Functional characterization of the vitellogenin promoter in the silkworm,Bombyx mori

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis‐regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798‐bp DNA sequence adjacent to the 5′‐end of the vitellogenin gene (Bmvg). PiggyBac‐based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex‐, tissue‐ and stage‐specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval?pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20‐hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis‐regulatory element in B. mori.     

Transgenic characterization of two silkworm tissue‐specific promoters in the haemocyte plasmatocyte cells

Haemocytes play crucial roles in insect metabolism, metamorphosis, and innate immunity. As a model of lepidopteran insects, the silkworm is a useful model to study the functions of both haematopoiesis and haemocytes. Tissue‐specific promoters are excellent tools for genetic manipulation and are widely used in fundamental biological research. Herein, two haemocyte‐specific genes, Integrin β2 and Integrin β3, were confirmed. Promoter activities of Integrin β2 and Integrin β3 were evaluated by genetic manipulation. Quantitative real‐time PCR and western blotting suggested that both promoters can drive enhanced green fluorescent protein (EGFP) specifically expressed in haemocytes. Further evidence clearly demonstrated that the transgenic silkworm exhibited a high level of EGFP signal in plasmatocytes, but not in other detected haemocyte types. Moreover, EGFP fluorescence signals were observed in the haematopoietic organ of both transgenic strains. Thus, two promoters that enable plasmatocytes to express genes of interest were confirmed in our study. It is expected that the results of this study will facilitate advances in our understanding of insect haematopoiesis and immunity in the silkworm, Bombyx mori.     

Two Tor genes in the silkworm Bombyx mori

Target of rapamycin (TOR), a member of the phosphatidylinositol kinase‐related kinase family, plays a critical role in the regulation of growth, metabolism, development and survival, at both the cellular and the organismal levels. Two paralogous Tor genes, BmTor1 and BmTor2, were identified as a pair of inverted repeats in the genome of the silkworm Bombyx mori. The synteny of BmTor1 and CG8360 indicates that BmTor1 is the orthologue while BmTor2 is a duplicate. Analyses of the two BmTor genes at both the nucleotide and amino acid levels reveal that they are evolutionally and structurally conserved. The two BmTor genes had similar expression patterns of tissue distribution with highest levels in the nervous system, and nearly identical developmental change profiles with maximal levels during the 4^th‐larval‐moulting and the larval–pupal transition stages. Furthermore, both BmTor genes were up‐regulated by either starvation or the moulting hormone 20‐hydroxyecdysone (20E), while BmTor2 was more sensitive to both treatments than BmTor1. For the first time, we have identified two copies of the Tor gene in a higher eukaryote, which are induced by starvation and 20E during the larval moulting and the larval–pupal transition stage.     

Identification and function of Abdominal-A in the silkworm, Bombyx mori

Abdominal-A ( adb-A ) is a key gene in the development of insects. To understand its function in the silkworm, we cloned 1193 bp of the abd-A gene of Bombyx mori ( Bmabd-A ), including the complete coding sequence and part of the 3' untranslated region sequence. Bmabd-A has at least three mRNA splice variants with coding sequences of lengths 1032, 1044 and 1059 bp, encoding 343, 347 and 352 amino acids, respectively. Each splice variant of Bmabd-A has three exons and differs only in second exon size. Bmabd-A was expressed at low levels in unfertilized eggs, but increased gradually in fertilized eggs after laying 22 h. Bmabd-A expression decreased in ant silkworms (newly hatched silkworms). After RNA interference for Bmabd-A , the embryos had two mutant phenotypes, either completely or partially absent abdominal feet from the third to sixth abdominal segments, suggesting that Bmabd-A is responsible for normal development of the third to sixth abdominal segments during embryonic development.     

20‐Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis‐related protein in the silkworm,Bombyx mori

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth‐instar ecdysis and larval–pupal metamorphosis. By immunoprecipitation with the anti‐BmNep1 antibody and liquid chromatography‐tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20‐hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval–pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.     

The effect of acetylation on the protein stability of BmApoLp‐III in the silkworm,Bombyx mori

Acetylation is an important, reversible posttranslational modification to a protein. In a previous study, we found that there were a large number of acetylated sites in various nutrient storage proteins of the silkworm haemolymph. In this study, we confirmed that acetylation can affect the stability of nutrient storage protein Bombyx mori apolipophorin‐III (BmApoLp‐III). First, the expression of BmApoLp‐III could be upregulated when BmN cells were treated with the deacetylase inhibitor panobinostat (LBH589); similarly, the expression was downregulated when the cells were treated with the acetylase inhibitor C646. Furthermore, the increase in acetylation by LBH589 could inhibit the degradation and improve the accumulation of BmApoLp‐III in BmN cells treated with cycloheximide and MG132 respectively. Moreover, we found that an increase in acetylation could decrease the ubiquitination of BmApoLp‐III and vice versa; therefore, we predicted that acetylation could improve the stability of BmApoLp‐III by competing for ubiquitination and inhibiting the protein degradation pathway mediated by ubiquitin. Additionally, BmApoLp‐III had an antiapoptosis function that increased after LBH589 treatment, which might have been due to the improved protein stability after acetylation. These results have laid the foundation for further study on the mechanism of acetylation in regulating the storage and utilization of silkworm nutrition.     

Molecular cloning and characterization of peroxiredoxin 4 involved in protection against oxidative stress in the silkworm Bombyx mori

Peroxiredoxins (Prxs) are a ubiquitous family of proteins that play important roles in insects in protection against oxidative stress through the detoxification of cellular peroxides. Here, we describe the cloning and characterization of a Prx4 cDNA of the silkworm Bombyx mori (BmPrx4). The BmPrx4 gene has an open reading frame of 744 bp encoding 248 amino acids and a conserved motif, VCP, involved in its presumed redox functions. The heterologously expressed proteins of the gene in Escherichia coli showed antioxidant activity, removed hydrogen peroxide and protected DnA. Western blotting analysis showed the presence of BmPrx4 in the haemolymph, suggesting that the protein is secretable. Moreover, BmPrx4 was expressed at all developmental stages. The expression level of BmPrx4 was relatively low during the feeding stage but high at the wandering stage. BmPrx4 was induced by quercetin or temperature stress. Immunohistochemical analysis revealed that BmPrx4 is present in the brain, neurones and olfactory organ of the head in silkworms. Overall, our results indicate that the expression profile of BmPrx4 correlates well with protection from oxidative damage. Our data provide clues for the development of control technology for agricultural and forestry pests as the silkworm is a representative of lepidopteran pests.     

Lipopolysaccharide elicits expression of immune-related genes in the silkworm, Bombyx mori

Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, was found to be unable to activate immune-related genes in Drosophila melanogaster . In contrast, highly purified LPS elicited immune-related gene expression in the fat body of Bombyx mori . However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune-related genes by LPS. Up-regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune-responsive cell line, NIAS-Bm-aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.     

Bombyx mori cyclin‐dependent kinase inhibitor is involved in regulation of the silkworm cell cycle

Cyclin‐dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin‐dependent kinase (CDK)‐cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654‐bp‐long BmCKI‐L (the longer splice variant) encoding a protein with 217 amino acids and a 579‐bp‐long BmCKI‐S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI‐L and BmCKI‐S contain the Cip/Kip family conserved cyclin‐binding domain and the CDK‐binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181–210. Overexpression of BmCKI‐L or BmCKI‐S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI‐L or BmCKI‐S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI‐L and BmCKI‐S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI‐L overexpression (BmCKI‐L‐OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI‐L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm.     

Cloning and characterization of a pyridoxine 5'-phosphate oxidase from silkworm, Bombyx mori

A cDNA encoding Pyridoxine 5'-phosphate oxidase (PNPO) from Bombyx mori was cloned and characterized (G en B ank accession number: DQ452398). The cDNA encodes a polypeptide of 257 amino acid residues. The recombinant enzyme purified from Escherichia coli exhibited maximal activity at pH 9.0, and the K _m values for the substrates of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate were determined as 0.65 and 1.15 µmol/l. It was found that B. mori PNPO shares 51.44% homology with humans, but several function-related, key amino acid residues in B. mori PNPO are different from the human and E. Coli gene. B. mori has a single copy of the PNPO gene, which spans a 3.5 kb region and contains five exons and four introns. B. mori PNPO is a homodimer, with each monomer containing nine antiparallel β-strands and five α-helical segments. The secondary structure was deduced from computational study.     

cDNA structure and characterization of a kinesin-like protein from the silkworm Bombyx mori

We have isolated a 1224 bp cDNA clone from a Bombyx mori embryonic cDNA library which contains sequences homologous to the kinesin-like protein gene, ncd , which is required for distribution of chromosomes at meiosis in Drosophila melanogaster females. This clone includes both a microtubule motor and the ATP-binding domains found in kinesin-like proteins. The motor domain is classified in the group of the BimC and cut7 , which have a role in spindle formation during mitosis of Aspergillus nidulans and Schizosaccharomyces pombe , respectively. However, the location of the domain at the carboxy terminus is not common in this family, except for ncd and KAR3.     

Characterization of an entomopathogenic fungi target integument protein,Bombyx mori single domain von Willebrand factor type C,in the silkworm,Bombyx mori

The insect cuticle works as the first line of defence to protect insects from pathogenic infections and water evaporation. However, the old cuticle must be shed in order to enter the next developmental stage. During each ecdysis, moulting fluids are produced and secreted into the area among the old and new cuticles. In a previous study, the protein Bombyx mori single domain von Willebrand factor type C (BmSVWC; BGIBMGA011399) was identified in the moulting fluids of Bo. mori and demonstrated to regulate ecdysis. In this study we show that in Bo. mori larvae, BmSVWC primarily locates to the integument (epidermal cells and cuticle), wing discs and head. During the moulting stage, BmSVWC is released into the moulting fluids, and is then produced again by epidermal cells after ecdysis. Fungal infection was shown to decrease the amount of BmSVWC in the cuticle, which indicates that BmSVWC is a target protein of entomopathogenic fungi. Thus, BmSVWC is mainly involved in maintaining the integrity of the integument structure, which serves to protect insects from physical damage and pathogenic infection.     

cDNA cloning,characterization and gene expression of nitric oxide synthase from the silkworm,Bombyx mori

Molecular cloning and nucleotide sequencing of cDNA encoding Bombyx mori nitric oxide synthase (BmNOS) was conducted to analyse its possible role in insect immunity. The amino acid sequence deduced from the BmNOS cDNA showed 84%, 54% and 53% identity with those of NOSs from Manduca sexta, Drosophila melanogaster and Rhodonius prolixus. Recombinant BmNOS produced in insect cells using baculovirus was found to require NADPH, Ca2+, calmodulin and tetrahydrobiopterin (BH4) for its activity. The BmNOS gene was constitutively expressed at a low level in the larval fat body, haemocyte, Malpighian tubule and midgut, and adult antenna, and induced strongly in the fat body by lipopolysaccharide (LPS), suggesting that the BmNOS gene plays different physiological roles in different tissues. Injection of NO donors that produce NO in vivo induced the gene expression of an antibacterial peptide, cecropin B, strongly suggesting that NO produced by BmNOS following LPS stimulation is involved in signal transduction as a signalling molecule for immune gene expression.     

Linkage analysis of maternal EST cDNA clones covering all twenty-eight chromosomes in the silkworm,Bombyx mori

A cDNA library of maternal messages was constructed from non-fertilized Bombyx mori eggs collected just after oviposition without mating. cDNA clones were identified by their nucleotide sequences and evaluated as probes for RFLP linkage analysis. Back crossed F1 segregants - by crossing an F1 female (RF02 x RF50) and a male (RF02) - were used, which takes advantage of the phenomenon that no crossing over occurs in Bombyx females. Fifteen ordered BF1 segregants gave either homozygous (homo) or heterozygous (hetero) RFLP patterns with each cDNA probe. cDNA probes on the same linkage group gave the same homo/hetero order. One hundred and fifty one out of 248 cDNA clones showed polymorphisms between RF02 and RF50, and were therefore suitable as probes for RFLP linkage analysis in the present BF1 cross. They were sorted into twenty-seven linkage groups and one independent group, by the homo/hetero pattern matrix, covering all twenty-eight chromosomes in B. mori.