Project description:Spider silk proteins are synthesized in the silk-producing glands, where the spidroins are produced, stored and processed into a solid fiber from a crystalline liquid solution. Despite great interest in the spider silk properties, that make this material suitable for biomedical and biotechnological applications, the mechanism of formation and spinning of the silk fibers has not been fully elucidated; and no combination of proteomic and transcriptomic study has been carried out so far in the spider silk-producing glands. Nephila clavipes is an attractive orb-web spider to investigate the spinning process of silk production, given the properties of strength, elasticity and biocompatibility of their silk fibers. Thus, considering that the combination of proteomic and transcriptomic analysis may reveal an extensive repertoire of novel proteins involved in the silk spinning process, and in order to facilitate and enable proteomics in this non-model organism, the current study aims to construct a high quality reference mRNA-derived protein database that could be used to identify tissue specific expression patterns in spider silk glands. Next-generation sequencing has offered a powerful and cost-efficient technique for the generation of transcriptomic datasets in non-model species using diverse platforms such as the Illumina HiSeq, Roche 454, Pacific Biosystems, and Applied Biosystems SOLiD; In the current study, the Illumina HiSeq 2000 platform will be used to generate a N. clavipes spider silk glands transcriptome-based protein database. The transcriptome data generated in this study will provide a comprehensive and valuable genomic resource for future research of the group of spider silk-producing glands, in order to improve our understanding of the overall mechanism of action involved in production, secretion, storage, transport, protection and conformational changes of spidroins during the spinning process, and prey capture; and the results may be relevant for scientists in material Science, biology, biochemistry, and environmental scientists.
Project description:To identify functions that distinguish the posterior and median cells producing fibroin and sericin in the silk gland of Bombyx mori, serial analysis of gene expression (SAGE) profiles from both silk gland regions were analyzed and compared. The construction of a B. mori reference tag collection extracted from a set of 38000 Bombyx EST sequenced from the 3’ side, helped annotating the SAGE libraries. Most of the tags appeared at similar relative concentration in the two libraries except for those corresponding to silk proteins that were found region-specific and highly abundant. Strikingly, besides tags from silk protein mRNAs, 19 tags were found in the class of high abundance in the median cell library, which were absent in the posterior cell tag collection. Except tags from SP1 mRNA, no PSG specific tags were found in the same class of abundance. The analysis of MSG-specific different transcripts led to suggest that middle silk gland cell realizes more diversified functions as those already known, of synthesis and secretion of the silk sericins.
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:To fully characterize the extent of genomic changes in senescent silk, we performed a time-course tissue harvesting. Material was collected at four time points: 3-, 7-, 11- and 15 days after silk emergence (DASE). Maize inbred line B104; 2 cm of basal part of silk from rings 6-10
Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous comparison of tissue-specific RNAseq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ~5% of these transcripts encode spidroins and the remaining predicted genes presumably encode other proteins associated with silk production. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands, and detect 16 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk associated proteins. Major ampullate and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major ampullate and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
Project description:The silk gland (SG) of the domesticated silkworm Bombyx mori, an economically important insect that has been used for silk production for over 5000 years, is a remarkable organ that produces vast amounts of silk with exceptional properties . Little is known about which SG cells execute silk protein synthesis and its precise spatiotemporal control. Here, we used single-cell RNA-seq to build a comprehensive cell atlas of the B. mori SG, consisting of 14,972 high-quality cells representing 10 distinct cell types, in three early developmental stages. We annotated all 10 cell types and determined their distributions in each region of the SG, decoded their developmental trajectory and gene-switch status, and discovered marker genes involved in the regulation of SG development and silk protein synthesis. Our study reveals the high heterogeneity of B. mori SG cells and their gene expression dynamics for the first time, affording a deeper understanding of silk-producing organs at the single-cell level .
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:In this study, we sequenced four small RNA libraries derived from mature pollens, in vitro germinated pollens, mature silks and pollinated silks of maize, respectively. In total, 161 known miRNAs belonging to 27 families and 82 novel miRNAs were identified. Of them, miRNAs involved in pollen-silk (pistil) interactions were analyzed. On the male side, miRNA differentially expressed between mature and germinated pollen were identified, some of them participate in pollen germination and tube growth. On the female side, silk-expressed miRNAs respond to pollination were also responsive to stresses, especially drought and fungal invasion. Furthermore, GO analysis of target genes revealed that members related to anxin signal transduction and gene expressional regulation were overrepresented.The results indicated that during pollen-silk interactions, miRNAs-mediated auxin signal transduction plays important roles, and miRNAs took part in complex transcriptional regulating network.