Transgenic characterization of two testis‐specific promoters in the silkworm,Bombyx mori

Sex‐specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female‐specific modification system whereas little success was reported on male‐specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene‐based, female‐specific lethality system has been established based on sex‐specific alternative splicing factors and a female‐specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male‐specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis‐specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta‐tubulin 4 gene (Bmβ4) were introduced using piggybac‐based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis‐specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis‐specific gene expression. Identification of these testis‐specific promoters not only contributes to a better understanding of testis‐specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.     

Comparative analysis of Bombyx mori nucleopolyhedrovirus responsive genes in fat body and haemocyte of B. mori resistant and susceptible strains

The infection profiles of the Bombyx mori nucleopolyhedrovirus (BmNPV) in B. mori larvae revealed that the virus invaded the fat body and haemocyte of both KN and 306 strains, which are highly resistant and susceptible, respectively, to BmNPV infection. However, viral proliferation was notably slowed in the resistant B. mori strain. Using suppression subtractive hybridization, two fat body cDNA libraries were constructed to compare BmNPV responsive gene expression levels between the two silkworm lines. In total, 96 differentially expressed genes were obtained. Real‐time quantitative PCR (qPCR) analysis confirmed that eight genes were significantly up‐regulated in the fat body and haemocyte of the KN strain following BmNPV injection. Our results suggest that these genes may have potential roles in B. mori antiviral infection mechanisms.     

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 analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm,Bombyx mori

The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome‐related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od^B) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ‐line transformation of the wild‐type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky–Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.     

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.     

Estrogen‐related receptor participates in regulating glycolysis and influences embryonic development in silkworm Bombyx mori

Estrogen‐related receptors (ERRs) play indispensable roles in development, energy metabolism, and cancers and are metabolic switches in Drosophila. However, the mechanism underlying their metabolic role is unknown in insects. This study analysed the expression profiles of Bombyx mori ERR (BmERR), hexokinase (BmHK), pyruvate kinase (BmPK) and phosphofructokinase (BmPFK) during embryonic development. The expression of BmERR tended to be similar to that of the other genes. We observed a regulatory association between BmERR and glycolytic rate‐limiting enzymes by BmERR overexpression, RNA interference (RNAi), and ERR inhibitors in B. mori embryo cells. Subsequently, ERR cis‐regulation elements (ERREs) were predicted and identified in the BmPFK promoter. Transfection assays, electrophoretic mobility shift assays and chromatin immunoprecipitation showed that BmERR can bind to one of these elements to regulate the expression of BmPFK. ERREs were also predicted in the BmHK and BmPK promoters. In the eggs, the expression of glycolytic rate‐limiting enzyme genes was suppressed when the expression of BmERR was interference by double‐stranded BmERR, the glucose levels also was increased. Meanwhile, the development of silkworm embryos was delayed by about 1 day. These results indicate that BmERR can bind to the ERREs of glycolytic gene promoters and regulate the expression of glycolytic genes, ultimately affecting embryonic development in silkworms.     

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern‐recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N‐acetylmuramoyl‐L‐alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP‐S4, a short‐form PGRP from the domesticated silkworm, Bombyx mori. The PGRP‐S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP‐S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP‐S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn²⁺. Scanning electron microscopy showed that PGRP‐S4 disrupted the bacterial cell surface. PGRP‐S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP‐S4 has multiple functions in immunity.     

Establishment of a specific cell death induction system in Bombyx mori by a transgene with the conserved apoptotic regulator,mouse Bcl‐2‐associated X protein (mouse Bax)

The induction of apoptosis in vivo is a useful tool for investigating the functions and importance of particular tissues. B‐cell leukaemia/lymphoma 2‐associated X protein (Bax) functions as a pro‐apoptotic factor and induces apoptosis in several organisms. The Bax‐mediated apoptotic system is widely conserved from Caenorhabditis elegans to humans. In order to establish a tissue‐specific cell death system in the domestic silkworm, Bombyx mori, we constructed a transgenic silkworm that overexpressed mouse Bax (mBax) in particular tissues by the Gal4‐upstream activation sequence system. We found that the expression of mBax induced specific cell death in the silk gland, fat body and sensory cells. Fragmentation of genomic DNA was observed in the fat body, which expressed mBax, thereby supporting apoptotic cell death in this tissue. Using this system, we also demonstrated that specific cell death in sensory cells attenuated the response to the sex pheromone bombykol. These results show that we successfully established a tissue‐specific cell death system in vivo that enabled specific deficiencies in particular tissues. The inducible cell death system may provide useful means for industrial applications of the silkworm and possible utilization for other species.     

Structure and expression of tandemly duplicated xanthine dehydrogenase genes of the silkworm (Bombyx mori)

Xanthine dehydrogenase (XDH) is a molybdoenzyme which catalyses oxidation of xanthine and hypoxanthine to uric acid. We isolated genomic clones of silkworm (Bombyx mori) XDH genes (BmXDH1 and BmXDH2). The BmXDH2 The BmXDH2 gene is located upstream from the BmXDH1 gene and they show a tandemly duplicated structure. Both BmXDH genes were expressed in the fat body and Malpighian tubules, whereas only the BmXDH1 gene was expressed in the midgut. Phylogenetic analysis indicates that BmXDH gene duplication occurred after the divergence of the silkworm and dipteran species. Intron insertion site comparison shows that some introns were lost during insect XDH gene evolution.     

Systemic disruption of the homeostasis of transfer RNA isopentenyltransferase causes growth and development abnormalities in Bombyx mori

Isopentenylation at A37 (i⁶A37) of some transfer RNAs (tRNAs) plays a vital role in regulating the efficiency and fidelity of protein synthesis. However, whether insects, which are well known for their highly efficient protein synthesis machinery, employ this regulatory mechanism remains uninvestigated. In the current study, a candidate tRNA isopentenyltransferase (IPT) gene with three alternative splicing isoforms (BmIPT1–BmIPT3) was identified in Bombyx mori (silkworm). Only BmIPT1 could complement a yeast mutant lacking tRNA IPT. Phylogenetic analysis showed that silkworm tRNA IPT is conserved in the Lepidoptera. BmIPT was expressed in all B. mori tissues and organs that were investigated, but was expressed at a significantly higher level in silk glands of the fourth instar compared to the first day of the fifth instar. Interestingly, BmIPT was expressed at a significantly higher level in the domesticated silkworm, B. mori, than in wild Bombyx mandarina in multiple tissues and organs. Knock‐down of BmIPT by RNA interference caused severe abnormalities in silk spinning and metamorphosis. Constitutive overexpression of BmIPT1 using a cytoplasmic actin 4 promoter in B. mori raised its messenger RNA level more than sixfold compared with nontransgenic insects and led to significant decreases in the body weight and cocoon shell ratio. Together, these results confirm the first functional tRNA IPT in insects and show that a suitable expression level of tRNA IPT is vital for silk spinning, normal growth, and metamorphosis. Thus, i⁶A modification at position A37 in tRNA probably plays an important role in B. mori protein synthesis.