Sericin Gene Research Articles

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.

Characterization of silk genes in Ephestia kuehniella and Galleria mellonella revealed duplication of sericin genes and highly divergent sequences encoding fibroin heavy chains.

Silk is a secretory product of numerous arthropods with remarkable mechanical properties. In this work, we present the complete sequences of the putative major silk proteins of E. kuehniella and compare them with those of G. mellonella, which belongs to the same moth family Pyralidae. To identify the silk genes of both species, we combined proteomic analysis of cocoon silk with a homology search in transcriptomes and genomic sequences to complement the information on both species. We analyzed structure of the candidate genes obtained, their expression specificity and their evolutionary relationships. We demonstrate that the silks of E. kuehniella and G. mellonella differ in their hydrophobicity and that the silk of E. kuehniella is highly hygroscopic. In our experiments, we show that the number of genes encoding sericins is higher in G. mellonella than in E. kuehniella. By analyzing the synteny of the chromosomal segment encoding sericin genes in both moth species, we found that the region encoding sericins is duplicated in G. mellonella. Finally, we present the complete primary structures of nine fibH genes and proteins from both families of the suborder Pyraloidea and discuss their specific and conserved features. This study provides a foundation for future research on the evolution of silk proteins and lays the groundwork for future detailed functional studies.

Sericin for Tissue Engineering

Sericin is a 10-to-400 kDa hydrophilic protein with high serine content and is a silk constituent together with fibroin. It is produced in the middle silk gland of the silkworm and encoded by four sericin genes. The molecular weight of sericin and its biological activity vary depending on the extraction method employed. Its chemical structure, in terms of random coil and β-sheet conformations, also differs with the extraction method, thereby extending its applications in various fields. Sericin, which was discarded in the textile industry in the past, is being applied and developed in the biomedical field, owing to its biological properties. In particular, many studies are underway in the field of tissue engineering, evaluating its applicability in burn dressing, drug delivery, bone regeneration, cartilage regeneration, and nerve regeneration.

Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera.

Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.

Identification of Bombyx mori sericin 4 protein as a new biological adhesive.

Sericins are large proteins with molecular weights >70 kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280 kDa and 260 kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks.

The expansion of genes encoding soluble silk components in the greater wax moth, Galleria mellonella

Lepidopteran silk is a complex assembly of proteins produced by a pair of highly specialized labial glands called silk glands. Silk composition has been examined only in a handful of species. Here we report on the analysis of silk gland-specific transcriptomes from three developmental stages of the greater wax moth, Galleria mellonella, combined with proteomics, Edman microsequencing and northern blot analysis. In addition to the genes known earlier, we identified twenty seven candidate cDNAs predicted to encode secretory proteins, which may represent novel silk components. Eight were verified by proteomic analysis or microsequencing, and several others were confirmed by similarity with known silk genes and their expression patterns. Our results revealed that most candidates encode abundant secreted proteins produced by middle silk glands including ten sericins, two seroins, one or more mucins, and several sequences without apparent similarity to known proteins. We did not detect any novel PSG-specific protein, confirming that there are only three fibroin subunits. Our data not only show that the number of sericin genes in the greater wax moth is higher than in other species thus far examined, but also the total content of soluble proteins in silk is twice as high in G. mellonella than in B. mori or A. yamamai. Our data will serve as a foundation for future identification and evolutionary analysis of silk proteins in the Lepidoptera.

Bmo-miR-275 down-regulates expression of Bombyx mori sericin gene 2 in vitro.

We hypothesized that bmo-miR-275 has a potential regulatory function regarding the expression of sericin gene 2 (BmSer-2). First, we examined the expression of bmo-miR-275 and its target gene BmSer-2 in seven different tissues from 5th instar day-3 silkworm larvae. The results showed that they were both specifically expressed in the middle silk gland, implying that spatio-temporal conditions are required for bmo-miR-275 to regulate the expression of BmSer-2. To test this hypothesis, we constructed a pri-bmo-miR-275 expressing plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and BmSer-2-3´UTR recombinant reporter plasmids pGL3.0 [A3-luc-Ser-2-3′ UTR-SV40]. Finally, BmN cells were harvested and luciferase activity was detected. Results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected with pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and pGL3.0 [A3-luc-Ser-2-3’UTR-SV40], suggesting that bmo-miR-275 can down-regulate the expression of BmSer-2 in vitro. Our results improve the understanding of the regulatory function of Bombyx mori miRNA on the expression of genes regulating silk formation.

Sericin Composition in the Silk of Antheraea yamamai.

The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well-known in Bombyx mori (Bombycidae) but have received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains five sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition.

Transcriptome sequencing and positive selected genes analysis of Bombyx mandarina.

The wild silkworm Bombyx mandarina is widely believed to be an ancestor of the domesticated silkworm, Bombyx mori. Silkworms are often used as a model for studying the mechanism of species domestication. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform. We produced 100,004,078 high-quality reads and assembled them into 50,773 contigs with an N50 length of 1764 bp and a mean length of 941.62 bp. A total of 33,759 unigenes were identified, with 12,805 annotated in the Nr database, 8273 in the Pfam database, and 9093 in the Swiss-Prot database. Expression profile analysis found significant differential expression of 1308 unigenes between the middle silk gland (MSG) and posterior silk gland (PSG). Three sericin genes (sericin 1, sericin 2, and sericin 3) were expressed specifically in the MSG and three fibroin genes (fibroin-H, fibroin-L, and fibroin/P25) were expressed specifically in the PSG. In addition, 32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. Comparison with the domesticated silkworm p50/Dazao identified 5,295 orthologous genes, among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research.

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.

Functional Conservation and Structural Diversification of Silk Sericins in Two Moth Species

Sericins are hydrophilic structural proteins produced by caterpillars in the middle section of silk glands and layered over fibroin proteins secreted in the posterior section. In the process of spinning, fibroins form strong solid filaments, while sericins seal the pair of filaments into a single fiber and glue the fiber into a cocoon. Galleria mellonella and the previously examined Bombyx mori harbor three sericin genes that encode proteins containing long repetitive regions. Galleria sericin genes are similar to each other and the protein repeats are built from short and extremely serine-rich motifs, while Bombyx sericin genes are diversified and encode proteins with long and complex repeats. Developmental changes in sericin properties are controlled at the level of gene expression and splicing. In Galleria , MG-1 sericin is produced throughout larval life until the wandering stage, while the production of MG-2 and MG-3 reaches a peak during cocoon spinning.

Identification of Ser2 proteins as major sericin components in the non-cocoon silk of Bombyx mori

Sericins are glue proteins of Bombyx mori silk fibers. They are produced in the middle silk gland (MSG) cells, stored in the lumen, and pushed out from the spinneret surrounding the fibroin fibers. The Ser2 gene was partly cloned from the anterior region of the MSG more than 20 years ago and is regarded as a sericin-encoding gene; however, Ser2 proteins do not appear to be major components of cocoon proteins. We used northern blotting to analyze the expression of three sericin genes— Ser1, Ser2, and Ser3—in the MSG of third to fifth instar larvae and measured the corresponding cDNA levels by competitive PCR. The results revealed that Ser2 gene expression dominated until the middle period of the fifth instar, while the expression of the other genes was mainly observed after the middle fifth instar. Protein analysis demonstrated that the two Ser2 proteins produced by alternative splicing were major coating proteins of larval silk threads spun during the growing stages. The molecular components of larval silk sericin were completely different from those of cocoon sericin, and the difference may be related to the functions of the two kinds of silk fibers.

The genome of a lepidopteran model insect, the silkworm Bombyx mori

Bombyx mori, the domesticated silkworm, is a major insect model for research, and the first lepidopteran for which draft genome sequences became available in 2004. Two independent data sets from whole-genome shotgun sequencing were merged and assembled together with newly obtained fosmid- and BAC-end sequences. The remarkably improved new assembly is presented here. The 8.5-fold sequence coverage of an estimated 432 Mb genome was assembled into scaffolds with an N50 size of ∼3.7 Mb; the largest scaffold was 14.5 million base pairs. With help of a high-density SNP linkage map, we anchored 87% of the scaffold sequences to all 28 chromosomes. A particular feature was the high repetitive sequence content estimated to be 43.6% and that consisted mainly of transposable elements. We predicted 14,623 gene models based on a GLEAN-based algorithm, a more accurate prediction than the previous gene models for this species. Over three thousand silkworm genes have no homologs in other insect or vertebrate genomes. Some insights into gene evolution and into characteristic biological processes are presented here and in other papers in this issue. The massive silk production correlates with the existence of specific tRNA clusters, and of several sericin genes assembled in a cluster. The silkworm's adaptation to feeding on mulberry leaves, which contain toxic alkaloids, is likely linked to the presence of new-type sucrase genes, apparently acquired from bacteria. The silkworm genome also revealed the cascade of genes involved in the juvenile hormone biosynthesis pathway, and a large number of cuticular protein genes.

Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori

Sericin is a group of proteins expressed in the middle silk gland that covers the surface of fibroin in the cocoon filament of Bombyx mori. Sericin consists of several serine-rich proteins with different molecular masses. Sericin A is one of the proteins and is produced in the anterior portion of the middle silk gland. To identify the gene coding for the protein, we determined the primary structures of its partial peptides, and the gene was searched using the silkworm genomic databases. Three contigs containing the corresponding nucleotide sequences were identified and categorized as one group. The gene structure covering the 5′ flanking and the 3′ end was determined by PCR fragments from genomic DNA, RT-PCR, and 5′ and 3′ RACE. The amino acid sequence deduced from the nucleotide sequence mainly consists of two serine-rich regions of 86-amino acid motif and 8-amino acid repeated sequence. The expression of the gene is limited to the anterior and middle parts of the middle silk gland. In addition, because the sericin gene appeared different from the sericin 1 and 2 genes reported earlier, we designated the newly discovered gene as sericin 3.

Silk gland specific cDNAs from Galleria mellonella L.

Four classes of Galleria mellonella silk gland specific cDNAs, designated PG-1, PG-2, MG-1 and MG-2, were cloned, sequenced and used as probes in Northern and Southern blots. Analysis of the results and their comparison with the data available for Bombyx mori reveal that the isolated cDNAs correspond to mRNAs for major silk proteins. Two small transcripts (1.1 and 1.2 kb), detected with PG-1 in the posterior region of Galleria silk glands, represent mRNAs for small proteins related to the light chain fibroin of Bombyx. A large transcript (⩾ 10 kb) homologous to PG-2 seems to be the mRNA for the heavy chain fibroin. Conceptual translation of available PG-2 sequence yields a product with similar amino acid composition as reported for the chemically analyzed fibroin. Hybridization to Southern blots of Galleria genomic DNA showed that PG-1 and PG-2 correspond to separate genes. In the middle silk gland region, multiple transcripts homologous to MG-1 are produced. They include 1.9 and 4.2 kb species, and in certain developmental periods also 3.2, 7.2 and ⩾ 10 kb products. Two different transcripts, one dominant (3.4 kb) and one rare (5.2 kb) were revealed with the MG-2 probe. MG-1 and MG-2 cDNAs contain somewhat similar repeats, and their deduced translation products resemble Bombyx sericins by the dominance of serine and the high content of glycine and asparagine. Southern blots indicated that MG-1 and MG-2 represent different sericin genes apparently corresponding to Ser-1 and Ser-2 genes of Bombyx.

Transcription signals and protein binding sites for sericin gene transcription in vitro

Using nuclear extracts from Bombyx mori middle silk glands (MSG) where the sericin-1 (Ser-1) mRNA is produced specifically, we have studied the in vitro transcription of the Ser-1 gene. To determine the sites required for the promoter function of the Ser-1 gene and characterize the factors binding to these sites, the upstream region was dissected. Footprinting assays revealed the presence of at least three specific protein binding sites, SA (-103 to -85), SB (-149 to -135) and SC (-204 to -183). Removal of SA and SC by 5'-deletions decreased the promoter activity. DNase I footprinting using four extracts of B. mori origin indicated that the factor binding to SA is specific to the silk gland extracts, and the factor binding to SC is very much abundant in the MSG extracts. Stimulation of the promoter activity with the region from -239 to -174 that accommodates the SC site, is stronger in MSG extracts than in the extracts from the posterior silk glands. These results suggest that the SC binding factor abundant in MSG may play an important role in the MSG-specific expression of the Ser-1 gene.

Temporal and spatial control of silk gene transcription analyzed by nuclear run-on assays

In vivo transcriptional activity of the Bombyx mori fibroin and sericin genes was estimated by nuclear run-on assay. Tissue-specific and developmental stage-specific expression of the fibroin and sericin genes through larval development was shown to be primarily regulated at the level of transcription. The expression patterns of these two genes are quite different in their developmental profile, suggesting that they are probably regulated by different underlying mechanisms. A finer spatial control of transcription could be found. The sericin gene transcription is mostly restricted to the posterior region of the middle silk gland throughout the fifth larval instar. Interestingly, the fibroin gene transcription is observed only in the anterior region of the posterior silk gland on the first day of the fifth instar, and spreads toward the posterior region as the fifth instar stage proceeds.

Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silkgland

Four mRNA of 10.5, 9.0, 4.0, and 2.8 kb are made from the sericin Ser1 gene by alternative maturation of a unique mRNA precursor. By means of RNA blots and in situ hybridization, we investigated variations in the distribution of these mRNA during the last larval instar in different territories of the middle silkgland. Taken together, the results from these two techniques show that 150 out of the 266 cells of this region of the organ express the Ser1 gene, but accumulate distinct mature mRNA species. Of these 150 cells 42 are specialized in a processing pathway resulting in the production of the 2.8-kb Ser1 mRNA throughout the larval instar. The 108 others perform successively three distinct splicing pathways leading to a development-dependent accumulation of, respectively, the 4.0-, the 10.5-, and the 9.0-kb mRNA. This suggests the occurrence of two switches in the splicing capacities of these cells during the fifth instar. The middle silkgland cells also express another sericin gene (Ser2) which encodes two mRNA of 5.4 and 3.1 kb, also arising by differential splicing. At the beginning of development, all the middle silkgland cells express this gene but, as development proceeds, expression becomes restricted to only the anterior cells. The biological consequence of this topological and temporal regulation of the mode of expression of these two genes is the sequential secretion and layering of the different sericins around the silk thread.

Changes of DNaseI Sensitivity of the Fibroin and Sericin Genes during the Silkworm Development: (DNaseI sensitivity/gene expression/silk gland/chromatin structure).

DNasel sensitivity assay of the fibroin gene and the sericin gene in different tissues along developmental stages have revealed unique patters of regulation. In the posterior silk gland the fibroin gene was more sensitive to DNaseI at the larval feeding stages, when the gene is transcriptionally active, than at the larval molting stages, when the gene is inactive. In the middle silk gland and the ovarian tissue where the fibroin gene is suppressed throughout development, the gene was found to be resistant to DNaseI. On the other hand the sericin gene remained rather DNaseI sensitive in the middle silk gland through larval development, where transcription activity of the gene changes drastically. Disappearance of DNaseI sensitivity of the fibroin gene at the fourth molting stage takes place earlier than the decline of the fibroin gene transcription. However, in the fifth feeding stagereappearance of DNaseI sensitivity occurs synchronously with resumption of the fibroin gene transcription. Thus a constitution of the DNaseI sensitivity may be required, but not sufficient to support an active state of genes.

Haemolymph control of sericin gene expression studied by organ transplantation

A factor that affects synthesis of sericin mRNAs of Bombyx mori was analyzed by organ transplantation and allatectomy. When silk glands of the third instar larvae were transplanted into the abdomen of fifth instar larvae, substantial amounts of sericin mRNAs were induced in the transplant. The induced sericin gene activity was suppressed upon re-transplantation into the abdomen of fourth instar larvae and induced again when the second hosts grew up to fifth instar larvae. An allatectomy performed on fourth instar larvae promoted production of these mRNAs, suggesting that the synthesis of sericin mRNA is regulated by the titer of juvenile hormone.