Map-based cloning and functional analysis revealed ABCC2 is responsible for Cry1Ac toxin resistance in Bombyx mori.

Bt toxins are parasporal crystals produced by Bacillus thuringiensis (Bt). They have specific killing activity against various insects and have been widely used to control agricultural pests. However, their widespread use has developed the resistance of many target insects. To maintain the sustainable use of Bt products, the resistance mechanism of insects to Bt toxins must be fully clarified. In this study, Bt-resistant and Bt-susceptible silkworm strains were used to construct genetic populations, and the genetic pattern of silkworm resistance to Cry1Ac toxin was determined. Sequence-tagged site molecular marker technology was used to finely map the resistance gene and to draw a molecular genetic linkage map, and the two closest markers were T1590 and T1581, indicating the resistance gene located in the 155 kb genetic region. After analyzing the sequence of the predicted gene in the genetic region, an ATP binding cassette transporter (ABCC2) was identified as the candidate gene. Molecular modeling and protein-protein docking result showed that a tyrosine insertion in the mutant ABCC2 might be responsible for the interaction between Cry1Ac and ABCC2. Moreover, CRISPR/Cas9-mediated genome editing technology was used to knockout ABCC2 gene. The homozygous mutant ABCC2 silkworm was resistant to Cry1Ac toxin, which indicated ABCC2 is the key gene that controls silkworm resistance to Cry1Ac toxin. The results have laid the foundation for elucidating the molecular resistance mechanism of silkworms to Cry1Ac toxin and could provide a theoretical basis for the biological control of lepidopteran pests.

Transcriptome reveal the response to Cry1Ac toxin in susceptible Bombyx mori.

Bombyx mori as a representative in Lepidoptera is an important economic insect in agriculture production. Bacillus thuringiensis (Bt) is a bacterial pathogen in silkworm production. Understanding how silkworm respond to Bt-toxin can provide guidance to cultivate resistant silkworm strains. Cry1Ac is one type of Bt-toxin. In current research, Dazao, a susceptible B. mori strain to Bt-toxin, was treated by Cry1Ac toxin and compared its transcriptome with untreated samples. This analysis detected 1234 differentially expressed genes (DEGs). Gene Ontology, KEGG, and UniProt keyword enrichment analysis showed that DEGs include ATP-binding cassette (ABC) transporter, stress response, cuticle, and protein synthesis, and folding process. Five ABC genes were upregulated after Cry1Ac treatment including ABCA2, ABCA3, and ABCC4. They are also known as the transporters of Bt-toxin in lepidopteran insect. Expression of cuticle proteins was significantly increased at 6 h after Cry1Ac treatment. Sex-specific storage-proteins and heat shock protein were also upregulated in Cry1Ac treated samples. Our data provide an expression profile about the response of Cry1Ac toxin in susceptible B. mori strain.

Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins.

Storage proteins are haemolymph-specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune-responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real-time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co-immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP-6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two-hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.

Analysis of reassortant and intragenic recombination in Cypovirus.

Cypoviruses (CPVs) are RNA viruses with segmented double-stranded genome and major pathogens of various insects, including economic insects like silkworms and pest insects for agricultural crops and forests. Genome reassortment and recombination are common phenomenon for viruses as a mechanism to expand host range and increase virulence. In the present study, we analyzed the reassortant and recombination events for CPVs. The results showed that two genome segments (S1 and S4) of BmCPV1-YN shared higher nucleotide identity with the corresponding segment of BmCPV1-I while others were all more closely to BmCPV1-SZ, suggesting BmCPV1-YN was originated from reassortant events between BmCPV1-I and BmCPV1-SZ. Recombination analyses revealed that S6 of BmCPV1-YN was a recombinant segment derived from BmCPV1-I and BmCPV1-SZ, and S10 of DpCPV1 was a recombinant segment emerged from BmCPV1-I and LdCPV1. Our findings provide the evidence for the fact that CPVs could undergo reassortant and recombinant events and enrich the knowledge about etiology and molecular epidemiology of CPVs.

Label-free LC-MS/MS proteomic analysis of the hemolymph of silkworm larvae infected with Beauveria bassiana.

Beauveria bassiana, a pathogen of the economically important silkworm (Bombyx mori), causes serious losses in the sericulture industry; however, the mechanisms underlying B. bassiana infection and the silkworm response are not fully understood. To obtain new insights into the interaction between B. bassiana and its host, hemolymph samples from fifth instar silkworm larvae infected with B. bassiana were analyzed at 36-h post-inoculation using a label-free LC-MS/MS proteomic technique. In total, 671 proteins were identified in the hemolymph, including 87 differentially expressed proteins, 42 up-regulated and 45 down-regulated in infected larvae. Six were detected only in infected larvae, and five were detected only in uninfected larvae. Based on GO annotations, 48 of the differentially expressed proteins were involved in molecular functions, 42 were involved in biological processes, and 39 were involved in cell components. A KEGG pathway analysis indicated that these differentially expressed proteins participate in 85 signal transduction pathways, including the amoebiasis, MAPK signaling, Hippo signaling, Toll and Imd signaling, and lysosome pathways. The silkworm hemolymph is the main site for B. bassiana replication. We identified differentially expressed proteins involved in the regulation of the host response to B. bassiana infection, providing important experimental data for the identification of key factors contributing to the interaction between the pathogenic fungus and its host.

Silkworm storage protein Bm30K-19G1 has a certain antifungal effects on Beauveria bassiana.

Storage proteins in the 30 K family are ubiquitous in the hemolymph of insects and play important roles in adult metamorphosis, development, egg formation, carrier transport and even host immunity. Some studies have shown that the 30 K proteins can inhibit apoptosis and have certain antifungal effects. The silkworm protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in hemolymph of fifth instar larvae. Our previous transcriptome sequencing, real-time PCR analysis and proteomic studies showed that the expression level of the 30 K protein gene was significantly up-regulated in the silkworm infected with Beauveria bassiana. In this study, the ORF sequence of Bm30K-19G1 was amplified by PCR. The sequence is 1311 bp in length and encodes a 436 amino acid peptide. Bm30K-19G1 was expressed in all tested tissues of fifth instar larvae, with highest expression in fat body and the lowest expression in the midgut. Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+) as vector and E. coli Arctic Express (DE3) as the expression bacterium strain. The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B. bassiana. Bm30K-19G1 also inhibited the growth and reproduction of B. bassiana in vivo; the median lethal time of infected silkworms was postponed by 6.4 h and the time for death of all infected larvae was postponed by 10 h. The results indicated that the silkworm storage protein 30K-19G1 is an antifungal protein against B. bassiana and help to elucidate the molecular mechanism of silkworm resistance against B. bassiana.

Physiological and Transcriptomic Changes during the Early Phases of Adventitious Root Formation in Mulberry Stem Hardwood Cuttings.

The initiation and induction of root primordia are of great importance for adventitious root (AR) formation in cutting propagation of horticultural and forestry crops. However, the underlying mechanisms orchestrating these early phases of AR formation remain largely unexplored. Here, we investigated the physiological and transcriptomic changes during the early AR phases in mulberry stem hardwood cuttings. The results showed that the concentrations of soluble proteins increased, whereas concentrations of soluble sugars and starch were decreased. Indole-3-acetic acid (IAA) and zeatin had a rapid transit peak at 6 h after planting (hAP) and declined thereafter. The activities of peroxidase and catalase persistently increased and indole-3-acetic acid oxidase was maintained at a higher stable level from 0 hAP, while the activities of polyphenol oxidase fluctuated with soluble phenolics and IAA levels. The comparative transcriptome identified 4276 common genes that were differentially regulated at -6, 0 and 54 hAP. They were separated into five clusters with distinct biological functions such as defense response and photosynthesis. Considerable common genes were assigned to pathways of sugar metabolism, mitogen-activated protein kinase, and circadian rhythm. The gene co-expression network analysis revealed three major co-expressed modules involved in stress responses, hormone signaling, energy metabolism, starch metabolism, and circadian rhythm. These findings demonstrate the positive effect of auxin on AR induction, and uncovered the crucial roles of stress responses, hormone signaling and circadian rhythm in coordinating the physiological changes during the early phases of AR formation in mulberry stem hardwood cuttings.

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus.

We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV immediate early-1 (ie-1) and me53 genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy.IMPORTANCE Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species.

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.

[Molecular mapping of test mapping strain for 18th linkage group recessive genes elp, ch-2 and mln in silkworm (Bombyx mori)].

The ellipsoid egg, the second recessive gene of chocolate larvae, and melanism are controlled by three recessive genes, elp, ch-2, and mln in silkworm, respectively. Their order and genetic distance have been scheduled in established linkage group. Owing to lack of crossing over in females, the reciprocal backcrossed F1(BC1) progenies were bred for linkage analysis using the wild type silkworm strain P50(+elp+ch-2+mln /+elp+ch-2+mln) and W18 with ellipsoid egg, the second recessive gene of chocolate larvae, and melanism (elp ch-2 mln / elp ch-2 mln). In this research, we mapped three mutant genes, elp, ch-2, and mln on the chromosome 18 based on the SSR linkage map and STS markers designed based on silkworm genome sequence. The established linkage group, molecular linkage group, and the physic map of chromosome 18 had been corresponded. The genetic distance for this chromosome in this research was 94.2 cM, and the order of the mutants and molecular markers were consistent with the established silkworm linkage maps and the fine genome sequences. This research will lay important bases for map-based cloning for other mutants on chromosome 18.

CRISPR/Cas9-based knockout reveals that the clock gene timeless is indispensable for regulating circadian behavioral rhythms in Bombyx mori.

Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.

iTRAQ-based quantitative proteomic analysis of silkworm infected with Beauveria bassiana.

Beauveria bassiana is a harmful pathogen to the economically important insect silkworm, always causes serious disease to the silkworm, which results in great losses to the sericulture industry. In order to explore the silkworm (Bombyx mori) response to B. bassiana infection, differential proteomes of the silkworm responsive to B. bassiana infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) at the different stage of the 3rd instar silkworm larvae. Among the 5040 proteins identified with confidence level of ≥95 %, total 937 proteins were differentially expressed, of which 488 proteins were up-regulated and 449 proteins were down-regulated. 23, 15, 250, 649 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the B. bassiana infected larvae at 18, 24, 36, 48 h post infection (hpi) respectively. Based on GO annotations, 6, 4, 128, 316 DEPs were involved in biological processes, 12, 5, 143, 376 DEPs were involved in molecular functions, and 6, 3, 108, 256 DEPs were involved in cell components at 18, 24, 36, 48 hpi respectively. KEGG pathway analysis displayed that 18, 12, 210, 548 DEPs separately participated in 63, 35, 201, 264 signal transduction pathways at different time of infection, and moreover a higher proportion of DEPs involved in metabolic pathways. The cluster analysis on the DEPs of different infection stages distinguished a co-regulated DEP, lysozyme precursor, which was up-regulated at both the mRNA level and the protein level, indicating that the lysozyme protein kept playing an important role in defending the silkworm against B. bassiana infection. This was the first report using an iTRAQ approach to analyze proteomes of the whole silkworm against B. bassiana infection, which contributes to better understanding the defense mechanisms of silkworm to B. bassiana infection and provides important experimental data for the identification of key factors involved in the interaction between the pathogenic fungus and its host.

Inhibitory effects of Bombyx mori antimicrobial peptide cecropins on esophageal cancer cells.

Bombyx mori antimicrobial peptides (BmAMPs) are important effectors in silkworm immune system. They can inhibit and kill a variety of bacteria and fungi. Recent studies have shown that some kinds of BmAMPs exert strong inhibitory effects on a variety of tumor cells. In the present study, the antitumor activity of BmAMP Cecropin A (BmCecA) and BmAMP Cecropin D (BmCecD) was investigated against human esophageal cancer cells and their antitumor mechanism preliminary explored. Cell Counting Kit-8 and colony formation assays indicated that BmCecA and BmCecD suppressed cell proliferation and reduced colony formation of both Eca109 and TE13 cells in a dose-dependent manner, but exhibited no inhibitory effect on normal human embryonic kidney 293T cells. Wound healing and invasion experiments indicated that both BmCecA and BmCecD inhibited migration and invasion of Eca109 and TE13 cells in vitro. Annexin V/propidium iodide staining and flow cytometry detection suggested that BmCecA induced the apoptosis of Eca109 cells in a dose-dependent manner. RT-qPCR and western blot analysis showed that BmCecA induced apoptosis of Eca109 cells through the activation of a mitochondria-mediated caspase pathway, the upregulation of B-cell lymphoma 2 (Bcl-2)-associated X protein and the downregulation of Bcl-2. In addition, BmCecA significantly inhibited the growth of xenograft tumors in Eca109-bearing mice. These results suggested that BmCecA and BmCecD might serve as potential therapeutic agents for the treatment of cancer in the future.

BmSd gene regulates the silkworm wing size by affecting the Hippo pathway.

Insect wings are developed from the wing disc during metamorphosis. Bombyx mori, a model lepidopteran insect, loses flight ability after long-term domestication from the wild silkworm, Bombyx mandarina. The mw mutant (u11 strain) shows minute wings compared to wild type (e.g., p50 strain) wings. RNA sequencing analysis previously revealed differential Hippo-pathway-related gene expression between the u11 and p50 strains. The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals. In this study, the function of BmSd which has been characterized as one of the Hippo-pathway-related genes was analyzed for silkworm wing development. We found that mats, warts, and hippo expression levels were higher in u11 compared to p50 wing discs. BmSd (scalloped) expression, which encodes a prominent transcriptional partner to Yorkie (Yki), gradually decreased during the wandering stage in u11, but exhibited the opposite expression pattern in p50. When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain, 57.9% of the individuals showed minute wings. Additionally, ex, kibra, and wingless expression levels decreased in the BmSd knockdown mutant. Further, BmSd deletion mediated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 induced 50% of individuals with minute wings, a phenotype similar to the mw mutant. This result demonstrates that BmSd plays pivotal roles in silkworm wing development. Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development, and our findings provide a basis for further research on B. mori wing development.

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori.

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified. However, molecular mechanisms of most of these mutants remain to be explored. Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing (mw) mutation which exhibits exceedingly small wings during pupal and adult stages. Compared with the wild type silkworm, relative messenger RNA expression of Bm-app is significantly decreased in the u11 mutant strain which shows mw phenotype. A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity. Furthermore, clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant, demonstrating that Bm-app controls wing development in B. mori. Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins, indicating that it is required for cell mitosis and growth during wing development. We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B. mori.

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.

Effects of insect viruses and pesticides on glutathione S-transferase activity and gene expression in Bombyx mori.

Glutathione S-transferase (GST) is a gene family generally associated with detoxification and plays an important role in detoxifying exogenous compounds. The silkworm Bombyx mori is an important economic animal for silk production. However, it is liable to infection by a number of viruses and chemical agents that can contaminate its food and growing environment. Here we conducted a comparative study of strain- and tissue-specific expressions of GST in the silkworm under infections by Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori densonucleosis virus (BmDNV) and under the stress of pesticides (insecticide and herbicide). BmDNV induced an increase of GST at 24 h and a decrease at 48 and 72 h in the midgut of the DNV-susceptible strain and a decrease at 24 h and increase at 48 and 72 h in the midgut of the DNV-tolerant strain. BmDNV induced a significant increase of GST from 24 to 72 h in the fat body of both DNV-susceptible and DNV-tolerant strains. In contrast, BmNPV induced a significant decrease of GST in both the fat body and midgut of the NPV-susceptible strain and induced increase of GST from 24 to 48 h in the midgut and at 72 h in the fat body of the NPV-tolerant strain. All of these results suggest that BmGST activity varies with the strain, tissue, feeding behavior, and developmental stage of the silkworm. On treating silkworm larvae with pesticides (insecticide and herbicide), expression of the BmGSTS2 gene increased noticeably in the midgut and reached a peak at 6 to 12 h. The mRNA level of BmGSTS2 in the midgut increased during administration of the chemicals, suggesting that the induction of BmGSTS2 is part of the defense mechanism against exogenous chemicals.

MicroRNA Expression Analysis of Naked Silkworms.

The silk gland (SG) is characterized by the synthesis and secretion of silk protein in the economically important silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Nd and Nd-s are two fibroin-secretion-deficient silkworm mutants. MicroRNA (miRNA) plays an important role in many biological processes, such as cell proliferation, differentiation, and apoptosis. Using the Dazao silkworm as a control, we explored the miRNA expression profiles in the SGs of u02 (Nd) and u05 (Nd-s) to reveal the potential functions of miRNAs in silk protein expression and SG development. Here, we sequenced small RNA libraries made from the whole SGs of three strains. There are 260, 236, and 233 known miRNAs and 20, 18, and 18 potential new miRNAs identified from Dazao, u02, and u05, respectively. Fifty-three miRNAs are differentially expressed between Dazao and u02, 51 between Dazao and u05, and 16 between u02 and u05. Gene ontology/KEGG analyses show that most of the predicted target genes of differentially expressed miRNAs were assigned to functional categories involved in cell proliferation, organ development, and cellular compartment structures. The miRNA expression profile of naked silkworms will pave the way for the understanding of SG development and the regulation of silk protein expression.

MicroRNA profile of silk gland reveals different silk yields of three silkworm strains.

Silk proteins are synthesized and secreted by the silk gland. The differential gene expression in it leads to different silk yield among various silkworm strains. As crucial factors, microRNAs (miRNAs) regulate protein synthesis at post-transcriptional level in silk gland. MiRNAs expression level in the silk gland of three silkworm strains (Jingsong, Lan10 and Dazao) was analyzed and 33 differentially expressed miRNAs (DEMs) were discovered between JingSong (JS) and Lan10 (L10), 60 DEMs between JS and Dazao, 54 DEMs between L10 and Dazao respectively. The DEMs target genes were predicted combing with two different methods and their functions were annotated according to gene ontology. Our previous studies showed that a batch of genes related to silk yield were identified in JS and L10 strains by comparative transcriptome and quantitative trait loci (QTL) method. Thirteen DEMs whose target genes are related to protein biosynthesis processes were screened by combining with these researches. Twelve DEMs potentially regulate nineteen genes which exist in our QTL results. Six common DEMs potentially regulate the genes in both of previous results. Finally, five DEMs were selected to verify their expression levels between JS and L10 by qRT-PCR, which showed similar difference as the results of small RNA-sequencing. MiRNAs in the silk gland may directly affect silk protein biosynthesis in different silkworm strains. In current work, we identified a batch of DEMs which potentially regulate the genes related to silk yield. Further functionally study of these miRNAs will contribute to improve varieties and boost the silk yield. Our research provides a basis for studying these miRNAs and their functions in silk production.

Analyzing genetic relationships in Bombyx mori using intersimple sequence repeat amplification.

Intersimple sequence repeat (ISSR) amplification was used to analyze genetic relationships among silkworm, Bombyx mori L., strains. Nineteen primers containing simple sequence repeat (SSR) motifs were tested for amplification on a panel of 42 strains, representative of the diversity of silkworm germplasm; 12 of the primers amplified distinct, reproducible bands. The primers amplified a total of 108 bands, of which 85 (78.7%) were polymorphic. The ISSR results suggested that within the dinucleotide class, the poly(CA) motif was more common than the poly(CT) motif. The ISSR amplification pattern was used to group the silkworm strains into seven subclusters based on their origin in an unweighted pair-group method with arithmetic average cluster analysis by using Nei's genetic distance. Seven major ecotypic silkworm groups were analyzed. Principal component analysis of the ISSR data supported the unweighted pair-group method with arithmetic average clustering. Therefore, ISSR amplification is a valuable method for determining genetic variability among silkworm varieties. This efficient genetic fingerprinting technique should be useful for characterizing the large numbers of silkworm strains held in national and international germplasm centers.