Silkworm is a holometabolous insect of Lepidoptera. During metamorphosis, significant morphological changes happen including the dissociation of old tissues and remodeling of new tissues. It has been reported that cathepsins are involved in these processes. Cathepsin is a kind of intracellular proteinase that exists in many species. It includes some subfamilies like cathepsin B, H and L. The studies on cathepsin are useful for clarifying the details of silkworm metamorphosis process. In total, 13 cathepsins were identified by screening the silkworm genome database. The basic information and the expression patterns about these genes were analyzed. Interestingly, an ovary-specific cathepsin L gene (Gene ID: BGIBMGAOO4622) was investigated by the data of silkworm microarray and real-time quantitative PCR (qPCR). The full-length cDNA is 1,209 bp, encoding a protein with 402 amino acids. Sequences alignment revealed that it has a high sequence similarity with cathepsin L of other species, and it is highly conserved in the active-site of the enzyme. The phylogenetic analysis showed that ovary-specific cathepsin L is clustered with other lepidopterous insects. Furthermore, this gene was cloned and prokaryotic expressed. Recombinant protein was present in inclusion body. Importantly, the qPCR result showed that the expression level of this gene is increasing during the early stage of pupal development and reaches the highest value at the 3rd day of pupal stage, which suggests that this gene may be involved in the process of development of the ovary and oocyte.
Animals ; Bombyx ; genetics ; Cathepsins ; genetics ; Insect Proteins ; genetics ; Phylogeny

The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.
Animals ; Bombyx/metabolism* ; CRISPR-Cas Systems/genetics* ; Endonucleases/genetics* ; Gene Editing ; RNA, Guide/genetics*

The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bml1721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm 11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm1 1721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes.
Animals ; Bombyx ; genetics ; metabolism ; Chitin ; metabolism ; Insect Proteins ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; Silk ; biosynthesis

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.
Animals ; Molting/genetics* ; Bombyx/genetics* ; Carboxypeptidases A/metabolism* ; Proteomics ; Larva/metabolism* ; Fluorescent Antibody Technique ; Insect Proteins/metabolism*

A pair of specific primers were designed according to the published 118 bp conserved sequence of polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) in GenBank and a serial dilutions of a recombinant plasmid were prepared and used to generate standard curves, to establish a real-time fluorescent quantitative PCR method for detection of BmCPV. The results showed that the linear relationship between virus concentration (X) and Ct (Y) was Y = -3. 582 lgX + 38.748 with the correlation coefficient R2 = 0999. The method was very sensitive, specific and reproducible. It can be applied in the rapid detection of BmCPV and the prevalence investigation of this disease.
Animals ; Bombyx ; virology ; Fluorescence ; Real-Time Polymerase Chain Reaction ; methods ; Reoviridae ; genetics ; isolation & purification

NHL proteins, which play important roles in regulation of cell proliferation and differentiation, have been extensively studied on mammals. Here, we cloned a member of NHL protein family namely BmBrat in silkworm. The full-length cDNA sequence of BmB rat was obtained by means of the rapid amplification of cDNA ends (RACE), including 3 614 bp. The ORF is 2 580 bp long, encoding a protein with 859 amino acid residues. The molecular weight is 94.3 kDa and the isoeledtric point (pI) is 6.65. The BmBrat expression profile was detected by RT-PCR at L5D3 larval stage, and it was expressed in all tissues, including silk gland, midgut, fat body and malpighian tubule. However, it was highly expressed in ovary and head. The expression profile was also detected at different stage of embryo development, and reached a peak at the 4th and 5th days of the embryonic period. Anti-BmBrat polyclonal antibody was generated f6llowing prokaryotic expression, protein purification and mice immunization, which is highly specific and effective for recognizing BmBrat protein through Western blotting and immunofluorescence staining. Subcellular localization of BmBrat in hemocytes revealed that it was specifically expressed in cytoplasm. This study provides a foundation for further research of the biological function of BmBrat gene.
Animals ; Bombyx ; Cloning, Molecular ; DNA, Complementary ; Insect Proteins ; genetics ; metabolism ; Larva ; Mice

The observation statistics suggested that the haemolymph melanization speed of larvae became fast and the growth inhibition of Escherichia coli was strong as the quantities of feeding on mulberry leaves increased. The RT-PCR result showed that the mRNA expressions of melanin biosynthesis enzyme BmTan, BmPo-1, BmYellow-f and BmDdc were high in the haemolyph of 5 L 3 d larvae. The qPCR analysis showed Bmtan, Bmddc, Bmyellow, Bmebony and Bmblack, especially Bmddc expression were significantly higher in black disease larvae than in normal larvae. Compared with control, Ddc inhibitors drastically inhibited the lipopolysaccharide-induced haemolymph melanization. In addition, the content of Dopa and Dopamine markedly rose after E. coli injection. These indicated that haemolymph melanization was linked to immune defenses and Bmddc may play a role in melanization response of haemolymph immune in silkworm.
Animals ; Bombyx ; enzymology ; genetics ; microbiology ; Escherichia coli ; Genes, Insect ; Hemolymph ; chemistry ; Larva ; Melanins ; biosynthesis

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.
Animals ; Bombyx/metabolism* ; Metamorphosis, Biological/genetics* ; Larva/metabolism* ; Gene Expression ; Insect Proteins/metabolism* ; Chitin

OBJECTIVETo construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.

METHODSHuman group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.

RESULTSThree genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.

CONCLUSIONWe have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.

Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; virology ; Capsid Proteins ; genetics ; Cell Line ; Gene Expression ; Genetic Vectors ; Rotavirus ; genetics ; metabolism

In order to make clear the packing mechanism of the BmNPV polyhedra, a polyhedrin gene negative recombinant baculovirus, vBmBac(polh-)-5B-EGFP, expressing EGFP was constructed, and used to infect BmN cells jointly with wild-type BmNPV. Fluorescent microscopic observation demonstrated that EGFP and polyhedrin were expressed simultaneously, and the EGFP expression and polyhedra formation occurred in most of the jointly infected cells. Analysis of the purified polyhedra from jointly infected BmN cells showed that the foreign proteins were present in the polyhedra. The results indicated that BmNPV polyhedrin could incorporate proteins other than viral proteins into the polyhedra. It implies that a nonspecific recognition mechanism exists in the embedment of BmNPV polyhedra.
Animals ; Bombyx ; Gene Expression ; Green Fluorescent Proteins ; genetics ; metabolism ; Nucleopolyhedrovirus ; genetics ; physiology ; Viral Structural Proteins ; genetics ; metabolism ; Virus Assembly