vector system (BEVS), secretory green fluorescent protein (sGFP) transcripts were expressed from day 2 to day 5 post-infection (p.i.), while transcripts of the endoplasmic reticulum (ER) molecular chaperone Bombyx mori protein disulfide isomerase (bPDI) were measurable in mock cells and in cells at day 1 p.i. The GFP was expressed from day 3 to day 5 p.i. whereas the levels of two ER chaperone proteins, bPDI and calnexin, decreased from day 3 p.i. and were not detected from day 4 p.i. These findings suggest that the rate-limited expression of ER molecular chaperones is strongly associated with the maximal expression of exogenous proteins in BEVS.

Revisiting the odorant-binding protein LUSH of Drosophila melanogaster: evidence for odour recognition and discrimination

LUSH is a soluble odorant-binding protein of the fruit fly Drosophila melanogaster. Mutants not expressing this protein have been reported to lack the avoidance behaviour, exhibited by wild type flies, to high concentrations of ethanol. Very recently, the three-dimensional structure of LUSH complexed with short-chain alcohols has been resolved supporting a role for this protein in binding and detecting small alcohols. Here we report that LUSH does not bind ethanol and that wild type flies are in fact attracted by high concentrations of ethanol. We also report that LUSH binds some phthalates and that flies are repelled by such compounds. Finally, our fluorescence data, interpreted in the light of the three-dimensional structure of LUSH, indicate that the protein undergoes a major conformational change, similar to that reported for the pheromone-binding protein of Bombyx mori, but triggered, in our case, by ligand.

Molecular Cloning and Expression of cDNAs Encoding Testis-Specific and Non-Specific ATPase Inhibitor-Like Proteins in Bombyx mori

Molecular Cloning and Expression of cDNAs Encoding Testis-Specific and Non-Specific ATPase Inhibitor-Like Proteins in Bombyx mori

NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori.

NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.