Hsp90 could promote BmNPV proliferation by interacting with Actin-4 and enhance its expression.

As a highly infectious pathogen, Bombyx mori nuclear polyhedrosis virus (BmNPV) has a high lethality rate in silkworm. Our previous study have confirmed that Hsp90 plays a positive role in BmNPV proliferation and Hsp90 inhibitor, geldanamycin (GA) can decrease the replication of BmNPV in vitro. However, its molecular mechanism is not fully understood. In the present study, first, we found that GA could inhibit the proliferation of BmNPV in a dose-dependent manner and delay the pathogenesis of BmNPV in vivo possibly by altering the transcript level of genes associated with cell apoptosis and immune pathways. Furthermore, by immunoprecipitation (IP) and mass spectrometry analysis, we identified a series of proteins potentially interacting with Hsp90 including two BmNPV encoded proteins. Subsequently, by Co-IP we confirmed the interaction between BmActin-4 and BmHsp90. Knocking down Bmhsp90 by small interfering RNA inhibited the protein expression level of BmActin-4. Over-expression of Bmactin-4 promoted the replication of BmNPV whereas knockdown of Bmactin-4 suppressed BmNPV replication. In addition, decrease of the transcript level of Bmhsp90 in Bmactin-4 knocking down BmN cells was also detected. Taken together, BmHsp90 can interact with BmActin-4 and promote its expression, thereby promoting BmNPV proliferation. Our findings may enrich the molecular mechanism of Hsp90 for promoting virus proliferation and provide new clues to elucidate the interact mechanism between silkworm and virus.

LincRNA_XR209691.3 could promote Bombyx mori nucleopolyhedrovirus replication by interacting with BmHSP70.

Long non-coding RNAs (lncRNAs), a class of transcripts exceeding 200 nucleotides and lacking protein coding potential, have been proven to play important roles in viral infection and host immunity. Bombyx mori nucleopolyhedrovirus (BmNPV) is an important pathogen, which causes the silkworm disease and leads to a huge challenge to the sericultural industry. At present, research on the roles of insect lncRNAs in host-virus interaction are relatively few. In this study, we explored the function of lincRNA_XR209691.3 that was significantly up-regulated in the silkworm fat body upon BmNPV infection. Firstly, the subcellular localization experiment confirmed that lincRNA_XR209691.3 was present in both the nucleus and cytoplasm. Enhancing the expression of lincRNA_XR209691.3 in BmN cells could promote the proliferation of BmNPV, while inhibition of lincRNA_XR209691.3 by RNA interference suppresses the proliferation of BmNPV. Combining RNA pull-down and mass spectrometry, we identified the host and BmNPV proteins that could interact with lincRNA_XR209691.3. Next, by using truncation experiment and RNA immunoprecipitation (RIP) assay, it was found that lincRNA_XR209691.3 could bind to the Actin domain of BmHSP70. Subsequently, overexpression of lncRNA_XR209691.3 in BmN cells promoted the expression of BmHSP70, while knockdown of BmHsp70 suppressed the replication of BmNPV. Based on the above results, it is speculated that lincRNA_XR209691.3 could promote the proliferation of BmNPV through interaction with BmHSP70, possibly by improving the stability of BmHSP70 and thereby enhancing the expression of BmHSP70. Our results shed light on the lncRNA function in insect-pathogen interactions and provide a new clue to elucidate the molecular mechanism of BmNPV infection.

Transcriptome of miRNA during inhibition of Bombyx mori nuclear polyhedrosis virus by geldanamycin in BmN cells.

Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of several viruses that cause great harm to the sericulture industry, and its pathogenic mechanism is still being explored. Geldanamycin (GA), a kind of HSP90 inhibitor, has been verified to suppress BmNPV proliferation. However, the molecular mechanism by which GA inhibits BmNPV is unclear. MicroRNAs (miRNAs) have been shown to play a key role in regulating virus proliferation and host-pathogen interactions. In this study, BmN cells infected with BmNPV were treated by GA and DMSO for 72 h, respectively, then transcriptome analysis of miRNA was performed from the GA group and the control group. As a result, a total of 29 miRNAs were differentially expressed (DE), with 13 upregulated and 16 downregulated. Using bioinformatics analysis, it was found that the target genes of DEmiRNAs were involved in ubiquitin-mediated proteolysis, phagosome, proteasome, endocytosis pathways, and so on. Six DEmiRNAs were verified by quantitative reverse-transcription polymerase chain reaction. DElong noncoding RNA (DElncRNA)-DEmiRNA-messenger RNA (mRNA) regulatory networks involved in apoptosis and immune pathways were constructed in GA-treated BmN cells, which included 12 DEmiRNA, 132 DElncRNA, and 69 mRNAs. This regulatory network enriched the functional role of miRNA in the BmNPV-silkworm interactions and improved our understanding of the molecular mechanism of HSP90 inhibitors on BmNPV proliferation.

Cellular Lnc_209997 suppresses Bombyx mori nucleopolyhedrovirus replication by targeting miR-275-5p in B. mori.

Long non-coding RNA (lncRNA) is a type of non-coding RNA molecule, which exceeds 200 nucleotides in length and participates in the regulation of a variety of life activities. Recent studies showed that lncRNAs play important roles in viral infection and host immunity. At present, the researches on insect lncRNAs are relatively few. In this study, we found the expression of Lnc_209997 was significantly down-regulated in silkworm fat body infected with Bombyx mori nucleopolyhedrosis virus (BmNPV). Inhibition of Lnc_209997 promoted BmNPV replication. Enhancing the expression of Lnc_209997 inhibited the proliferation of BmNPV. miR-275-5p was up-regulated in silkworm fat body infected with BmNPV. Dual luciferase reporter gene system confirmed the interaction between Lnc_209997 and miR-275-5p. Over-expression of Lnc_209997 inhibited the expression of miR-275-5p, while inhibition of Lnc_209997 enhanced the expression of miR-275-5p. Further, over-expression of miR-275-5p can facilitate the replication of BmNPV. These results suggested that BmNPV could increase the expression of miR-275-5p by inhibiting cellular Lnc_209997 expression to promote their own proliferation. Our results are helpful for better understanding the role of lncRNAs in BmNPV infection, and provide insights into elucidating the molecular mechanism of interaction between Bombyx mori and virus.

Functional analysis of a miRNA-like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus-encoded microRNAs (miRNAs) have been proven to play important roles in host-pathogen interactions. In this study we identified a BmCPV-derived miRNA-like 21 nt small RNA, BmCPV-miR-1, from the small RNA deep sequencing of BmCPV-infected silkworm larvae by stem-loop quantitative real-time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV-miR-1 at the 5' untranslated region. It was found that the expression of BmCPV-miR-1 and its target gene BmIAP were both up-regulated in BmCPV-infected larvae. At the same time, it was confirmed that BmCPV-miR-1 could up-regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV-miR-1 mimics could up-regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV-infected larvae, BmCPV-miR-1 mimics could be further up-regulated and inhibitors could lower the virus-mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV-miR-1 mimics could up-regulate and inhibitors down-regulate their replication in the infected silkworm. These results implied that BmCPV-miR-1 could inhibit cell apoptosis in the infected silkworm through up-regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.

Identification and characterization of two putative microRNAs encoded by Bombyx mori cypovirus.

Viral microRNAs (miRNAs) have been demonstrated to play important roles in virus-host interactions. Some RNA virus-encoded miRNAs have been reported to promote viral replication and may be used as potential drug targets. Bombyx mori cypovirus (BmCPV), an important pathogen of silkworm, is a double-stranded RNA virus frequently causing serious damages in sericulture. Research on miRNA encoded by BmCPV may be useful to elucidate the BmCPV-host interaction and to develop new anti-viral methods. In our previous study, small RNA libraries of the midgut of BmCPV-infected silkworm have been generated by deep sequencing and several BmCPV-encoded putative miRNAs were predicted. In this study, two putative miRNAs encoded by BmCPV were identified and then validated by stem-loop qRT-PCR and northern blot. They are BmCPV-miR-3 encoded by the third genomic RNA segment of BmCPV (478-497bp) and BmCPV-miR-5 encoded by the fifth genomic RNA segment (2481-2500bp), both are 20bp and encoded by ORF regions. miRNA expression could be detected as early as 5h after BmCPV infection, and the expression level of BmCPV-miR-3 is much higher than that of BmCPV-miR-5 in the course of infection. Three potential target genes were predicted in the host genome, two for BmCPV-miR-3 and one for BmCPV-miR-5, but just one in the virus genome for BmCPV-miR-3 only, with the binding sites all in coding regions. Dual luciferase assay and qRT-PCR indicated that BmCPV-miR-3 could down-regulate the expression of both its two target genes, but no regulatory effect by BmCPV-miR-5 on its target gene was detected. In contrast, BmCPV-miR-3 could up-regulate the viral target. This is the first report that an insect double stranded RNA virus may generate miRNAs and the results obtained will benefit the future study of the functions of BmCPV-encoded miRNAs on viral replication and virus-host interaction.

iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. SIGNIFICANCE: The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes the silkworm to death, which negatively affects the sericulture industry. Studies on insect antiviral immunity and on interactive mechanisms between host cells and BmCPV are in their infancy and remain insufficient. In order to obtain an overall view of silkworm response to BmCPV infection, we performed a proteomic analysis of the midgut of silkworm responses to BmCPV infection by iTRAQ. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection.

JAK/STAT signaling pathway-mediated immune response in silkworm (Bombyx mori) challenged by Beauveria bassiana.

Innate immunity was critical in insects defensive system and able to be induced by Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway. Currently, it had been identified many JAK/STAT signaling pathway-related genes in silkworm, but little function was known on insect innate immunity. To explore the roles of JAK/STAT pathway in antifungal immune response in silkworm (Bombyx mori) against Beauveria bassiana infection, the expression patterns of B. mori C-type lectin 5 (BmCTL5) and genes encoding 6 components of JAK/STAT signaling pathway in silkworm challenged by B. bassiana were analyzed using quantitative real time PCR. Meanwhile the activation of JAK/STAT signaling pathway by various pathogenic micro-organisms and the affect of JAK/STAT signaling pathway inhibitors on antifungal activity in silkworm hemolymph was also detected. Moreover, RNAi assay of BmCTL5 and the affect on expression levels of signaling factors were also analyzed. We found that JAK/STAT pathway could be obviously activated in silkworm challenged with B. bassiana and had no response to bacteria and B. mori cytoplasmic polyhedrosis virus (BmCPV). However, the temporal expression patterns of JAK/STAT signaling pathway related genes were significantly different. B. mori downstream receptor kinase (BmDRK) might be a positive regulator of JAK/STAT signaling pathway in silkworm against B. bassiana infection. Moreover, antifungal activity assay showed that the suppression of JAK/STAT signaling pathway by inhibitors could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm to B. bassiana infection, indicating that JAK/STAT signaling pathway might be involved in the synthesis and secretion of antifungal substances. The results of RNAi assays suggested that BmCTL5 might be one pattern recognition receptors for JAK/STAT signaling pathway in silkworm. These findings yield insights for better understand the molecular mechanisms of JAK/STAT signaling pathway in antifungal immune response in silkworm.

dsRNA interference on expression of a RNA-dependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens in silkworm. Its infection often results in significant losses to sericulture. Studies have demonstrated that RNAi is one of the important anti-viral mechanisms in organisms. In this study, three dsRNAs targeting the RNA-dependent RNA polymerase (RDRP) gene of BmCPV were designed and synthesized with 2'-F modification to explore their interference effects on BmCPV replication in silkworm larvae. The results showed that injecting dsRNA in the dosage of 4-6 ng per mg body weight into the 5th instar larvae can interfere with the BmCPV-RDRP expression by 93% after virus infection and by 99.9% before virus infection. In addition, the expression of two viral structural protein genes (genome RNA segments 1 and 5) was also decreased with the decrease of RDRP expression, suggesting that RNAi interference of BmCPV-RDRP expression could affect viral replication. The study provides an effective method for investigating virus replication as well as the virus-host interactions in the silkworm larvae using dsRNA.

Cloning and expression analysis of a peptidoglycan recognition protein in silkworm related to virus infection.

In this study, the full-length cDNA of a peptidoglycan recognition protein named BmPGRP-S3 was identified from the silkworm, Bombyx mori by rapid amplification of cDNA ends. It is 807 bp and comprises the following: a 5'-untranslated region (UTR) with a length of 112 bp, a 3'-UTR with a length of 92 bp including a poly-adenylation signal sequence (AATAAA) and a poly(A) tail. The longest open reading frame (ORF) of BmPGRP-S3 is 603 bp and encodes a polypeptide of 200 amino acids with a predicted molecular weight of 22.3 kDa including a PGRP domain. Sequence similarity and phylogenic analysis results indicated that BmPGRP-S3 belongs to the group of insect PGRPs and is closer to BmPGRP-S4 with the highest identity of 68%. Fluorescent quantitative real-time PCR results revealed that the mRNA transcripts of BmPGRP-S3 were presented in all of the tissues, but were highest in the midgut. In the silkworm larvae infected with B. mori cytoplasmic polyhedrosis virus (BmCPV), the relative expression level of BmPGRP-S3 was upregulated. The DNA segment of a mature BmPGRP-S3 peptide was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. Western blot results revealed that mature BmPGRP-S3 could be detected in the hemolymph and midgut which were the most important immune tissues in silkworm. All the results suggested that BmPGRP-S3 may play an important role in the immune response of silkworm to BmCPV infection and provided helpful information for further studying the function of BmPGRP-S3 in silkworm.

Core promoter regulates the expression of cathepsin B gene in the fat body of Bombyx mori.

Bombyx mori cathepsin B (BmCatB) is involved in the programmed cell death of the fat body during B. mori metamorphosis. For a better understanding of the functional regulatory mechanism, the promoter region of BmCatB in the transcriptional regulation has been identified and analyzed in the present study. BmCatB promoter region performed by the 5' truncation or mutagenesis of EcREs was inserted in the pFA3Luc-A3RL double fluorescence expression vector to activate the fireflies luciferase (FLuc) gene. The results indicated that the dual-luciferase activity of BmCatB gene in the silkworm larval fat body is regulated by the length of promoter. Site-directed mutagenesis of EcRE experiment has shown that the EcREs are up-regulated significantly in the regulation of the BmCatB promoter. A 142bp region (-1165 to -1023) and EcREs are the mainly fat-body tissue-specificity related region and could function as a core promoter element.

Cytoplasmic polyhedrosis virus-induced differential gene expression in two silkworm strains of different susceptibility.

Digital gene expression (DGE) was performed to investigate the gene expression profiles of 4008 and p50 silkworm strains at 48 h after oral infection with BmCPV. 3,668,437 clean tags were identified in the BmCPV-infected p50 silkworms and 3,540,790 clean tags in the control p50. By contrast, 4,498,263 clean tags were identified in the BmCPV-infected 4008 silkworms and 4,164,250 clean tags in the control 4008. A total of 691 differentially expressed genes were detected in the infected 4008 DGE library and 185 were detected in the infected p50 DGE library, respectively. The expression profiles identified some important differentially expressed genes involved in signal transduction, enzyme activity and apoptotic changes, some of which were verified using quantitative real-time PCR (qRT-PCR). These results provide important clues on the molecular mechanism of BmCPV invasion and resistance mechanism of silkworms against BmCPV infection.

Identification and functional analysis of the cathepsin D gene promoter of Bombyx mori.

The gene encoding cathepsin D of silkworm, Bombyx mori (BmCatD) is specifically expressed in the larval fat body and pupal gut, and plays an important role in the programmed cell death during metamorphosis. To identify element involved in this transcription-dependent spatial restriction, truncation and deletion of the 5' terminal from the BmCatD promoter were conducted in vivo. The recombinant AcMNPV vector (Autographa californica multiple nucleopolyhedrovirus) with a dual-luciferase quantitative assay system was used as the transfer. A 289 bp DNA sequence (-1,214 to -925) upstream of the transcriptional start site is found to be responsible for promoting tissue-specific transcription. Further analysis of a series of deletion within the 289 bp region of overlapping deletion showed that a 33 bp region (-1,071 to -1,038) sequence suppresses the ectopic expression of the BmCatD promoter. These results suggest that this 33 bp region could function as a promoter element in the tissue-specificity expression.

Digital gene expression analysis in the midgut of 4008 silkworm strain infected with cytoplasmic polyhedrosis virus.

Digital Gene Expression was performed to investigate the midgut transcriptome profile of 4008 silkworm strain orally infected with BmCPV. A total of 4,498,263 and 4,258,240 clean tags were obtained from the control and BmCPV-infected larvae. A total of 752 differentially expressed genes were detected, of which 649 were upregulated and 103 were downregulated. Analysis results of the Kyoto Encyclopedia of Genes and Genomes pathway showed that 334 genes were involved in the ribosome and RNA transport pathways. Moreover, 408 of the 752 differentially expressed genes have a GO category and can be categorized into 41 functional groups according to molecular function, cellular component and biological process. Differentially expressed genes involved in signaling, gene expression, metabolic process, cell death, binding, and catalytic activity changes were detected in the expression profiles. Quantitative real-time PCR was performed to verify the expression of these genes. The upregulated expression levels of Calreticulin, FK506-binding protein, and protein kinase c inhibitor gene probably led to a calcium-dependent apoptosis in the BmCPV-infected cells. The results of this study may serve as a basis for future research not only on the molecular mechanism of BmCPV invasion but also on the anti-BmCPV mechanism of silkworm.

[Development of real-time fluorescent quantitative PCR method for detecting Bombyx mori cytoplasmic polyhedrosis virus].

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.

Differentially expressed genes in the cuticle and hemolymph of the silkworm, Bombyx mori, injected with the fungus Beauveria bassiana.

The most important pathogenic fungus of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is Beauveria bassiana (Balsamo-Crivelli ) Vuillemin (Hypocreales: Clavicipitaceae), which causes significant damage to sericulture production. Therefore, diagnosing fungal disease and developing new control measures are crucial to silk production. To better understand the responsive and interactive mechanisms between the host silkworm and this fungus, variations in silkworm gene expression were investigated using the suppression subtractive hybridization method following the injection of B. bassiana conidia. Two cDNA libraries were constructed, and 140 cDNA clones were isolated. Of the 50 differentially expressed genes identified, 45 (112 clones) were identified in the forward library, and 5 (28 clones) were identified in the reverse library. Expression profiling of six of these genes by quantitative polymerase chain reaction (qPCR) verified that they were induced by the fungal challenge. The present study provides insight into the interaction between lepidopteran insects and pathogenic fungi.

Enhancement of the selective enzymatic biotransformation of rutin to isoquercitrin using an ionic liquid as a co-solvent.

An ionic liquid (IL)-containing buffer system was first applied in the conversion of rutin to isoquercitrin. High substrate solubility was achieved to enhance the selectivity and efficiency of hesperidinase-catalyzed reaction. Ten ILs were selected as co-solvents to assist catalytic reactions in this biotransformation process. The transformed products of rutin were identified by LC-MS. The [Bmim][BF(4)]-glycine-sodium hydroxide buffer (pH 9) (10:90, v/v) was found to be the best medium for the biotransformation of isoquercitrin from rutin with higher selectivity and efficiency. The reaction time was reduced by 0.33-fold while the conversion of rutin and the yield of isoquercitrin were increased by 1.67-fold and 2.33-fold. The results suggest that IL co-solvents have great potential to enhance the selectively enzymatic hydrolysis of rutin for isoquercitrin production.

cDNA cloning and characterization of LASP1 from silkworm, Bombyx mori, involved in cytoplasmic polyhedrosis virus infection.

Full-length cDNA of a LIM and SH3 contained protein 1 (named BmLASP1) was identified from the silkworm, Bombyx mori, for the first time by rapid amplification of cDNA ends. The full-length cDNA of BmLASP1 is 2094 bp, consisting of a 5'-terminal untranslated region (UTR) of 117 bp, and a 3'-UTR of 610 bp with two poly-adenylation signal sequence AATAAA and a poly (A) tail. The BmLASP1 cDNA encodes a polypeptide comprising 455 amino acids, including a LIM domain, two nebulin domains and an SH3 domain. The theoretical isoelectric point is 7.07 and the predicted molecular weight is 51.8 kDa. BmLASP1 has no signal peptide but three potential N-glycosylation sites. Sequence similarity and phylogenic analyses indicated that BmLASP1 belonged to the group of insect LASP1 with a longer linker region which is different from vertebrate LASP1. The LASP1 in silkworm contained eight exons in its coding regions, and the last exon-intron boundary was conserved the same as in mammalian and Ciona intestinalis LASP1 genes. By fluorescent quantitative real-time polymerase chain reaction, the mRNA transcripts of BmLASP1 were mainly detected in the gonad, head, and spiracle, and slightly in the silk gland, vasa mucosa, midgut, fat body, and hemocytes. After silkworm larvae were infected by B. mori cytoplasmic polyhedrosis virus (BmCPV), the relative expression level of BmLASP1 was down-regulated in the midgut. This result suggested that BmLASP1 may play an important role in the response of silkworm to BmCPV infection.

Identification of ecdysone response elements (EcREs) in the Bombyx mori cathepsin D promoter.

Bombyx mori Cathepsin D (BmCatD) is specifically expressed in the fat body, and plays a critical role for the programmed cell death of the larval fat body and pupal gut during metamorphosis. To better understand the transcriptional control of BmCatD expression, we conducted this study to identify the ecdysone response elements (EcREs) in the BmCatD promoter and clarify their regulational functions. We inserted EcREs into a recombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector and performed luciferase assay with a dual-luciferase quantitative assay system. Three putative EcREs were located at positions -109 to -99, -836 to -826 and -856 to -846 relative to the transcription start site. Overlapping deletion studies of this EcRE region showed that the three EcREs could suppress the ectopic expression of the BmCatD promoter. EcRE mutations resulted in the loss of the fat body-specific expression of the BmCatD gene. These results suggest that the EcREs are vital for activation of the promoter by 20-hydroxyecdysone (20E) in the larval fat body and further support the crucial role of ecdysone signaling to control cathepsin D gene transcription. It may suggest that the heterodimeric complex EcR/USP mediates the activation of ecdysone-dependent BmCatD transcription in the larval fat body of B. mori.