m6A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkworm (Bombyx mori).

BACKGROUND: Research has shown that epigenetic modification are involved the regulation of diapause in bivoltine silkworms (Bombyx mori), but it remains unclear how epigenetic modification in response to environmental signals precisely to regulate the diapause processing of bivoltine B. mori. METHODS AND RESULTS: In this study, the diapause terminated eggs of bivoltine B. mori, Qiufeng (QF) were divided into two groups: a QFHT group incubated at 25 °C with a natural day/night cycle to produce diapause eggs, and a QFLT group incubated at 16.5 °C in darkness to produce non-diapause eggs. On the 3rd day of the pupal stage, the total RNAs of the eggs were extracted and their N6-adenosine methylation (m6A) abundances were analyzed to explore the effects of m6A methylation on diapause in the silkworm. The results showed that 1984 m6A peaks are shared, 1563 in QFLT and 659 in QFHT. The m6A methylation level of the QFLT group was higher than that of the QFHT one in various signaling pathways. The m6A methylation rate of mevalonate kinase (MK) in the insect hormone synthesis pathway was significantly different between the two groups. The knockdown of MK by RNA interference in the pupae of QFLT resulted in females laying diapause eggs rather than non-diapause eggs after mating. CONCLUSIONS: m6A methylation involves in the diapause regulation of bivoltine B. mori by changing the expression levels of MK. This result provides a clearer image of the environmental signals on the regulation of diapause in bivoltine silkworms.

RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathway.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

Exorista sorbillans (Diptera: Tachinidae) parasitism shortens host larvae growth duration by regulating ecdysone and juvenile hormone titers in Bombyx mori (Lepidoptera: Bombycidae).

The tachinid fly, Exorista sorbillans, is a notorious ovolarviparous endoparasitoid of the silkworm, Bombyx mori, causing severe damage to silkworm cocoon industry. Silkworm larvae show typically precocious wandering behavior after being parasitized by E. sorbillans; however, the underlying molecular mechanism remains unexplored. Herein, we investigated the changes in the levels of 20-hydroxyecdysone (20E) and juvenile hormone (JH) titer, and they both increased in the hemolymph of parasitized silkworms. Furthermore, we verified the expression patterns of related genes, which showed an upregulation of 20E signaling and biosynthesis genes but a significant downregulation of ecdysone oxidase (EO), a 20E inactivation enzyme, in parasitized silkworms. In addition, related genes of the JH signaling were activated in parasitized silkworms, while related genes of the JH degradation pathway were suppressed, resulting in an increase in JH titer. Notably, the precocious wandering behavior of parasitized silkworms was partly recoverable by silencing the transcriptions of BmCYP302A1 or BmCYP307A1 genes. Our findings suggest that the developmental duration of silkworm post parasitism could be shortened by regulation of 20E and JH titers, which may help silkworm to resist the E. sorbillans infestation. These findings provide a basis for deeper insight into the interplay between silkworms and E. sorbillans and may serve as a reference for the development of a novel approach to control silkworm myiasis.

The Pseudogene BMEA_B0173 Deficiency in Brucella melitensis Contributes to M-epitope Formation and Potentiates Virulence in a Mice Infection Model.

It is widely acknowledged that pseudogenes play important roles in bacterial diversification and evolution and participate in gene regulation and RNA interference (RNAi). However, the function of most pseudogenes in Brucella spp remains poorly understood, warranting further studies.To comprehensively analyze the function of the pseudogenes BMEA_B0173 in Brucella melitensis strain 63/9, a BMEA_B0173 in-frame deleted mutant strain was constructed. Then, the phenotypes of the mutant strain, such as growth characteristics and bacterial virulence, were assessed in mice infection models. Finally, iTRAQ analysis was performed to investigate the gene expression profile affected by the pseudogenes BMEA_B0173. In this study, we found that BMEA_B0173 deletion exhibited increased agglutination with M monospecific sera. In a mouse model of chronic infection, the BMEA_B0173 deletion strain displayed increased colonization in the spleen compared to the wild-type pathogen. The iTRAQ assay revealed that 252 proteins were differentially expressed between the BMEA_B0173 deletion and the wild-type strains. In addition, deletion of BMEA_B0173 significantly increased the expression of proteins involved in the denitrification pathway, iron metabolism, and several transcriptional regulators, which might cause increased virulence of the mutant strain. In conclusion, this study preliminary uncovered the function of the pseudogene BMEA_B0173 in Brucella melitensis 63/9 and provided novel insights for studying the pathogenesis of Brucella strains.

18 Additional Amino Acids of the Signal Peptide of the Bombyx mori Nucleopolyhedrovirus GP64 Activates Immunoglobulin Binding Protein (BiP) Expression by RNA-seq Analysis.

GP64 is the key membrane fusion protein of Group I baculovirus, and while the Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 contains a longer n-region (18 amino acid) of the signal peptide than does the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the function of the n-region has not been determined. In this study, we first showed that n-region is required for membrane protein localization in BmN cells, then the transcriptome sequencing was conducted on proteins guided by different signal peptide regions, and the results were analyzed and validated by quantitative PCR and luciferase assays. The results indicated that 1049 differentially expressed genes (DEGs) were identified among the different region of signal peptides and the control. With the n-region, the protein export pathway was upregulated significantly, the Wnt-1 signaling pathway was downregulated, and BiP was significantly activated by the GP64 full-length signal peptide. Furthermore, RNA interference on BiP efficiently increased luciferase secretion. These results indicate that the GP64 n-region plays a key role in protein expression and regulation.

Bmo-miR-2780a regulates the expression of the sericin-1 gene of Bombyx mori.

Silk production in Bombyx mori L. is largely determined by the expression of genes encoding fibroin and sericin. Here, we examined the regulatory function of a microRNA (miRNA) on silk gene expression using the sericin-1 gene (BmSer-1). First, we downloaded whole mature miRNAs of silkworm from miRBase and identified bmo-miR-2780a as a candidate miRNA for the regulation of BmSer-1 expression. We used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with stem-loop primers to investigate the expression profile of bmo-miR-2780a and its predicted target gene BmSer-1 in seven different tissues from 5th instar day-3 larvae, including head, fat body, anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland (PSG), middle gut, and hemolymph. Our results showed that bmo-miR-2780a was specifically expressed in the MSG and that the expression level of BmSer-1 was significantly higher in the MSG than in other tissues. Recombinant plasmids carrying both pri-mir-2780a and Ser1-3'UTR were constructed and then used to cotransfect BmN cells. We further detected the effect of bmo-miR-2780a on Ser-1 in vivo. These results showed that the target gene was significantly decreased by miR-2780a compared with the control group (p < .05), thus indicating that bmo-miR-2780a might negatively regulate the expression of Ser-1.

Bom-miR-2805 upregulates the expression of Bombyx mori fibroin light chain gene in vivo.

MicroRNAs (miRs) are inner regulatory RNAs mainly by regulating expression of genes at the posttranscriptional level. To investigate the regulatory function of Bombyx mori (B. mori) fibroin protein genes, the mRNA 3'-untranslated region (3'-UTR) of fibroin light chain gene (BmFib-L) was used as the target and one miRNA, miR-2805 was predicted by using the Software. miR-2805 expression plasmid pcDNA3.0[ie1-egfp-pre-miR-2805-SV40] and BmFib-L 3'-UTR plasmid pGL3.0[A3-luc-Fib-L-3'-UTR-SV40] were constructed, respectively. The mentioned plasmids were cotransfected in BmN cells, and the regulatory function of miR-2805 on BmFib-L was detected by assay of dual luciferase activities, as well as synthesized mimic and inhibitor of miR-2805. The results revealed that miR-2805 significantly downregulated the expression of BmFib-L in BmN cells. To validate the function of miR-2805 in vivo, cultured silk glands or larvae were injected with solution containing pcDNA3.0[ie1-egfp-SV40], pcDNA3.0[ie1-egfp-pre-miR-2805-SV40], mimic, inhibitor respectively. BmFib-L expression was analyzed by quantitative reverse transcription polymerase chain reaction using total RNAs extracted from silk glands. The results showed that miR-2805 significantly upregulated the expression of BmFib-L in both cultured tissues and individuals. To find out how miR-2805 differentially regulates BmFib-L expression in cells and tissues or individuals, we analyzed the expression level of transcription factors (TFs) involved in expression of silk protein genes. The results showed that miR-2805 upregulated the expression of TFs BmAwh and Bmdimm. These results suggest that miR-2805 may up-regulate the expression of BmFib-L interaction with BmAwh and/or Bmdimm in vivo. These findings are beneficial to clarify the molecular mechanism of miRNAs in regulating B. mori silk protein biosynthesis.

Expression analysis and functional identification of several genes related to diapause in Bombyx mori.

Diapause is an important characteristic of insects used to adapt to extreme changes in environmental conditions. Embryonic diapause of the bivoltine silkworm (Bombyx mori) is determined by environmental conditions experienced by the mother while in the embryo stage. If they are incubated at 25°C with natural light, their progenies will be diapause-destined. If they are incubated at 17°C in darkness, their progenies will be non-diapause-destined. The molecular mechanism of diapause remains unknown. In the present study, we analyzed two downregulated genes (BGIBMGA003835, BGIBMGA012335) and two upregulated genes (BGIBMG012996, BGIBMG002426) related to carbohydrate metabolism, verified differentially expressed in ovaries and heads of 1-day-old fifth-instar larvae to 6-day pupae by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). In line with published data, the expression level of these genes in larvae were generally lower than in pupae. We further analyzed the expression levels of the four genes in BmN cells that had been treated with various concentrations of diapause hormone (DH). It demonstrated that the expression of these genes was affected by DH. Knockdown of the selected genes in non-diapause-destined female pupae changed the fate of the progeny from non-diapause- to daipause-destined, as seen by the appearance of diapause eggs. Our study provides insight into the molecular mechanism of diapause in B. mori.

The role of N6-methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism.

N6-methyladenosine (m6 A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m6 A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m6 A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m6 A-modification-related gene expression and m6 A content were greater in diapause-destinated compared to nondiapause-destined strains. Our findings suggest that m6 A modification may provide significant epigenetic regulation of diapause-related genes in the silkworm.

An amino acid duplication/insertion in the Bm126 gene of Bombyx mori nucleopolyhedrovirus alters viral gene expression as shown by differential gene expression analysis.

Open reading frame (ORF) 126 (Bm126) of Bombyx mori nucleopolyhedrovirus (BmNPV) is not essential for viral replication, and two subtypes of this gene have been identified in China. The Bm126-SX subtype encodes a protein with a simple amino acid duplication/insertion relative to the Bm126-GD subtype; however, significant differences in the cytopathic effect and infectivity of viruses carrying these variant genes have been observed. To elucidate the cause of these differences, differential gene expression analysis was performed at the early stage of infection with viruses harbouring variants of Bm126. Differential expression was observed for 103, 209, and 313 host genes and 9, 44, and 67 viral genes in vGD126 samples relative to the control samples (vSX126) at 6, 12, and 24 h postinfection, respectively. These results indicated that the duplication/insertion in Bm126 altered the viral expression pattern. The differentially expressed host genes were found to be related to ribosome, spliceosome, and proteasome pathways, and several factors involved in signal transduction were also identified. The differential expression of these viral and host genes was confirmed by qPCR. This study indicates that the amino acid duplication/insertion in the Bm126 gene has a biological function related to the regulation of viral gene expression and serves as a basis for further characterization of Bm126 gene function.

Preincubation with a low concentration of methyl-ß-cyclodextrin enhances baculovirus expression system productivity.

OBJECTIVES: To enhance the productivity of foreign protein in culture cells using baculovirus expression system. RESULTS: A low concentration of MßCD, with the optimal application concentration of 0.25 mM and the appropriate preincubation time range from 10 to 120 min, can efficiently enhance expression levels in both the AcMNPV and BmNPV expression systems. CONCLUSIONS: Preincubation with a low concentration MßCD enhance baculovirus infection and foreign protein expression productivity.

Inhibition of expression of BmNPV cg30 by bmo-miRNA-390 is a host response to baculovirus invasion.

Bombyx mori larvae exhibit in vivo defensive reactions immediately after invasion by a virus. One of these defense systems is to express appropriate microRNAs (miRNAs) to respond to the infection. A novel Bombyx mori-encoded miRNA, bmo-miR-390, was identified previously by high-throughput sequencing. Based on bioinformatic predictions, the Bombyx mori nuclear polyhedrosis virus cg30 gene (BmNPV-cg30) is one of the target genes of bmo-miR-390. In this study, expression vectors with an enhanced green fluorescence protein (EGFP) or a luciferase (luc) reporter gene together with bm-miR-390 or the cg30 3' UTR were constructed and used to co-transfect BmN cells. Using a dual luciferase reporter (DLR) assay, we found that bmo-miR-390 significantly downregulates the expression of BmNPV-cg30 (P < 0.05) in vitro. Moreover, artificially synthesized bmo-miR-390 mimics enhanced the regulatory effect of bmo-miR-390, while an inhibitor eliminated the inhibitory effect. These results show for the first time that bmo-miR-390 can effectively downregulate the expression of BmNPV-cg30 in BmNPV-infected BmN cells.

Bmo-miR-2758 Targets BmFMBP-1 (Lepidoptera: Bombycidae) and Suppresses Its Expression in BmN Cells.

MicroRNAs (miRNAs) are an abundant family of endogenous noncoding small RNA molecules. They play crucial roles on regulation of life processes both in plants and animals. Fibroin modulator binding protein-1 (FMBP-1) is a silk gland transcription factor of Bombyx mori, which is considered as a trans-activator of fibroin genes. And bioinformatics prediction showed that at the 3' untranslated region (3' UTR) of BmFMBP-1 there were binding sites for three bmo-miRNAs, bmo-miR-2b*, bmo-miR-305, and bmo-miR-2758, separately. In order to validate whether these bmo-miRNAs involved in the regulation of BmFMBP-1 expression, the expression levels of three bmo-miRNAs and BmFMBP-1 in the middle silk gland (MSG) and posterior silk gland (PSG) during the fourth- and fifth-larval stages of B. mori were measured by semi-quantitative reverse transcription polymerase chain reaction. The results revealed that the expression level of bmo-miR-2758 was the highest in the three, and it expressed higher in the PSG than in the MSG with a similar expression pattern as BmFMBP-1, implying that bmo-miR-2758 may involved in regulation of BmFMBP-1. To validate the regulation function of bmo-miR-2758 on BmFMBP-1, recombinant plasmids pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] and pGL3 [A3-luc-FMBP-1 3' UTR-SV40] were constructed and co-transfected in BmN cells. The dual-luciferase reporter assay system was used for assay of transient expression. The results showed that the expression of the luciferase reporter was significantly decreased when pGL3 [A3-luc-FMBP-1 3' UTR-SV40] co-transfected with pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] (P < .01). Furthermore, when the artificial antisense RNA of bmo-miR-2758 (inhibitor) was added to the above co-transfection, the expression of the luciferase reporter was recovered significantly (P < 0.01). These results suggest that bmo-miR-2758 represses the expression of BmFMBP-1 in vitro.

Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.

Overview of research on Bombyx mori microRNA.

MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs.

[Genetic analysis and gene mapping of two novel quail-like mutants from the silkworm (Bombyx mori)].

Two novel body marking mutants were discovered during silkworm (Bombyx mori) breeding. The mutants have no obvious eye-spots compared with normal marking (+) individuals, but their star spots and semilunar markings on dorsal sides are normal, and there are dots and lines with longitudinal wave markings on dorsal sides of the 6th to 7th abdominal segments which consist quail markings in between star spots and semilunar markings. The whole body markings are very similar to that of quail mutant (q); thus these mutants are named as quail-like mutants (q-l). Young larvae of one mutant are in brown color, and develop normally. Their cocoons are regular and uniform in size. Thus, this mutant is designated as brown quail-like (q-lb). Another mutant's larvae are in light purple skin; thus this mutant is named as purple quail-like (q-lp). They take little amount of mulberry leaves, and are weak and develop slowly and unevenly. Their larval bodies and cocoons are small. Genetic analysis revealed that both q-lb and q-lp were recessive genes, and they were allelic, with q-lb recessive to q-lp. These genes are different from quail mutant (q) and located on the chromosome 8 after tested by the morphological markers, P3(2), p(2), Ze(3), L(4), re(5), E(6), q(7), I-a(9), ms(12), ch(13), oa(14), cts(16), mln(18), msn(19), rb(21) and so(26) and SSR markers.

Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori).

Genetic mutations leading to premature termination codons are known to have detrimental effects. Using the Lepidoptera model insect, the silkworm (Bombyx mori), we explored the genetic compensatory response triggered by mutations with premature termination codons. Additionally, we delved into the molecular mechanisms associated with the nonsense-mediated mRNA degradation pathway. CRISPR/Cas9 technology was utilized to generate a homozygous bivoltine silkworm line BmTrpA1-/- with a premature termination. Transcript levels were assessed for the BmTrpA paralogs, BmPyrexia and BmPainless as well as for the essential factors Upf1, Upf2, and Upf3a involved in the nonsense-mediated mRNA degradation (NMD) pathway. Upf2 was specifically knocked down via RNA interference at the embryonic stage. The results comfirmed that the BmTrpA1 transcripts with a 2-base deletion generating a premature termination codon in the BmTrpA1-/- line. From day 6 of embryonic development, the mRNA levels of BmPyrexia, BmPainless, Upf1, and Upf2 were significantly elevated in the gene-edited line. Embryonic knockdown of Upf2 resulted in the suppression of the genetic compensation response in the mutant. As a result, the offspring silkworm eggs were able to hatch normally after 10 days of incubation, displaying a non-diapause phenotype. It was observed that a genetic compensation response does exist in BmTrpA1-/-B. mori. This study presents a novel discovery of the NMD-mediated genetic compensation response in B. mori. The findings offer new insights into understanding the genetic compensation response and exploring the gene functions in lepidopteran insects, such as silkworms.

Bmo-miR-9A down regulates the expression of Bm-ase gene in vitro.

MicroRNAs (miRNAs) are a class of non-protein coding small RNAs of 18-24 nucleotides in length that regulate expression of genes at post-transcriptional levels and play multiple roles in biological processes. Bm-ase plays an important role in the course of nerve development of the silkworm, Bombyx mori. Bmo-miR-9a is a conservative miRNA. By using target prediction software RNA22 and RNAhybrid, we found a target site of Bmo-miR-9a in the 3'UTR of Bm-ase gene. To verify the regulation function of Bmo-miR-9a on the expression of Bm-ase gene, a Bmo-miR-9a over-expressing vector and Bm-ase 3'UTR fused firefly luciferase gene reporter plasmid were constructed, respectively. Then they were used to co-transfect the BmN cells. The result showed that luciferase activity in the co-transfected cells was suppressed compared with the control. A similar result was obtained when BmN cells were co-transfected with artificial synthetic Bmo-miR-9a mimics and Bm-ase 3'UTR fused luciferase reporter plasmid. These results suggest that Bmo-miR-9a can down regulate the expression of Bm-ase gene.

Metabolomics analysis of dietary restriction results in a longer lifespan due to alters of amino acid levels in larval hemolymph of Bombyx mori.

Dietary restriction (DR) has been a very important discovery in modern aging biology research. Its remarkable anti-aging effect has been proved in a variety of organisms, including members of Lepidoptera, but mechanisms by which DR increases longevity are not fully understood. By using the silkworm (Bombyx mori), a model of lepidopteran insect, we established a DR model, isolated hemolymph from fifth instar larvae and employed LC-MS/MS metabolomics to analyze the effect of DR on the endogenous metabolites of silkworm, and tried to clarify the mechanism of DR to prolong lifespan. We identified the potential biomarkers by analyzing the metabolites of the DR and control groups. Then, we constructed relevant metabolic pathways and networks with MetaboAnalyst. DR significantly prolonged the lifespan of silkworm. The differential metabolites between the DR and control groups were mainly organic acids (including amino acid), and amines. These metabolites are involved in metabolic pathways such as amino acid metabolism. Further analysis showed that, the levels of 17 amino acids were significantly changed in the DR group, indicating that the prolonged lifespan was mainly due to changes in amino acid metabolism. Furthermore, we identified 41 and 28 unique differential metabolites in males and females, respectively, demonstrating sex differences in biological responses to DR. The DR group showed higher antioxidant capacity and lower lipid peroxidation and inflammatory precursors, with differences between the sexes. These results provide evidence for various DR anti-aging mechanisms at the metabolic level and novel reference for the future development of DR-simulating drugs or foods.

Determination of Key Components in the Bombyx mori p53 Apoptosis Regulation Network Using Y2H-Seq.

The apoptosis pathway is highly conserved between invertebrates and mammals. Although genes encoding the classical apoptosis pathway can be found in the silkworm genome, the regulatory pathway and other apoptotic network genes have yet to be confirmed. Consequently, characterizing these genes and their underlying mechanisms could provide critical insights into the molecular basis of organ apoptosis and remodeling. A homolog of p53, a key apoptosis regulator in vertebrates, has been identified and cloned from Bombyx mori (Bmp53). This study confirmed via gene knockdown and overexpression that Bmp53 directly induces cell apoptosis and regulates the morphology and development of individuals during the metamorphosis stage. Furthermore, yeast two-hybrid sequencing (Y2H-Seq) identified several potential apoptotic regulatory interacting proteins, including the MDM2-like ubiquitination regulatory protein, which may represent an apoptosis factor unique to Bmp53 and which differs from that in other Lepidoptera. These results provide a theoretical basis for analyzing the various biological processes regulated by Bmp53 interaction groups and thus provide insight into the regulation of apoptosis in silkworms. The global interaction set identified in this study also provides a basic framework for future studies on apoptosis-dependent pupation in Lepidoptera.