Adaptive Changes in Detoxification Metabolism and Transmembrane Transport of Bombyx mori Malpighian Tubules to Artificial Diet.

The high adaptability of insects to food sources has contributed to their ranking among the most abundant and diverse species on Earth. However, the molecular mechanisms underlying the rapid adaptation of insects to different foods remain unclear. We explored the changes in gene expression and metabolic composition of the Malpighian tubules as an important metabolic excretion and detoxification organ in silkworms (Bombyx mori) fed mulberry leaf and artificial diets. A total of 2436 differentially expressed genes (DEGs) and 245 differential metabolites were identified between groups, with the majority of DEGs associated with metabolic detoxification, transmembrane transport, and mitochondrial function. Detoxification enzymes, such as cytochrome P450 (CYP), glutathione-S-transferase (GST), and UDP-glycosyltransferase, and ABC and SLC transporters of endogenous and exogenous solutes were more abundant in the artificial diet group. Enzyme activity assays confirmed increased CYP and GST activity in the Malpighian tubules of the artificial diet-fed group. Metabolome analysis showed increased contents of secondary metabolites, terpenoids, flavonoids, alkaloids, organic acids, lipids, and food additives in the artificial diet group. Our findings highlight the important role of the Malpighian tubules in adaptation to different foods and provide guidance for further optimization of artificial diets to improve silkworm breeding.

Non-classical digestive lipase BmTGL selected by gene amplification reduces the effects of mulberry inhibitor during silkworm domestication.

Efficient utilization of dietary lipids is crucial for Bombyx mori, also known as domesticated silkworms. However, the effects of domestication on the genes encoding lipases remain unknown. In this study, we investigated the expression difference of one triacylglycerol lipase (BmTGL) between B.mori and wild (ancestor) silkworm strains (Bombyx mandarina). An immunofluorescence localization analysis showed that BmTGL was present in all parts of the gut and was released into the intestinal lumen. BmTGL expression was significantly enhanced in different domesticated silkworm strains compared to that in the B. mandarina strains. The BmTGL copy numbers in the genomes of the domesticated silkworm strains were 2-to-3 folds that of the B. mandarina strains and accounted for the enhanced expression of BmTGL in the domesticated silkworm strains. The Ser144Asn substitution in the Ser-Asp-His catalytic triads of BmTGL resulted in relatively lower lipase activity and reduced sensitivity to the lipase inhibitor morachalcone A. Moreover, BmTGL overexpression significantly increased the weights of the B. mori silkworms compared to those of the non-transgenic controls. Thus, the selection of BmTGL by gene amplification may be a trade-off between maintaining high enzymatic activity and reducing the effects of mulberry inhibitors during silkworm domestication.

Biochemical characterization and overexpression of an α-amylase (BmAmy) in silkworm, Bombyx mori.

Silkworm (Bombyx mori) is the only fully domesticated insect. As an economically important insect, nutrition utilization is important for its productivity. Hence, the present study investigated the expression pattern of BmAmy, an α-amylase, in B. mori. BmAmy protein purification and biochemical characterization were performed, and effects of BmAmy overexpression were assessed. Real-time quantitative reverse transcription polymerase chain reaction indicated that BmAmy transcription was positively correlated with the silkworm's food intate. Moreover, enzymatic activity assay results showed that BmAmy had significant α-amylase activity of about 1 mg/min/mg protein. Furthermore, treatment with mulberry amylase inhibitors MnAI1 and MnAI2 resulted to 89.92% and 93.67% inhibition in BmAmy activity, respectively, and the interaction between BmAmy and MnAI was also confirmed by protein docking analysis. A silkworm line that specifically overexpressed BmAmy in the midgut was generated through piggyBac-based transgenic technology, and compared to those of non-transgenic silkworms, the whole cocoon and cocoon shell weights of these transgenic silkworms increased by 10.13% and 18.32%, respectively, in the female group, and by 5.83% and 6.00%, respectively, in the male group. These results suggested that BmAmy may be a suitable target for breeding better silkworm varieties in the future.

Genome-Wide Identification and Characterization of GASA Gene Family in Nicotiana tabacum.

The gibberellic acid stimulated Arabidopsis (GASA) gene family is critical for plant growth, development, and stress response. GASA gene family has been studied in various plant species, however, the GASA gene family in tobacco (Nicotiana tabacum) have not been characterized in detail. In this study, we identified 18 GASA genes in the tobacco genome, which were distributed to 13 chromosomes. All the proteins contained a conserved GASA domain and highly specific 12-cysteine residues at the C-terminus. Phylogenetic analysis divided the NtGASA genes into three well-conserved subfamilies. Synteny analysis suggested that tandem and segmental duplications played an important role in the expansion of the NtGASA gene family. Cis-elements analysis showed that NtGASA genes might influence different phytohormone and stress responses. Tissue expression analysis revealed that NtGASA genes displayed unique or distinct expression patterns in different tissues, suggesting their potential roles in plant growth and development. We also found that the expression of NtGASA genes were mostly regulated by abscisic and gibberellic acid, signifying their roles in the two phytohormone signaling pathways. Overall, these findings improve our understanding of NtGASA genes and provided useful information for further studies on their molecular functions.

Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication.

Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 °C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5'-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding.

Comparative Fecal Metabolomes of Silkworms Being Fed Mulberry Leaf and Artificial Diet.

Metabonomics accurately monitors the precise metabolic responses to various dietary patterns. Metabolic profiling allows simultaneous measurement of various fecal metabolites whose concentrations may be affected by food intake. In this study, we analyzed the fecal metabolomes of silkworm (Bombyx mori) larvae reared on fresh mulberry leaves and artificial diets. 57 differentially expressed metabolites were identified by gas chromatography-mass spectrometry. Of these, 39 were up-regulated and 18 were downregulated in the mulberry leaf meal group. Most of the amino acids, carbohydrates and lipids associated with physical development and silk protein biosynthesis were enriched in silkworms reared on mulberry leaves. In contrast, the urea, citric acid, D-pinitol, D-(+)-cellobiose and N-acetyl glucosamine levels were relatively higher in the silkworm feeding on the artificial diets. The findings of this study help clarify the association between diet and metabolic profiling.

Comparative transcriptomic analysis reveals that multiple hormone signal transduction and carbohydrate metabolic pathways are affected by Bacillus cereus in Nicotiana tabacum.

Bacillus cereus is thought to be a beneficial bacterium for plants in several aspects, such as promoting plant growth and inducing plant disease resistance. However, there is no detailed report on the effect of Bacillus cereus acting on Nicotiana tabacum. In the present study, RNA-based sequencing (RNA-seq) was used to identify the molecular mechanisms of the interaction between B. cereus CGMCC 5977 and N. tabacum. A total of 7345 and 5604 differentially expressed genes (DEGs) were identified from leaves inoculated with Bacillus cereus at 6 and 24 hpi, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the most DEGs could be significantly enriched in hormone signal transduction, the MAPK signaling pathway, photosynthesis, oxidative stress, and amino sugar, and nucleotide sugar metabolism. Furthermore, glycolysis/gluconeogenesis was severely affected by inoculation with Bacillus cereus. In the hormone signal pathway, multiple DEGs were involved in plant defense-related major hormones, including activation of jasmonic acid (JA), salicylic acid (SA), and ethylene (Eth). Further analyses showed that other hormone-related genes involved in abscisic acid (ABA), gibberellin (GA), auxin (AUX), and cytokinin (CK) also showed changes. Notably, a large number of genes associated with glycolysis/gluconeogenesis, catabolism of starch and oxidative stress were induced. In addition, the majority of DEGs related to nucleic acid sugar metabolism were also significantly upregulated. Biochemical assays showed that the starch content of B. cereus-treated leaves was reduced to 2.51 mg/g and 2.38 mg/g at 6 and 24 hpi, respectively, while that of the control sample was 5.42 mg/g. Overall, our results demonstrated that multiple hormone signal transduction and carbohydrate metabolic pathways are involved in the interaction of tobacco and B. cereus.

Genome-Wide Identification and Expression Analysis of HD-ZIP I Gene Subfamily in Nicotiana tabacum.

The homeodomain-leucine zipper (HD-Zip) gene family, whose members play vital roles in plant growth and development, and participate in responding to various stresses, is an important class of transcription factors currently only found in plants. Although the HD-Zip gene family, especially the HD-Zip I subfamily, has been extensively studied in many plant species, the systematic report on HD-Zip I subfamily in cultivated tobacco (Nicotiana tabacum) is lacking. In this study, 39 HD-Zip I genes were systematically identified in N. tabacum (Nt). Interestingly, that 64.5% of the 31 genes with definite chromosome location information were found to originate from N. tomentosoformis, one of the two ancestral species of allotetraploid N. tabacum. Phylogenetic analysis divided the NtHD-Zip I subfamily into eight clades. Analysis of gene structures showed that NtHD-Zip I proteins contained conserved homeodomain and leucine-zipper domains. Three-dimensional structure analysis revealed that most NtHD-Zip I proteins in each clade, except for those in clade η, share a similar structure to their counterparts in Arabidopsis. Prediction of cis-regulatory elements showed that a number of elements responding to abscisic acid and different abiotic stresses, including low temperature, drought, and salinity, existed in the promoter region of NtHD-Zip I genes. The prediction of Arabidopsis ortholog-based protein-protein interaction network implied that NtHD-Zip I proteins have complex connections. The expression profile of these genes showed that different NtHD-Zip I genes were highly expressed in different tissues and could respond to abscisic acid and low-temperature treatments. Our study provides insights into the evolution and expression patterns of NtHD-Zip I genes in N. tabacum and will be useful for further functional characterization of NtHD-Zip I genes in the future.

Isolation and functional characterization of a novel FLOWERING LOCUS T homolog (NtFT5) in Nicotiana tabacum.

The phosphatidyl ethanolamine-binding proteins (PEBPs) function primarily in regulating flowering in plants. Here, we report a genome-wide identification of PEBPs and functional characterization of a novel FLOWERING LOCUS T homolog (NtFT5) in tobacco, Nicotiana tabacum. Seven new PEBPs were identified by a genome-wide analysis from N. tabacum. Expression profile showed that NtFT5 was mainly expressed in flowers. Overexpression of NtFT5 conferred an early flowering phenotype. By optimizing rooting medium, heritable short life-cycle tobacco lines were obtained by overexpression of NtFT5. Several orthologs of flowering genes downstream of FT gene were up-regulated in the NtFT5-overexpression transgenic plant lines. The NtFT5-overexpressing tobaccos formed fewer flowers and seeds per capsule compared with wild type. The seed-to-seed life cycle of NtFT5 overexpressing tobacco lines was about 2.5 months. Gene identification was effectively undertaken in the short life-cycle tobacco line by a second transformation via a gusA reporter gene and transient expression of Ros1 via PVX (Potato Virus X)system. Our findings indicate that NtFT5 is a novel FT homolog that has potential to induce flowering, which will improve our understanding of the mechanism underlying flowering control in N. tabacum. In addition, the results show that the generated heritable short life-cycle transgenic tobacco line is an effective and stable host system to accelerate gene function study, which promises to provide a better tobacco research model for plants.

Identification and expression analysis of EDR1-like genes in tobacco (Nicotiana tabacum) in response to Golovinomyces orontii.

ENHANCED DISEASE RESISTANCE1 (EDR1) encodes a Raf-like mitogen-activated protein kinase, and it acts as a negative regulator of disease resistance and ethylene-induced senescence. Mutations in the EDR1 gene can enhance resistance to powdery mildew both in monocotyledonous and dicotyledonous plants. However, little is known about EDR1-like gene members from a genome-wide perspective in plants. In this study, the tobacco (Nicotiana tabacum)EDR1-like gene family was first systematically analyzed. We identified 19 EDR1-like genes in tobacco, and compared them to those from Arabidopsis, tomato and rice. Phylogenetic analyses divided the EDR1-like gene family into six clades, among them monocot and dicot plants were respectively divided into two sub-clades. NtEDR1-1A and NtEDR1-1B were classified into clade I in which the other members have been reported to negatively regulate plant resistance to powdery mildew. The expression patterns of tobacco EDR1-like genes were analyzed after plants were challenged by Golovinomyces orontii, and showed that several other EDR1-like genes were induced after infection, as well as NtEDR1-1A and NtEDR1-1B. Expression analysis showed that NtEDR1-13 and NtEDR1-16 had exclusively abundant expression patterns in roots and leaves, respectively, and the remaining NtEDR1-like members were actively expressed in most of the tissue/organ samples investigated. Our findings will contribute to further study of the physiological functions of EDR1-like genes in tobacco.

Selection of reference genes for tissue/organ samples on day 3 fifth-instar larvae in silkworm, Bombyx mori.

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth-instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori.

Functional analysis of NtMPK2 uncovers its positive role in response to Pseudomonas syringae pv. tomato DC3000 in tobacco.

Mitogen-activated protein kinase cascades are highly conserved signaling modules downstream of receptors/sensors and play pivotal roles in signaling plant defense against pathogen attack. Extensive studies on Arabidopsis MPK4 have implicated that the MAP kinase is involved in multilayered plant defense pathways. In this study, we identified tobacco NtMPK2 as an ortholog of AtMPK4. Transgenic tobacco overexpressing NtMPK2 markedly enhances resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) virulent and avirulent strains. Transcriptome analysis of NtMPK2-dependent genes shows that possibly the basal resistance system is activated by NtMPK2 overexpression. In addition to NtMPK2-mediated resistance, multiple pathways are involved in response to the avirulent bacteria based on analysis of Pst-responding genes, including SA and ET pathways. Notably, it is possible that biosynthesis of antibacterial compounds is responsible for inhibition of Pst DC3000 avirulent strain when programmed cell death processes in the host. Our results uncover that NtMPK2 positively regulate tobacco defense response to Pst DC3000 and improve our understanding of plant molecular defense mechanism.

Genome-wide patterns of genetic variation among silkworms.

Although the draft genome sequence of silkworm is available for a decade, its genetic variations, especially structural variations, are far from well explored. In this study, we identified 1,298,659 SNPs and 9,731 indels, of which 32 % of SNPs and 92.2 % of indels were novel compared to previous silkworm re-sequencing analysis. In addition, we applied a read depth-based approach to investigate copy number variations among 21 silkworm strains at genome-wide level. This effort resulted in 562 duplicated and 41 deleted CNV regions, and among them 442 CNV were newly identified. Functional annotation of genes affected by these genetic variations reveal that these genes include a wide spectrum of molecular functions, such as immunity and drug detoxification, which are important for the adaptive evolution of silkworms. We further validated the predicted CNV regions using q-PCR. 94.7 % (36/38) of the selected regions show divergent copy numbers compared to a single-copy gene OR2. In addition, potential presence/absence variations are also observed in our study: 11 genes are present in the reference genome, but absent in other strains. Overall, we draw an integrative map of silkworm genetic variation at genome-wide level. The identification of genetic variations in this study improves our understanding that these variants play important roles in shaping phenotypic variations between wild and domesticated silkworms.

Cloning and characterization of a novel Nicotiana tabacum ABC transporter involved in shoot branching.

The ATP-binding cassette (ABC) superfamily is a large protein family with diverse physiological functions in all kingdoms of life. One distinguished subfamily, the pleiotropic drug resistance (PDR) transporters, has only been identified in plants and fungi. Here, we identified a Nicotiana tabacum PDR gene, NtPDR6, which is a homolog of Petunia hybrida PDR1. The full-length cDNA of NtPDR6 had a 4482-bp open reading frame encoding a full-size ABC transporter with 1493 amino acids. Sequence comparison showed that NtPDR6 had high homology with plant PDR proteins. NtPDR6 was strongly induced by phosphate starvation as well as by 1-naphthalene acetic acid. Tissue expression pattern analysis showed that NtPDR6 was detected in all surveyed tissues but preferentially in roots. We cloned the 1.3-kb NtPDR6 promoter and found that there was one phosphate starvation response-related element Pho-like and several root-specific expression-related elements rootmotiftapox1 in the NtPDR6 promoter. A tissue-specific pattern of NtPDR6 promoter-ß-glucuronidase expression was dominantly observed in subepidermal cells and the elongation zone of lateral roots. RNA interference technology was used to knock down NtPDR6 expression, and there was a significantly increased branching phenotype in the NtPDR6 knockdown plants. These data suggest that NtPDR6 plays a key role in regulation of shoot branching processes.

CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

Characterization of Argonaute family members in the silkworm, Bombyx mori.

The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgo1 and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5' end of the small RNA guide strand and of the conserved (aspartate, aspartate and histidine [DDH]) motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.

A transgenic animal with antiviral properties that might inhibit multiple stages of infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms, causing severe economic losses in sericulture. To create antiviral silkworm strains, we constructed a transgenic vector in which the dsRNA for five tandem BmNPV genes was controlled by the BmNPV hr3 enhancer and IE1 promoter. The antivirus gene Bmlipase-1 was driven by B. mori midgut-specific promoter P2. Transgenic strains (SW-H) were generated via embryo microinjection using the practical silkworm strain SW. After infection with a high dose of BmNPV, the survival rates of SW-H and non-transgenic SW were 64% and 13%, respectively. SW-H could be the first transgenic animal that is highly antiviral and that might inhibit the virus at multiple stages of infection.

Comparison of factors that may affect the inhibitory efficacy of transgenic RNAi targeting of baculoviral genes in silkworm, Bombyx mori.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen affecting B. mori. This virus could be combated via RNAi of BmNPV genes in transgenic silkworm. However, several factors may affect the resistance of transgenic RNAi silkworm, such as the connection pattern of gene fragments and spacers ("head to head" or "tail to tail"), and the selection of promoters and target genes. In this study, we constructed several transgenic RNAi vectors using different phase genes (ie-1, helicase, gp64, and vp39) and promoters (BmNPV IE1 promoter (IE1P), IE1P combined with hr3 enhancer of BmNPV, and B. mori A4 promoter (A4P)). Transgenic lines were generated via embryo microinjection using a practical silkworm strain. We analyzed the anti-BmNPV ability, virus gene mRNA level, and BmNPV content of these transgenic larvae. The results showed that "head to head" was better than "tail to tail," IE1P combined with hr3 was better than IE1P and A4P, and an immediate early gene was the best target for RNAi.

Cloning and evolutionary analysis of tobacco MAPK gene family.

The mitogen-activated protein (MAP) kinase cascade is an important signaling module which is involved in biotic and abiotic stress responses as well as plant growth and development. In this study, we identified 17 tobacco MAPKs including 11 novel tobacco MAPK genes that have not been identified before. Comparative analysis with MAPK gene families from other plants, such as Athaliana thaliana, rice and poplar, suggested that tobacco MAPKs (such as NtMPK1, NtMPK3 and NtMPK8) might play similar functions in response to abiotic and biotic stresses. QRT-PCR analysis revealed that a total of 14 NtMPKs were regulated by SA and/or MeJA, suggesting their potential roles involved in plant defense response. In addition, 6 NtMPKs were induced by drought treatment, implying their roles in response to drought stress. Our results indicated that most of tobacco MAPK might be involved in plant defense response, which provides the basis for further analysis on physiological functions of tobacco MAPKs.

Resistance to BmNPV via overexpression of an exogenous gene controlled by an inducible promoter and enhancer in transgenic silkworm, Bombyx mori.

The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 10(5) occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.