Project description:Background: MicroRNA (miRNA) and other small regulatory RNAs contribute to the modulation of a large number of cellular processes. We sequenced three total RNA libraries prepared from the whole body, and the anterior and posterior silk glands of Bombyx mori, with a view to expanding the repertoire of silkworm miRNAs and exploring transcriptional differences in miRNAs between segments of the silk gland. Results: With the aid of large-scale Solexa sequencing technology, we validated 244 unique miRNA genes, including 191 novel and 53 previously reported genes, corresponding to 309 loci in the silkworm genome. Interestingly, 24 unique miRNAs were widely conserved from invertebrates to vertebrates; 12 unique ones were limited to invertebrates and 33 were confined to insects; whereas the majority of the newly identified miRNAs were silkworm-specific. We identified 21 clusters and 42 paralogs of miRNAs in the silkworm genome. However, sequence tags showed that paralogs or clusters are not prerequisites for coordinated transcription and accumulation. The majority of silkworm-specific miRNAs are located in transposable elements, and display significant differences in abundance between the anterior and posterior silk glands. Conclusions: Conservative analysis revealed that miRNAs serve as phylogenetic markers and function in evolutionary signaling. The newly identified miRNAs greatly enriched the repertoire of insect miRNAs, and provide insights into miRNA evolution, biogenesis, and expression in insects. The differential expression of miRNAs in the anterior and posterior silk glands supports their involvement as new layers in the regulation of the silkworm silk gland. Sequencing three total RNA pools of the whole silkworm body from 5th-instar day-3 larvae, and anterior and posterior silkworm silk glands, using the latest sequencing Solexa technology
Project description:This study aims to bridge the gap in our knowledge of Philippine-reared silkworm by analyzing the gene expression profiles in the silkworm silk glands through next generation sequencing. RNA was isolated from the silk glands of 5th instar larvae and mRNA-enriched libraries were sequenced with NextSeq 500 (Illumina). To compare gene expression profiles of strains from CAR (Benguet) and TCMO (Misamis Oriental), DESeq2 analysis was performed. DESeq2 found 476 differentially expressed genes (222 upregulated, 254 downregulated) in CAR strains when compared to TCMO strains. Genes were mapped to protein IDs from the NCBI nr database and GO terms were assigned by mapping to the latest annotation data from KAIKObase. Enrichment of GO terms was analyzed using R package goseq. Among the top DEGs are myrosinase, heat shock proteins, serine protease inhibitors, dehydrogenases, and regulators of juvenile hormone. GO term enrichment analysis reveals overrepresentation of GO terms related to the biological processes nucleotide metabolism and biosynthesis, lipid and carbohydrate metabolic processes, regulation of transcription, and molecular functions related to nucleotide binding, protein binding, and metal binding, catalytic activity, oxidoreductase activity, and hydrolase activity. This study provides for the first time valuable information on the transcriptome of B. mori strains in the Philippines, which are adapted to the tropical environment. The transcriptome assemblies may serve as a resource for studies intended to improve local strains, particularly for increasing silk production.
Project description:Background: The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production. Results: Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, from the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the genes into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing. Conclusion: The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs in silk production. Sequencing 10 total RNA samples from the posterior silk gland of different strains and developmental stage using Illumina Solexa technology. Four strains of silkworm (Q, B, QB and BQ) with different two development stages (stage 1: fourth instar molting to day 2 of fifth instar; stage 2: fifth instar day 3 to day 8 before spinning, according to our previous genes expression cluster analysis), and two strains (R1 and J1) from entire period (stage 1 + stage 2).
Project description:The anterior silk gland in the silkworm plays an important role in the process of liquid fibroin to solid silk fiber .In view of this,the proteomics analysis was applied to to study the relationship between the function of proteins in the anterior silk gland and the mechanism of spinning. The anterior silk glands on the 3rd day of fifth instar were dissected.Aftter 1D SDS-PAGE ,one gel lane was cut into 10 bands and each band further sliced into small pieces was subjected to in-gel tryptic digestion for 20 hours.The digested peptides were separated by RP nanoscale capillary liquid chromatography and analyzed using a surveyor LC system (Thermo Figgigan, San Jose, CA).The eluate from the RP column was analyzed by Finnigan LTQ(Thermo Electron Corporation)linear ion trap Mass equipped with a nanospray souce in the positive ion mode. The MS analysis was performed with one full MS scan followed by three MS/MS scans on the most intense ions from the MS spectrum with the dynamic exclusion settings: repeat count 2, repeat duration 30s, exclusion duration 90s. Data were acquired in data-dependent mode using Xcalibur software.Ten raw datasets from LC-MS/MS were searched against the predicted silkworm database by Xia.et al which consists of 21312 silkworm proteins.The searching was carried out with the Turbo SEQUEST(Bioworks version 3.2, Thermo Electron).
Project description:Background: MicroRNA (miRNA) and other small regulatory RNAs contribute to the modulation of a large number of cellular processes. We sequenced three total RNA libraries prepared from the whole body, and the anterior and posterior silk glands of Bombyx mori, with a view to expanding the repertoire of silkworm miRNAs and exploring transcriptional differences in miRNAs between segments of the silk gland. Results: With the aid of large-scale Solexa sequencing technology, we validated 244 unique miRNA genes, including 191 novel and 53 previously reported genes, corresponding to 309 loci in the silkworm genome. Interestingly, 24 unique miRNAs were widely conserved from invertebrates to vertebrates; 12 unique ones were limited to invertebrates and 33 were confined to insects; whereas the majority of the newly identified miRNAs were silkworm-specific. We identified 21 clusters and 42 paralogs of miRNAs in the silkworm genome. However, sequence tags showed that paralogs or clusters are not prerequisites for coordinated transcription and accumulation. The majority of silkworm-specific miRNAs are located in transposable elements, and display significant differences in abundance between the anterior and posterior silk glands. Conclusions: Conservative analysis revealed that miRNAs serve as phylogenetic markers and function in evolutionary signaling. The newly identified miRNAs greatly enriched the repertoire of insect miRNAs, and provide insights into miRNA evolution, biogenesis, and expression in insects. The differential expression of miRNAs in the anterior and posterior silk glands supports their involvement as new layers in the regulation of the silkworm silk gland.
Project description:Spinneret, which locates at the end of the silk gland, is an important tissue for silk spinning. Although it has been discovered for hundreds of years, its particular roles in silk spinning and fiber formation are still unclear. Here we report the first proteome profile of silkworm spinnerets by LC-MS/MS. Totally, 1572 non-redundant proteins and 232 differential expressed proteins were identified in the spinneret from the spinning larvae and the third day of fifth instar larvae. Silk fiber formation related proteins, such as cuticle proteins, ion-transporting proteins, muscular proteins, and energy metabolic proteins, were abundant in the spinneret. By analyzing the signal pathways, we discovered that the processes associated with energy metabolism were active in spinneret. GO enrichment analysis revealed that the differential expressed proteins were involved in energy metabolism, chitin binding, and cuticle construction. The active energy metabolism might provide abundant energy for the muscle contraction, ion and water transporting. The chitin binding and cuticle construction process might provide sufficient shear forces for silk formation. This dataset suggests that the spinneret provided a suitable physiological and biochemical environment for silk formation, and will be helpful for elucidating the functions of spinneret.
Project description:The silk gland development has a greater impact on silk yields in silkworms. Silk glands from three pure silkworm strains (A798, A306, and XH) with different silk gland weight phenotypes were compared using transcriptome, proteomics, and WGCNA. Five genes (BGIBMGA002524, BGIBMGA002629, BGIBMGA005659, BGIBMGA005711, and BGIBMGA010889) may be strongly associated with the growth of silk glands to be confirmed. These DEGs encoded alkylglycerol monooxygenase (AGMO), glucose dehydrogenase (GDH), zonadhesin (ZAN), odorant binding protein (OBPs), and β-fructofuranosidase (INV), respectively. PCR and ELISA were used to verify the mRNA and protein expression of five genes in the silk glands and tissues of 18 silkworm strains. The GO results showed that four genes have higher levels of expression and participate in glycogen metabolism, fatty acid synthesis, and branched-chain amino acid metabolism, thus, promoting growth and silk proteins synthesis.
Project description:Background: The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production. Results: Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, from the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the genes into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing. Conclusion: The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs in silk production.
Project description:The silkworm, Bombyx mori, is a complete metamorphosis insect and an economically important for silk production, the model to study insect physiology and biochemistry. Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the silkworm and its host range is restricted to silkworm larvae, requiring interaction with silkworm larvae to accomplish virus replication. Prothoracic glands (PGs) are a model for synthetic ecdysone with regulating insect growth and development. In this study, day-4 fifth instar silkworm larvae were infected by BmNPV, the wandering silkworms appeared in the infected groups were 12 hours earlier than that in the control groups, and the ecdysone titer in infected larvae was significantly higher than that of the control larvae. Then, we used RNA sequencing (RNA-seq) to analyze silkworm PGs 48 h after BmNPV infection. The classifications of the 15 differential expression genes (DEGs) were mainly involved in the metabolic processes and pathways. The RT-qPCR results of the DEGs in the PGs of BmNPV-infected at 24, 48, and 72 h were generally consistent with the transcriptome data. The transcripts of BmTrypsin-1 and BmACSS3 were significantly increased from 24 to 72 h after BmNPV infection that they may be involved in the maturation process in the latter half of silkworm fifth instar larvae. These findings will help to address the interactions between BmNPV infection and host developmental response.
Project description:We have performed the first systematic identification and analysis of intermediate size ncRNAs (50-500 nt) in the silkworm genome. We identified 194 novel ncRNAs, the expression profiles of them during the transitions from the egg to the first instar larva and from the fifth instar larva to the pupa were anlyzed by dual-channel microarray. Results showed that 12 ncRNAs had significantly differential expression during the two developmental transitions.