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.     

Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx mori

Class B scavenger receptors (SR‐Bs) are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagocytosis of xenobiotics, and signaling. But little information is available about silkworm SR‐Bs; it is necessary to study these SR‐Bs for revealing their function. In this study, we cloned the full‐length coding sequence of BmSCRBQ4, a SR‐B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA (mRNA) was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.     

D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.     

Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector

The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human granucyto-macrophage colony-stimulating factor (hGM-CSF) in the posterior silk glands of gene-targeted silkworms, a targeting vector pSK-FibL-L-A3GFP-PH-GMCSF-LPA-FibL-R was constructed, harboring a 1.2 kb portion of the left homogenous arm (FibL-L), a 0.5 kb portion of the right homogenous arm (FibL-R), fibroin H-chain-promoter-driven hGM-CSF and silkworm actin 3-promoter-driven gfp. The targeting vector was then introduced into the eggs of silkworm, and the transgenic silkworms were verified by PCR and DNA hybridization after being screened for the gfp gene. Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kD in the silk glands of the G3 generation transgenic silkworms. The level of expression of hGM-CSF in the posterior silk glands of the G3 generation transgenic silkworms was approximately 2.70 ng/g of freeze-dried powdered posterior silk gland. These results showed that the heterologous gene could be introduced into the silkworm genome and expressed successfully. Further more, the exogenous genes existing in the G5 transgenic silkworm identified by PCR confirmed its integration stability. In addition, the silk glands containing expressed hGM-CSF performed the function of significantly increasing leukocyte count of CY-treated mice in a time-and-dose-dependent manner.     

Germ line transformation of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, using the transposable element <Emphasis Type="Italic">Minos</Emphasis>

We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used. This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects.     

Oxidative stress,DNA damage,and histological alterations in Bombyx mori exposed orally to pesticide dimethoate

Pesticides are an essential part of agricultural practices that ward off pathogens and diseases from the agricultural crop. However, apart from target organisms, these chemicals also have adverse effects on non-target organisms. Dimethoate is an insecticide used extensively in agriculture and horticulture practices worldwide. We used the silkworm Bombyx mori as a model organism to study the effect of commercial formulation of dimethoate (Dimethoate-30% EC) on the gut, silk gland, and fat body tissues. LD₅₀ of dimethoate-30% EC on silkworm (B. mori) was 997 ppm, as reported in a previous study. We used concentrations of 25, 50, and 100 ppm in our experiments. Our results showed that sub-lethal doses of dimethoate caused weight loss and induced damage at the histological level to the mid-gut, silk gland, and fat body of B. mori. It also caused a decrease in the level of antioxidants like CAT, SOD, GPx, GSH, and GST, indicating that dimethoate has produced a shift of ROS balance towards free radical generation and therefore resulted in overall damage to this organism. Sub-lethal doses of this pesticide also caused lipid peroxidation in the silk gland, gut, and fat body of B. mori, damaging these tissues. The disruption was also seen in the mid-gut and middle silk gland at the DNA level, where it caused single-strand breaks, as was revealed by single cell gel electrophoresis studies. Damage at histological, biochemical, and molecular levels was most extreme at a concentration of 100 ppm, the highest sub-lethal concentration given to B. mori.     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.     

A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.