Peng Lü

In silico Identification of Novel Chitinase-Like Proteins in the Silkworm, Bombyx mori, Genome

Abstract In insects, chitinases participate in the periodic shedding of old exoskeletons and the turnover of peritrophic membranes. Chitinase family members have been identified in dozens of species, including Tribolium castaneum, Drosophila melanogaster, and Anopheles gambiae. In this study, nine chitinases and three hypothetical chitinases have been identified in Bombyx mori L. (Lepidoptera: Bombycidae) through genome-wide searching. Phylogenetic analyses revealed that seven of them belong to the seven chitinase groups, respectively. BmCht25 and BmCht26 could not be grouped into the known chitinase groups, and might belong to two new groups of the chitinase family. BmCht10, BmCht25, and BmIDGF have glutamate amino acid substitutions in the active catalytic domain. Only BmCht5 and BmCht10 contain CBD domain and PEST sequences (rich in proline, glutamic acid, serine, and threonine). BmCht5 and BmCht26 are located on chromosome 7, and others (BmCht6, BmCht7, BmCht10, BmCht11, BmCht20, BmIDGF) are located on separate chromosomes of Bombyx mori, respectively. The present study provides important background information for future studies using Bombyx mori as a model organism for insect development and virus and host interaction.

V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus

Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.

Molecular Cloning, Expression and Characterization of BmIDGF Gene from Bombyx mori

Imaginal disc growth factors (IDGF) play a key role in insect development, but their mechanism remains unclear. In this study, we cloned a novel IDGF gene in Bombyx mori and designated it as BmIDGF. We found that the BmIDGF gene contains eight exons and seven introns, encoding a peptide of 434 amino-acid residues. The protein was predicted to contain one conserved motif of the glycosyl hydrolases family 18 and fall into group V chitinases. Sequence alignment showed that BmIDGF shares extensive homology with other invertebrate IDGF. RT-PCR analysis showed that BmIDGF is expressed in all developmental stages of silkworm larvae and various larvae tissues, which was further confirmed by Western blot analysis. Subcellular localization analysis indicated that BmIDGF is located in the extracellular space. We also successfully expressed it in E. coli and further characterized it by SDS-PAGE and mass spectrometry. Taken together, our data suggests that BmIDGF is a chitinase-like extracellular protein, and provides an excellent platform for subsequent studies on its enzyme activity and role in B. mori development

Molecular and Physiological Characterization of Two Novel Multirepeat β-Thymosins from Silkworm, Bombyx mori

β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

Toxic effects of fluoride on organisms

Abstract Accumulation of excess fluoride in the environment poses serious health risks to plants, animals, and humans. This endangers human health, affects organism growth and development, and negatively impacts the food chain, thereby affecting ecological balance. In recent years, numerous studies focused on the molecular mechanisms associated with fluoride toxicity. These studies have demonstrated that fluoride can induce oxidative stress, regulate intracellular redox homeostasis, and lead to mitochondrial damage, endoplasmic reticulum stress and alter gene expression. This paper reviews the present research on the potential adverse effects of overdose fluoride on various organisms and aims to improve our understanding of fluoride toxicity. Graphical abstract Figure. Unlabelled Image

Discovery of anti-viral molecules and their vital functions in Bombyx mori

The silkworm Bombyx mori (B. mori), a lepidopteran model organism, has become an important model for molecular biology researches with its genome completely sequenced. Silkworms confront different types of virus diseases, mainly including those caused by Bombyx mori nucleopolyhedrovirus (BmNPV), Bombyx mori densovirus type 1 (BmDNV-1), Bombyx mori bidesovirus (BmBDV) which was termed as Bombyx mori densovirus type 2 (BmDNV-2) or Bombyx mori parvo-like virus (BmPLV) before in sericulture. B. mori offers excellent models to study the molecular mechanisms of insect innate immune responses to viruses. A variety of molecules and pathways have been identified to be involved in the immune responses in the silkworm to viruses, such as the antimicrobial peptides, prophenoloxidase-activating system, apoptosis, ROS, small RNA and related molecules. Here in this review, we summarize the current research advances in molecules involved in silkworm anti-virus pathways. Moreover, taking BmNPV as an example, we proposed a schematic model of molecules and pathways involved in silkworm immune responses against virus infection. We hope this review can facilitate further study of antiviral mechanisms in silkworm, and provide a reference for virus diseases in other organisms.

A characterization of structural proteins expressed by Bombyx mori bidensovirus.

Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5kb (viral DNA 1, VD1) and 6.0kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22-24nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism.

Comparative transcriptomic analysis of Bombyx mori fat body tissue following dietary restriction

Dietary restriction (DR) refers to a reduction in food intake to induce undernutrition but not malnutrition, which extends the lifespan of multiple species. Although there are invertebrate aging models, such as the Caenorhabditis elegans and Drosophila melanogaster, aging studies in Lepidoptera are few in number and the underlying life-extending molecular mechanisms are not clear. Research on a broader range of animals is necessary to support generalizations on mechanisms of aging and rates of aging. The aim of this study was to further investigate genes and pathways associated with DR in Bombyx mori. Here, we used mRNA deep sequencing (RNA-seq) to further investigate genes and pathways associated with DR. The transcriptome profiles showed that most of the differentially expressed genes were upregulated following DR, and genes involved in amino acid and protein metabolism, RNA metabolism and translation, energy metabolism, nitrogen metabolism, and juvenile hormone pathway-related proteins were particularly affected. DR also affects the metabolism of uric acid and urea, which accumulated in silkworm following DR. We speculate that this may not be due to activation of uric acid biosynthesis, but rather by downregulating the degradation of uric acid and urea. These results may help us to understand the mechanisms by which DR prolong lifespan in insects and other animals.

Cloning and rescue of the genome of Bombyx mori bidensovirus, and characterization of a recombinant virus

BackgroundBombyx mori bidensovirus (BmBDV), which belongs to the Bidnaviridae family established by the International Committee on Taxonomy of Viruses in 2011, was the first bidensovirus identified in insects. The structure of BmBDV is similar to that of parvoviruses, while its replication is similar to that of adenoviruses. Although BmBDV has the potential to be used as a tool in biological pest control and as an expression vector, virus rescue has been a bottleneck in the application of this virus.MethodsIn this study, we constructed a full-length genomic clone of BmBDV and showed that its terminal structure was restored. A recombinant BmBDV that expressed the green fluorescence protein (GFP) gene was constructed. Then, BmN cells, which are an ovarian cell line, were co-transfected with the linearized genome using continuous culture and expanded cell culture methods.ResultsThe results showed that the GFP gene was expressed successfully, and that cell lesions occurred in virus-infected cells. Furthermore, typical densonucleosis viruses were observed in reinfected silkworm larvae and larval midgut tissues infected by BmBDV, as evidenced by the emission of green fluorescence.ConclusionsOverall, these results suggest that the virus could be rescued from the infected BmN cells after co-transfection with the linear full length virus genome.

Proteomic Study on the New Potential Mechanism and Biomarkers of Diabetes

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia. So far, the pathogenesis of diabetes has not been fully elucidated. Identifying new potential molecule mechanisms and biomarkers in this process could contribute to the understanding of pathophysiology.