Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L

The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc.     

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.     

Analysis of expressed sequence tags and characterization of a novel gene, Slmg7, in the midgut of the common cutworm, Spodoptera litura

Background

Out of total 3,081 assembled expressed sequence tags (ESTs) sequences representing 6,815 high-quality ESTs identified in three cDNA libraries constructed with RNA isolated from the midgut of Spodoptera litura, 1,039 ESTs showed significant hits and 1,107 ESTs did not show significant hits in BLAST searches. It is of interest to clarify whether or not these ESTs that did not show hits function in S. Litura.

Results

Twenty “no-hit” ESTs containing at least one putative open reading frame were selected for further expression analysis. The results from northern blot analysis showed that six of the selected ESTs are expressed in the larval midgut of this insect at different levels, suggesting that these ESTs represent true mRNA products, whereas the other 14 ESTs could not be detected. Homologues of the four larval midgut-predominant genes (Slmg2, Slmg7, Slmg9 and Slmg17) were detected in the genomes of other lepidopteran insects but not in Drosophila melanogaster. A novel gene, Slmg7, is expressed at a high level specifically in the midgut during each of the larval stages. Slmg7 is a single copy gene and encodes a 143-amino acids protein. The SLMG7 protein was localized to the cytoplasm of Spli-221 cells.

Conclusions

Six ESTs from the no hit list are transcribed into mRNA and are mainly expressed in the midgut of S. litura. Slmg7 is a novel gene that is localized to the cytoplasm.     

Expression profiles of glutathione <Emphasis Type="Italic">S</Emphasis>-transferase genes in larval midgut of <Emphasis Type="Italic">Bombyx mori</Emphasis> exposed to insect hormones

Glutathione S-transferases (GSTs) are believed to play a role in the detoxification of xenobiotics, resistance to insect viruses and pesticides, intracellular transport, biosynthesis of hormones and protection against oxidative stress. In this study, we used quantitative real time RT-PCR to examine expression profiles of the silkworm Bombyx mori GST-Sigma (BmGSTS2) and GST-Delta (BmGSTD2) genes in the larval midgut of the silkworm after exposure to 2-hydroxyecdysone (20E) and juvenile hormone analog (JHA). In concentration-course study, 20E at higher concentrations (1.0 and 2.0 μg/μl) caused significant upregulation of BmGSTD2, and all concentrations (0.5–2.0 μg/μl) of 20E caused significant upregulation of BmGSTS2. However, JHA in all concentrations downregulated the expression of BmGSTD2 and BmGSTS2. When exposed to either 20E (2.0 μg/μl) or JHA (2.0 μg/μl) on the third day of the fifth instar, the silkworm had higher BmGSTD2 at later time points: 15, 18, and 24 h for 20E and 24 h for JHA. BmGSTS2 expression was downregulated within 24 h after exposure to JHA and showed a time-dependent response after exposure to 20E. We also did a stage-dependent study, in which JHA downregulated BmGSTD2 expression and upregulated BmGSTS2 expression significantly at both day 1 and day 3 of the fifth instar. 20E upregulated the expression of BmGSTD2 and BmGSTS2 at the two stages. These findings imply that hormones have an important role in the regulation of basal GST expression. However, further validation and field trials should be carried out on the regulatory elements relevant to BmGSTD2 and BmGSTS2 gene expression.     

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer’s tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.     

BmSUC1 is essential for glycometabolism modulation in the silkworm, Bombyx mori

Sucrose is the most commonly transported sugar in plants and is easily assimilated by insects to fulfill the requirement of physiological metabolism. BmSuc1 is a novel animal β-fructofuranosidase (β-FFase, EC 3.2.1.26)-encoding gene that was firstly cloned and identified in silkworm, Bombyx mori. BmSUC1 was presumed to play an important role in the silkworm-mulberry enzymatic adaptation system by effectively hydrolyzing sucrose absorbed from mulberry leaves. However, this has not been proved with direct evidence thus far. In this study, we investigated sucrose hydrolysis activity in the larval midgut of B. mori by inhibition tests and found that sucrase activity mainly stemmed from β-FFase, not α-glucosidase. Next, we performed shRNA-mediated transgenic RNAi to analyze the growth characteristics of silkworm larvae and variations in glycometabolism in vivo in transgenic silkworms. The results showed that in the RNAi-BmSuc1 transgenic line, larval development was delayed, and their body size was markedly reduced. Finally, the activity of several disaccharidases alone in the midgut and the sugar distribution, total sugar and glycogen in the midgut, hemolymph and fat body were then determined and compared. Our results demonstrated that silencing BmSuc1 significantly reduced glucose and apparently activated maltase and trehalase in the midgut. Together with a clear decrease in both glycogen and trehalose in the fat body, we conclude that BmSUC1 acts as an essential sucrase by directly modulating the degree of sucrose hydrolysis in the silkworm larval midgut, and insufficient sugar storage in the fat body may be responsible for larval malnutrition and abnormal petite phenotypes.     

Ligand carrier protein genes expressed in larval chemosensory organs of Bombyx mori

Expressed sequence tags (ESTs) of the maxillary galea of the silkworm were analyzed to identify proteins involved in food selection systems. From the 1251 redundant genes of the ESTs, we identified 7 odorant-binding protein-like genes (bmObpL), 6 takeout-like genes (bmToL), and 6 chemosensory protein genes (bmCsp). Quantitative RT-PCR analysis indicated that bmObpL1, bmObpL2, bmObpL3, bmObpL5, bmToL1, bmToL3, and bmorCsp15 were predominantly expressed in the larval oral appendages, such as the maxilla, labrum, labium and antenna. Immunocytochemical analysis indicated that the proteins of bmObpL1, bmObpL3, and bmToL1 were localized in the gustatory chemosensilla on the maxillary galea and olfactory sensilla in the antenna. The proteins encoded by bmObpL1 and bmObpL3 were detected in the gustatory chemosensilla of the epipharynx. However, bmObpL1 and bmToL1 were also detected in tactile hairs and in the epidermis of several chemosensory organs. The bmObpL2 protein was localized inside and in the epidermis around the chemosensilla, tactile hairs, and wide surface of the epipharynx. From these results, bmObpL3 is the most likely to have a dedicated role in chemoreception in the silkworm, Bombyx mori.     

Characterization of a novel chromodomain-containing gene from the silkworm, Bombyx mori

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species, and is crucial in the establishment and maintenance of heterochromatin. HP1 proteins have two distinct functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), which are required for the selective binding of HP1 proteins to modified histones. During our screen for HP1-like proteins in the Bombyx mori genome, we found a novel silkworm gene, Bombyx mori chromodomain protein 1 (BmCdp1), encoding a putative chromobox protein with only two CDs. The BmCdp1 family proteins are closely related to the HP1 proteins, and most of them belong to insect lineages. qRT-PCR analysis indicated that BmCdp1 mRNA was most abundantly expressed in early embryos, and relatively higher expression was observed in larval testes, hemocytes, and pupal ovaries. Western blot and immunostaining experiments showed that BmCdp1 was localized mainly in the nucleus of BmN4 cells. We searched BmCdp1-bound loci in the Bombyx genome by ChIP-seq analysis using Flag-tagged BmCdp1-expressing BmN4 cells. Combined with ChIP-qPCR experiments, we identified two reliable BmCdp1-bound loci in the genome. siRNA-mediated knockdown of BmCdp1 in BmN4 cells and early embryos did not affect the expression of the gene located close to the BmCdp1-bound locus.     

Shotgun analysis on the peritrophic membrane of the silkworm Bombyx mori

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori. [BMB Reports 2012; 45(11): 665-670]     

Expression pattern of immunoglobulin superfamily members in the silkworm, Bombyx mori

Immunoglobulin superfamily (IgSF) proteins are involved in cell adhesion, cell communication and immune functions. In this study, 152 IgSF genes containing at least one immunoglobulin (Ig) domain were predicted in the Bombyx mori silkworm genome. Of these, 145 were distributed on 25 chromosomes with no genes on chromosomes 16, 18 and 26. Multiple sequence alignments and phylogenetic evolution analysis indicated that IgSFs evolved rapidly. Gene ontology (GO) annotation indicated that IgSF members functioned as cellular components and in molecular functions and biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that IgSF proteins were involved in signal transduction, signaling molecules and interaction, and cell communication. Microarray-based expression data showed tissue expression for 136 genes in anterior silkgland, middle silkgland, posterior silkgland, testis, ovary, fat body, midgut, integument, hemocyte, malpighian tubule and head. Expression pattern of IgSF genes in the silkworm ovary and midgut was analyzed by RNA-Seq. Expression of 105 genes was detected in the ovary in strain Dazao. Expression in the midgut was detected for 74 genes in strain Lan5 and 75 genes in strain Ou17. Expression of 34 IgSF genes in the midgut relative to the actin A3 gene was significantly different between strains Lan5 and Ou17. Furthermore, 1 IgSF gene was upregulated and 1 IgSF gene was downregulated in strain Lan5, and 4 IgSF genes were upregulated and 2 IgSF genes were downregulated in strain Ou17 after silkworms were challenged with B. mori cypovirus (BmCPV), indicating potential involvement in the response to BmCPV-infection. These results provide an overview of IgSF family members in silkworms, and lay the foundation for further functional studies.     

Expression Analysis of Bombyx mori Bidensovirus Structural Proteins and Assembly of Virus-like Particles in Insect Cells

Bombyx mori bidensovirus (BmBDV) is a new designated species of the new genus Bidensovirus in the new family Bidnaviridae, which contains two single-stranded linear DNAs (VD1 and VD2) and causes the chronic densonucleosis disease of silkworm. Previous researches revealed that VD1-ORF3 encodes the major structural proteins VPs. In this work, through western blot, we found that VPs expressed from 48 h post-inoculation and kept increasing until 120 h post-inoculation in midgut of Bombyx mori. In order to further investigate the translation of vp gene, the ORFs (vp1 and vp2) of the VP started just up-stream of the first two candidate initiation codons were expressed in Sf9 cells by a baculovirus expression system. The expression products were purified by gradient density centrifugation and analyzed by Western blot and electron microscopy. The results showed that the expressions of vp1 yielded three proteins (VP1, VP1′, and VP2), which are the same with the viral VPs expression in midgut of Bombyx mori, and vp2 generated two VPs with the molecular weights of about 51 kDa (VP2) and 37 kDa. The observation by electron microscopy indicated that these VPs can auto-assemble into virus-like particles that could not be distinguished from virus particles. These findings will provide materials for studying the structure of BmBDV and be helpful in the studies on BmBDV-based disease in silkworms.