Expression of the hGM-CSF in the silk glands of germline of gene-targeted silkworm

To express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene in the silk glands of transformation silkworm (Bombyx mori) based on gene-targeting, two fragments from fibroin heavy chain gene (fib-H) of silkworm were cloned and sequenced. One fragment contains the 1st exon and its downstream 1st intron’s partial sequence; and the other fragment contains the 1st intron’s partial sequence and the 2nd exon’s partial sequence. Then the two fragments, as homologous arm, were inserted into pSK to generate a gene-targeted vector, pSK-HL-A3GFP-FLP-GM-CSF-FLPA-HR in which a gfp gene driven by A3 promoter and an hGM-CSF gene under the control of fibroin light chain (fib-L) promoter were included. The vector was transferred into the silkworm eggs using sperm-mediated gene transfer. After being screened for green fluorescent, the transformation silkworm was obtained, whose genome was verified by PCR and dot hybridization to confirm whether the target genes had been integrated into the silkworm genome. Furthermore, in the posterior silk glands of the G4 generation transformation silkworms, a specific band with the molecular weight of 22 kDa could be detected by Western blotting with an antibody against hGM-CSF, and the expression level of the hGM-CSF estimated by ELISA was approximately 1.26 ng per gram fresh posterior silk gland.     

Determination of the multiplicity of the silk fibroin gene and detection of fibroin gene-related DNA in the genome of Bombyx mori.

The number of silk fibroin genes per genome in the silkworm Bombyx mori has been determined by hybridization using fibroin [¹²⁵I]mRNA. The purified [¹²⁵I]mRNA had an oligonucleotide pattern after RNAase T₁ digestion which was characteristic of fibroin mRNA (Suzuki &; Brown, 1972) and it hybridized specifically to DNA with a G + C content expected for a fibroin gene. Thermal denaturations indicated that these hybrids were mismatched by about 3%, which probably indicates some variation among the sequences encoding the internal repetitions of the fibroin protein.The concentration of fibroin gene sequences in B. mori DNA was measured by saturation hybridization of [¹²⁵I]mRNA to filter bound DNA. The same saturation level of 1.8 × 10^?5 μg mRNA per μg DNA was calculated from data obtained with unfractionated DNA and with fibroin gene sequences which had been separated from bulk B. mori DNA by actinomycin $\text{D}\text{CsCl}$ centrifugation. Scatchard plots of the subsaturation data extrapolated to an identical saturation value. Internal reiteration of the fibroin mRNA molecule was apparent from the high association constant of hybridization. An exhaustive hybridization experiment showed that such repetitions comprise at least 90% of each mRNA molecule. The saturation value, in conjunction with the genome DNA content and the mRNA size, indicated the presence of only one fibroin gene per haploid B. mori genome.Hybridization of actinomycin $\text{D}\text{CsCl}$ fractionated DNA indicated that fibroin mRNA can form hybrids with DNA that bands with bulk B. mori DNA. These hybrids appear to involve DNA which is related to, but distinguishable from, true fibroin gene sequences. The fibroin gene-related sequences form mismatched hybrids with the mRNA, are much shorter than the fibroin gene and are dispersed in B. mori DNA of much lower G + C content, and there are many copies of these sequences per B. mori genome.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Reducing blood glucose level in TIDM mice by orally administering the silk glands of transgenic hIGF-I silkworms

To realize the secretory expression of human insulin-like growth factor-I (hIGF-I) in the posterior silk glands (PSGs) of transgenic silkworms, the piggyBac transposon vector pigA3GFP-fibHS-hIGF-i.e.-neo containing a neomycin-resistance gene (neo), green fluorescent protein gene (gfp) and human insulin-like growth factor I (hIGF-I) gene controlled by the Bombyxmori fibroin heavy chain gene (fib-H) promoter with its downstream signal peptide sequence, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 gene (A3) promoter were transferred into silkworm eggs by sperm-mediated gene transfer. Transformed silkworms were obtained after being screened for green fluorescence and by the antibiotic G418. In the PSGs of the transformed silkworms, a specific band representing hIGF-I could be detected by Western blotting, and the content of the hIGF-I estimated by ELISA was approximately 1.84 μg/gram of cocoon and 19.18 μg/gram of freeze-dried PSG powder. To further estimate the biological activity of the expressed hIGF-I, streptozotocin-induced TIDM mice were orally administered with the PSG powder of the transgenic silkworms, the results showed the blood glucose levels of mice were significantly reduced, suggesting that the the PSGs powder of transgenic hIGF-I silkworms could possibly be used as a perorally administered medicine.     

Phylogeny and evolutionary history of the silkworm

The silkworm, Bombyx mori, played an important role in the old Silk Road that connected ancient Asia and Europe. However, to date, there have been few studies of the origins and domestication of this species using molecular methods. In this study, DNA sequences of mitochondrial and nuclear loci were used to infer the phylogeny and evolutionary history of the domesticated silkworm and its relatives. All of the phylogenetic analyses indicated a close relationship between the domesticated silkworm and the Chinese wild silkworm. Domestication was estimated to have occurred about 4100 years ago (ya), and the radiation of the different geographic strains of B. mori about 2000 ya. The Chinese wild silkworm and the Japanese wild silkworm split about 23600 ya. These estimates are in good agreement with the fossil evidence and historical records. In addition, we show that the domesticated silkworm experienced a population expansion around 1000 ya. The divergence times and the population dynamics of silkworms presented in this study will be useful for studies of lepidopteran phylogenetics, in the genetic analysis of domestic animals, and for understanding the spread of human civilizations.     

Molecular phylogeny of the domesticated silkworm,Bombyx mori, based on the sequences of mitochondrialcytochrome b genes

Pupae from the Chinese wild mulberry silkworm,Bombyx mandarina, and 11 representative strains of the domesticated silkworm,Bombyx mori were selected for preparation of mitochondrial DNA. The 5′-end fragments ofcytochrome b genes (Cytb) were generated by polymerase chain reaction products and sequenced directly. The homologous sequences of the JapaneseB. mandarina and three strains ofB. mori were from the GenBank database. The sequences of the 16 silkworm strains were analysed with DNASTAR software and a phylogenic tree was constructed using PHYLIP software. The result showed that: (i) The sequence divergence between the strains ofB. mori and the JapaneseB. mandarina was larger (5.4% ≈ 5.8%) compared with that between strains ofB. mori and the ChineseB. mandarina (0.8% ≈ 1.9%). Analysis of clustering also showed that the sequences ofB. mori strains and ChineseB. mandarina clustered into group (B group), while that of JapaneseB. mandarina (A group) was outside this cluster. This may be evidence for the hypothesis thatB. mori originated from ChineseB. mandarina. (ii) Among 14 strains ofB. mori, sequence divergence was small and the most divergence was seen between strains Yanhe-1 and Chuxiong, whose sequences branched off from those of the otherB. mori strains on the phylogenetic tree. From this and from historical records, we infer that the strains Yanhe-1 and Chuxiong originated independently from southwest China.     

Molecular identification of the strains of the domestic silkworm,Bombyx mori (Lepidoptera: Bombycidae), which are endemic to Korea,based on single nucleotide polymorphisms in mitochondrial genome sequences

The domestic silkworm, Bombyx mori (Lepidoptera: Bombycidae), has been diversified into various strains over a long period. However, methods to distinguish silkworm strains remain limited partially owing to the genetic similarity caused by the long history of domestication. In this study, we developed molecular identification methods to distinguish three domestic silkworm strains, which are endemic to Korea. By comparing publicly available complete mitochondrial genome (mitogenome) sequences of five endemic strains and 34 stock silkworm strains analyzed in a previous study, we detected 15 single nucleotide polymorphisms (SNPs; SNP1–SNP15), which distinguished the following three endemic strains: Sun7ho (SN7), Sandongsammyeon (SDS), and Sammyeonhonghoeback (SMH). We used two SNPs for each strain to identify the three endemic strains. To distinguish each SN7 and SDS from the remaining four endemic and 34 stock strains, the PCR-restriction fragment length polymorphism method was employed using Acu I and Hpa I restriction enzymes, which recognize SNP1 and SNP8, respectively. Additionally, the tetra-primer amplification refractory mutation system PCR method was used to determine the regions containing SNP3, SNP11, and both SNP14 and SNP15 to distinguish SN7, SDS, and SMH, respectively, from the remaining strains. A validation test with additional individuals showed that each target strain was clearly recognized, suggesting that mitogenome SNP-based methods can be used to identify three endemic silkworm strains during culture and breeding.     

Non-uniqueness of factors constraint on the codon usage in Bombyx mori

Background

The analysis of codon usage is a good way to understand the genetic and evolutionary characteristics of an organism. However, there are only a few reports related with the codon usage of the domesticated silkworm, Bombyx mori (B. mori). Hence, the codon usage of B. mori was analyzed here to reveal the constraint factors and it could be helpful to improve the bioreactor based on B. mori.

Results

A total of 1,097 annotated mRNA sequences from B. mori were analyzed, revealing there is only a weak codon bias. It also shows that the gene expression level is related to the GC content, and the amino acids with higher general average hydropathicity (GRAVY) and aromaticity (Aromo). And the genes on the primary axis are strongly positively correlated with the GC content, and GC3s. Meanwhile, the effective number of codons (ENc) is strongly correlated with codon adaptation index (CAI), gene length, and Aromo values. However, the ENc values are correlated with the second axis, which indicates that the codon usage in B. mori is affected by not only mutation pressure and natural selection, but also nucleotide composition and the gene expression level. It is also associated with Aromo values, and gene length. Additionally, B. mori has a greater relative discrepancy in codon preferences with Drosophila melanogaster (D. melanogaster) or Saccharomyces cerevisiae (S. cerevisiae) than with Arabidopsis thaliana (A. thaliana), Escherichia coli (E. coli), or Caenorhabditis elegans (C. elegans).

Conclusions

The codon usage bias in B. mori is relatively weak, and many influence factors are found here, such as nucleotide composition, mutation pressure, natural selection, and expression level. Additionally, it is also associated with Aromo values, and gene length. Among them, natural selection might play a major role. Moreover, the “optimal codons” of B. mori are all encoded by G and C, which provides useful information for enhancing the gene expression in B. mori through codon optimization.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1596-z) contains supplementary material, which is available to authorized users.     

Syntheses of glycine and L-serine by their interconversion in the posterior silkgland of the silkworm, Bombyx mori

Summary. It was observed by solution-state ¹³C NMR spectroscopy that a great portion of the ¹³C of [1-¹³C]L-serine fed to the 5th instar larvae of the silkworm, Bombyx mori was incorporated into C1 of glycine in silk fibroin. [1-¹³C]Glycine was detected along with [1-¹³C]serine in fibroin of the posterior silkgland cultured in a medium containing [1-¹³C]serine. This formation of [1-¹³C]glycine was inhibited by addition of aminopterin to the culture medium. These findings suggest that an active conversion from serine to glycine, which needs tetrahydrofolate, occurs in the posterior silkgland for fibroin synthesis. Moreover, the solid-state ¹³C CP/MAS spectrum of the fibroin prepared from cocoons spun by larvae fed with [¹³C]formate revealed that serine C3 was labelled specifically with ¹³C, suggesting that the reverse conversion from glycine to serine took place in the silkworm. The posterior silkgland has the ability to synthesize not only fibroin but also its major materials, glycine and serine. Received May 4, 1999 Accepted December 10, 1999     

Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori

ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.     

A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran insects

A second-generation linkage map was constructed for the silkworm, Bombyx mori, focusing on mapping Bombyx sequences appearing in public nucleotide databases and bacterial artificial chromosome (BAC) contigs. A total of 874 BAC contigs containing 5067 clones (22% of the library) were constructed by PCR-based screening with sequence-tagged sites (STSs) derived from whole-genome shotgun (WGS) sequences. A total of 523 BAC contigs, including 342 independent genes registered in public databases and 85 expressed sequence tags (ESTs), were placed onto the linkage map. We found significant synteny and conserved gene order between B. mori and a nymphalid butterfly, Heliconius melpomene, in four linkage groups (LGs), strongly suggesting that using B. mori as a reference for comparative genomics in Lepidotera is highly feasible.     

Diversity analysis of Beauveria bassiana isolated from infected silkworm in southwest China based on molecular data and morphological features of colony

Beauveria bassiana is an important entomopathogenic fungus that not only often causes infection and epidemics of wild insects but some strains also show pathogenicity to the silkworm, Bombyx mori. The present study is about diversity of B. bassiana isolated from the silkworm in southwest China. Five strains of B. bassiana were isolated from infected silkworm. Two isolates, GXtr1009 and GXtr1010, were isolated from infected silkworms treated with two kinds of biological pesticides applied in Guangxi province, and three isolates, SCsk1006, YNsk1106 and GXsk1011, were collected from naturally infected silkworms from different geographical locations in Yunnan and Sichuan. All of the isolates showed highly similar conidia and conidial fructification, but the colony characteristics demonstrated great differences among the isolates. The ITS and 18S rDNA sequence analysis was sufficient to identify all five isolates as B. bassiana. However, the dendrogram, based on the ISSR data, produced two large genetic groups. GXtr1009 and GXtr1010 comprised one group, and SCsk1006, YNsk1106 and GXsk1011 converged in a different large group. The results suggested that, although all of these five B. bassiana strains were pathogenic to silkworms, strains of biological pesticides could be differentiated from strains of naturally infected silkworm via ISSR analysis.     

Advanced technologies for genetically manipulating the silkworm Bombyx mori,a model Lepidopteran insect

Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed.     

Study of gut bacterial diversity of Bombyx mandarina and Bombyx mori through 16S rRNA gene sequencing

The wild silkworm B. mandarina is living in the natural environment has a strong stress resistance and adaptability after harsh natural selection. The indoor rearing or domestication of the wild silkworm under artificial custody for long period deteriorates stress resistance and ecological adaptability. Therefore, we aimed to investigate the effects of artificial domestication and evolutionary pressure on the gut bacterial diversity of B. mandarina and B. mori. The intestinal content of 6th day of fifth instar B. mandarina and B. mori larvae were analyzed by sequencing of the 16S rRNA gene through Illumina miseq sequencing technology. The outcome of the study revealed that abundance of predominant bacteria of phylum Firmicutes were respectively 81.40% and 81.85% in the late fifth instar silkworm larvae (6th day) of B. mandarina and B. mori. In Firmicutes, abundance of predominant bacterial genus Enterococcus in B. mandarina (69.73%) was comparatively higher than B. mori (48.99%). The genus Advenella belongs to phylum Proteobacteria was recorded only in B. mandarina (11.54%). The abundance of Unclassified_Peptostreptococcaceae, Methanobrevibacter, Ignatzschineria, Petrimonas and Proteiniphilum in B. mandarina were between 0.12 and 0.17%, nevertheless, these bacterial genera were not detected in B. mori. The abundance of genera Lactococcus, Bacillus and Pseudomonas in B. mori (17.73%, 5.02%, and 1.61%) were remarkably higher than B. mandarina (0.15%, 0.54% and 0.45%). These results indicated that substantial difference was observed between the intestinal bacteria of B. mori and B. mandarina population, and structure of the intestinal bacteria could be affected by the artificial domestication and evolutionary pressure.