Genome-wide survey and characterization of transcription factors in the silk gland of the silkworm, Bombyx mori

Transcription factors (TFs) are key proteins that modulate gene transcription and thereby lead to changes in the gene expression profile and the subsequent alteration of cellular functions. In the silk gland (SG) of silkworm Bombyx mori, an important silk-producing insect, TFs are of vital importance in the regulation of silk protein synthesis in this organ. However, which TFs exist and express in the SG remains largely unknown. Here, we report the large-scale identification of TFs in the SG based on available full-length transcript sequences and the most recent version of silkworm genome data. In total, 348 candidate TFs were identified by strict filtration and were classified into 56 TF families. Chromosomal distribution, motif composition, and phylogenetic relationship analyses revealed the typical characteristics of these TFs. In addition, the expression patterns of 348 TFs in various tissues of B. mori, especially the SG of fourth-molt (4LM) and day-3 and day-4 fifth-instar (5L3D and 5L4D) larvae, were investigated based on public RNA-seq data and gene microarray data, followed by spatiotemporal verification of TF expression levels by quantitative real-time PCR (qRT-PCR). This report describes the first comprehensive analysis of TFs in the B. mori SG. The results can serve as a baseline for further studies of the roles of TFs in the B. mori SG.

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori

Background With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5′ GG motif for efficient initiation. The resulting transcripts bear a 5′ GG motif, which significantly constrains the number of targetable sites in the silkworm genome. Results In this study, we used the T7 promoter to add two supernumerary G residues to the 5′ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5′ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5′ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5′ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5′ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5′ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. Conclusions In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

Expansion of Targetable Sites for the Ribonucleoprotein-Based CRISPR/Cas9 System in the Silkworm Bombyx Mori

Background:With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5’ GG motif for efficient initiation. The resulting transcripts bear a 5’ GG motif, which significantly constrains the number of targetable sites in the silkworm genome. Results:In this study, we used the T7 promoter to add two supernumerary G residues to the 5’ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5’ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5’ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5’ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5’ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5’ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. Conclusions: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

SilkDB 3.0: visualizing and exploring multiple levels of data for silkworm

SilkDB is an open-accessibility database and powerful platform that provides comprehensive information on the silkworm (Bombyx mori) genome. Since SilkDB 2.0 was released 10 years ago, vast quantities of data about multiple aspects of the silkworm have been generated, including genome, transcriptome, Hi-C and pangenome. To visualize data at these different biological levels, we present SilkDB 3.0 (https://silkdb.bioinfotoolkits.net), a visual analytic tool for exploring silkworm data through an interactive user interface. The database contains a high-quality chromosome-level assembly of the silkworm genome, and its coding sequences and gene sets are more accurate than those in the previous version. SilkDB 3.0 provides a view of the information for each gene at the levels of sequence, protein structure, gene family, orthology, synteny, genome organization and gives access to gene expression information, genetic variation and genome interaction map. A set of visualization tools are available to display the abundant information in the above datasets. With an improved interactive user interface for the integration of large data sets, the updated SilkDB 3.0 database will be a valuable resource for the silkworm and insect research community.

SGID: a comprehensive and interactive database of the silkworm

Although the domestic silkworm (Bombyx mori) is an important model and economic animal, there is a lack of comprehensive database for this organism. Here, we developed the silkworm genome informatics database, SGID. It aims to bring together all silkworm related biological data and provide an interactive platform for gene inquiry and analysis. The function annotation in SGID is thorough and covers 98% of the silkworm genes. The annotation details include function description, gene ontology, KEGG, pathway, subcellular location, transmembrane topology, protein secondary/tertiary structure, homologous group and transcription factor. SGID provides genome scale visualization of population genetics test results based on high depth resequencing data of 158 silkworm samples. It also provides interactive analysis tools of transcriptomic and epigenomic data from 79 NCBI BioProjects. SGID is freely available at http://sgid.popgenetics.net. This database will be extremely useful to silkworm research in the future.

Genome-Wide Analysis and Hormone Regulation of Chitin Deacetylases in Silkworm

Chitin deacetylases (CDAs) are a group of enzymes involved in chitin metabolism in insects; they play a critical role in molting, pupation, and the modification of chitin. In this study, we identified several CDAs in the silkworm, Bombyx mori (BmCDA), and investigated the effect of various hormones on their expression in B. mori larvae and embryo cell lines (BmE). Eight genes encoding BmCDAs were identified in the silkworm genome. They showed different expression patterns in different tissues, and were classified into three types based on where they were expressed: the exoskeleton, digestive organs, and genital organs. Moreover, we found that some BmCDAs showed upregulated expression during the molting period, especially during the fourth molting period in larvae. We also verified that the expression of BmCDA1–6 was upregulated by treatment with 20-hydroxyecdysone not only in larvae, but also in BmE cells. Interestingly, juvenile hormone analog treatment also upregulated the expression of some BmCDAs. The overexpression of several transcription factors revealed that the POU transcription factor POUM2 may play a major role in the regulation of BmCDA expression. Finally, the silencing of BmCDA1 and BmCDA2 did not lead to abnormal phenotypes or death, but may have led to delays in silkworm pupation. These results provide important information about lepidopteran insects in terms of chitin deacetylases and the regulation of their expression.

SGID: a comprehensive and interactive database of the silkworm

Although the domestic silkworm (Bombyx mori) is an important model and economic animal, there is a lack of comprehensive database for this organism. Here, we developed the silkworm genome informatics database (SGID). It aims to bring together all silkworm-related biological data and provide an interactive platform for gene inquiry and analysis. The function annotation in SGID is thorough and covers 98% of the silkworm genes. The annotation details include function description, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, subcellular location, transmembrane topology, protein secondary/tertiary structure, homologous group and transcription factor. SGID provides genome-scale visualization of population genetics test results based on high-depth resequencing data of 158 silkworm samples. It also provides interactive analysis tools of transcriptomic and epigenomic data from 79 NCBI BioProjects. SGID will be extremely useful to silkworm research in the future.

Artificial Selection on Storage Protein 1 Contributes to Increase of Hatchability during Silkworm Domestication

Like other domesticates, efficient utilization of nitrogen resource is also important for the domestic insect, the silkworm. Deciphering how artificial selection act on silkworm genome for improved utilization of nitrogen resource and further human-favored domestication traits will provide unique cues from the insect scenario for understanding general rules of Darwin’s evolutionary theory on domestication. Storage proteins (SP), which belong to a hemocyanin superfamily, basically serve as a source of amino acids and nitrogen during metamorphosis and reproduction in insects. Here through genomic search and further screening of artificial selection signature on silkworm SPs, we discovered a candidate domestication gene, i.e. the methionine-rich storage protein1 (SP1), which is uniquely diverged from the others and showed increased expression in the ova of domestic silkworms. Knockout of SP1 via CRISPR/Cas9 approach resulted in dramatic decrease in egg hatchability, without obvious impact on egg production, which was similar to the case in the wild silkworm compared with domestic one. Larval development or metamorphosis were not affected by SP1 knockout. Comprehensive ova comparative transcriptomes indicated a general repression of gene expression, specifically vitellogenin, chorion proteins and structural component proteins in the extracellular matrix (ECM)-interaction pathway, as well as enzymes in folate biosynthesis, in both the mutant and the wild silkworm with the mutated allele, compared to the wild type domestic silkworm. Wild silkworms with the wild allele also showed generally down-regulated expression of genes enriched in structural constituent of ribosome and amide and peptide biosynthesis. This study exemplified a novel case that artificial selection could directly act on nitrogen resource protein to affect egg nutrient and eggshell formation, and activate ribosome for improved biosynthesis and increased hatchability during domestication. The findings shed new light on both understanding of artificial selection and silkworm breeding from the angle of nitrogen and amino acid resource.Author summaryLike other domesticates, nitrogen resource is also important for the domestic insect, the silkworm. Deciphering how artificial selection act on silkworm genome for improved utilization of nitrogen resource and further human-favored domestication traits, will provide unique cues from insect scenario, for understanding general rules of Darwin’s evolutionary theory. However, mechanism of domestication in the silkworm is largely unknown to date. Here we focused on one important nitrogen resource, i.e, the storage proteins (SP). We discovered that the methionine-rich storage protein1 (SP1) which is divergent from the other SPs are the only target of the artificial selection. We proposed based on functional evidence together with the key findings of comprehensive comparative transcriptome, that artificial selection, on one hand favored higher expression of SP1 in the domestic silkworm, which would subsequently up-regulate the genes or pathways vital for egg development and eggshell formation. On the other hand, artificial selection consistently favored activated ribosome activities and improved amide and peptide biosynthesis and in the ova, as it might act in the silk gland for increased silk-cocoon yield. We here exemplified a novel case that artificial selection could directly act on nitrogen resource protein for human desired domestication trait.

Genome-wide identification and analysis of elongase of very long chain fatty acid genes in the silkworm, Bombyx mori

Very long chain fatty acids (VLCFAs), such as sphingolipids, are components of cellular lipids, which are essential for cell proliferation. Mutations in the genes that encode proteins participating in VLCFA biosynthesis may cause inherited diseases, such as macular degeneration. Elongases of very long chain fatty acid (ELOVL) are enzymes that are involved in the biosynthesis of VLCFAs. Here, a total of 13 ELOVL genes, distributed across three chromosomes, were identified in the silkworm genome; all the ELOVL members contain a distinct ELO domain and a conserved HXXHH motif. Phylogenetic reconstruction was performed to analyze the evolutionary relationships among different species and to predict gene functions. The 13 ELOVL genes were assigned to the ELOVL3/6, ELOVL1/7, and ELOVL4 clades. Microarray and semiquantitative PCR analyses indicated that these genes are differentially expressed among various tissues, in turn suggesting functional divergence in the growth and development of each tissue. Further investigation showed that the expression level of the BGIBMGA000424 gene is significantly negatively correlated with the cocoon-shell weight among different silkworm strains. Taken together, the present study is the first comprehensive analysis of ELOVL genes in silkworm, and the results may serve as a foundation for further analysis of the physiological functions of ELOVL genes in silkworm.