The architecture of silk-secreting organs during the final larval stage of silkworms revealed by single-nucleus and spatial transcriptomics.

Natural silks are renewable proteins with impressive mechanical properties and biocompatibility that are useful in various fields. However, the cellular and spatial organization of silk-secreting organs remains unclear. Here, we combined single-nucleus and spatially resolved transcriptomics to systematically map the cellular and spatial composition of the silk glands (SGs) of mulberry silkworms late in larval development. This approach allowed us to profile SG cell types and cell state dynamics and identify regulatory networks and cell-cell communication related to efficient silk protein synthesis; key markers were validated via transgenic approaches. Notably, we demonstrated the indispensable role of the ecdysone receptor (ultraspiracle) in regulating endoreplication in SG cells. Our atlas presents the results of spatiotemporal analysis of silk-secreting organ architecture late in larval development; this atlas provides a valuable reference for elucidating the mechanism of efficient silk protein synthesis and developing sustainable products made from natural silk.

An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms.

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

Chloroplast Genome Sequences and Phylogenetic Analysis of Eight Newly Sequenced Caryophyllaceae Species.

BACKGROUND: Caryophyllaceae is a big family composed of many economic and medicinal species. However, the phylogeny of the family is insufficient and genome data are lacking for many species. OBJECTIVE: Using next-generation sequencing (NGS) to acquire the chloroplast (cp) genomes of Eremogone acicularis (F.N.Williams) Ikonn., E. brevipetala (Tsui & L.H.Zhou) Sadeghian & Zarre, E. bryophylla (Fernald) Pusalkar & D.K.Singh, E. kansuensis (Maxim.) Dillenb. & Kadereit, Shivparvatia glanduligera (Edgew.) Pusalkar & D.K.Singh, Silene atsaensis (Marq.) Bocquet, S. caespitella Williams, and S. lhassana (Williams) Majumdar. METHODS: Bioinformatic software was used to conduct the comparative genome and phylogeny analysis of these cp genomes. RESULTS: The eight cp genomes were 132 188-151 919 bp in length, containing 130-132 genes. A/T was dominant in simple sequence repeats (SSRs). Forward repeats and palindromic repeats were the most frequent in long terminal repeats (LTRs). Compared with the four species of Eremogone Fenzl, the inverted repeat (IR) boundaries of S. caespitella, S. atsaensis, S. lhassana, and Sh. glanduligera were significantly expanded. Four and one mutational hotspots were identified in the large single copy (LSC) region and small single copy (SSC) region, respectively. The ratio of nonsynonymous substitution to synonymous substitution (Ka/Ks ratio) showed these cp genomes may have undergone strong purifying selection. In the phylogenetic trees, both Silene L. and Eremogone were monophyletic groups. However, Sh. glanduligera was closely related to Amaranthus hypochondriacus. CONCLUSION: These results have provided new evidence and useful information for species identification, evolution, and genetic research on the Caryophyllaceae. HIGHLIGHTS: In this study, eight newly sequenced cp genomes of Caryophyllaceae species were reported for the first time.

Panax quinquefolium L. and Salvia miltiorrhiza Bunge. Enhances Angiogenesis by Regulating the miR-155-5p/HIF-1α/VEGF Axis in Acute Myocardial Infarction.

Background: Combination of Panax quinquefolium L and Salvia miltiorrhiza Bunge. (PS) has been widely used in the clinical treatment of ischemic heart disease. The purpose of this study was to explore the therapeutic effect and mechanism of PS on angiogenesis in rats after acute myocardial infarction (AMI). Methods: A rat model of AMI was established by ligating the left anterior descending (LAD) artery. The grouping and administration scheme were as follows: sham group, model group, PS low-dose (PS-L) group, PS high-dose (PS-H) group, PX-478 group and angiotensin converting enzyme inhibitor (ACEI) group. After 28 days of treatment, echocardiography, myocardial infarct size, some angiogenesis markers and the miR-155-5p/HIF-1α/VEGF axis were measured. Results: PS improved cardiac structure and function, reduced infarct size, and alleviated myocardial fibrosis and inflammatory cell infiltration in AMI rats. Mechanistically, PS enhanced the expression of HGF and bFGF in serum, increased the levels of MVD and CD31 in myocardial tissues, and inhibited the activation of the miR-155-5p/HIF-1α/VEGF pathway, which ultimately promoted angiogenesis. In addition, the regulatory effect of PS on angiogenesis was partly abolished by PX-478. Conclusion: PS increased the expression of MVD and CD31 in the myocardium and stimulated angiogenesis. The above effects of PS may be associated with the inhibition of the miR-155-5p/HIF-1α/VEGF axis.

Effects of cadmium on the synthesis of active ingredients in Salvia miltiorrhiza.

Cadmium (Cd) could pose threats to human health by affecting Salvia miltiorrhiza (SM) safety. Cd enrichment trait and its effects on the active ingredient synthesis in SM remain unknown. Here we investigated the Cd concentration using ICP-MS-based method, physiologies (contents of malondialdehyde and proline, and activities of superoxide dismutase, peroxidase [POD], and catalase [CAT]), and LC-MS/MS-based metabolites of SM under 25, 50, and 100 mg kg-1 Cd stress. The results revealed that Cd concentrations, as it rose in soil, increased in roots and leaves of SM with transfer factors and bioconcentration factors below 1 in Cd-treated groups; POD and CAT activities and proline content increased and then declined. Amino acids and organic acids (especially d-glutamine [d-Gln], l-aspartic acid [l-Asp], l-phenylalanine [l-Phe], l-tyrosine [l-Tyr], geranylgeranyl-PP [GGPP], and rosmarinic acid [RA]) contributed more in discriminating SM roots of different groups. GGPP was negatively related to l-Tyr and l-Phe, and RA was positively related to d-Gln and l-Asp in SM. These results revealed that SM belonged to a non-Cd-hyperaccumulator with most Cd accumulated in roots, Cd could enhance phenolic acid synthesis via regulating amino acid metabolism and might inhibit tanshinone synthesis by declining the GGPP content, and proline, POD, and CAT played vital roles in resisting Cd stress. These provided new ideas and theoretical basis for further study on medical plants' response to heavy metals.

Role of Baduanjin exercise-based cardiac rehabilitation in coronary heart disease after percutaneous coronary intervention: A protocol for systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: This study aimed to explore the effects of Baduanjin-based cardiac rehabilitation on cardiac function and quality of life in patients with coronary heart disease who have undergone percutaneous coronary intervention. METHODS: PubMed, the Excerpta Medica Database, the Cochrane Library, Web of Science, the Wanfang, SINOMED, the China Science and Technology Journal Database and China National Knowledge Infrastructure were searched for appropriate articles from their respective inception until March 30, 2021. Meta-analysis was conducted with the RevMan 5.3 software. RESULTS: A total of 11 studies including 1025 patients were considered. Compared with conventional Western medicine, Baduanjin improved the left ventricular ejection fraction of patients [mean difference (MD) = 2.83, 95% confidence interval (CI) (2.05, 3.61), P < .00001], increased the Seattle angina questionnaire and SF-36 health survey scale scores [MD = 6.67, 95% CI (4.09, 9.26), P < .00001; standard mean difference  = 0.73, 95% CI (0.55, 0.91), P < .00001, respectively] and decreased the scores of Zung self-rating anxiety scale and self-rating depression scale [MD = -6.64, 95% CI (-7.69, -5.22), P < .00001; MD = -6.63, 95% CI (-7.60, -5.66), P < .00001, respectively]. CONCLUSION: Our findings showed that Baduanjin exercise improved cardiac function and quality of life and alleviated patients' anxiety and depression.

Untargeted LC-MS/MS-Based Metabolomic Profiling for the Edible and Medicinal Plant Salvia miltiorrhiza Under Different Levels of Cadmium Stress.

Salvia miltiorrhiza, a medicinal and edible plant, has been extensively applied to treat cardiovascular diseases and chronic hepatitis. Cadmium (Cd) affects the quality of S. miltiorrhiza, posing serious threats to human health. To reveal the metabolic mechanisms of S. miltiorrhiza's resistance to Cd stress, metabolite changes in S. miltiorrhiza roots treated with 0 (CK), 25 (T1), 50 (T2) and 100 (T3) mg kg-1 Cd by liquid chromatography coupled to mass spectrometry (LC-MS/MS) were investigated. A total of 305 metabolites were identified, and most of them were amino acids, organic acids and fatty acids, which contributed to the discrimination of CK from the Cd-treated groups. Among them, S. miltiorrhiza mainly upregulated o-tyrosine, chorismate and eudesmic acid in resistance to 25 mg kg-1 Cd; DL-tryptophan, L-aspartic acid, L-proline and chorismite in resistance to 50 mg kg-1 Cd; and L-proline, L-serine, L-histidine, eudesmic acid, and rosmarinic acid in resistance to 100 mg kg-1 Cd. It mainly downregulated unsaturated fatty acids (e.g., oleic acid, linoleic acid) in resistance to 25, 50, and 100 mg kg-1 Cd and upregulated saturated fatty acids (especially stearic acid) in resistance to 100 mg kg-1 Cd. Biosynthesis of unsaturated fatty acids, isoquinoline alkaloid, betalain, aminoacyl-tRNA, and tyrosine metabolism were the significantly enriched metabolic pathways and the most important pathways involved in the Cd resistance of S. miltiorrhiza. These data elucidated the crucial metabolic mechanisms involved in S. miltiorrhiza Cd resistance and the crucial metabolites that could be used to improve resistance to Cd stress in medicinal plant breeding.

A single-cell transcriptomic atlas characterizes the silk-producing organ in the silkworm.

The silk gland of the domesticated silkworm Bombyx mori, is a remarkable organ that produces vast amounts of silk with exceptional properties. Little is known about which silk gland cells execute silk protein synthesis and its precise spatiotemporal control. Here, we use single-cell RNA sequencing to build a comprehensive cell atlas of the silkworm silk gland, consisting of 14,972 high-quality cells representing 10 distinct cell types, in three early developmental stages. We annotate all 10 cell types and determine their distributions in each region of the silk gland. Additionally, we decode the developmental trajectory and gene expression status of silk gland cells. Finally, we discover marker genes involved in the regulation of silk gland development and silk protein synthesis. Altogether, this work reveals the heterogeneity of silkworm silk gland cells and their gene expression dynamics, affording a deeper understanding of silk-producing organs at the single-cell level.

Cardiac rehabilitation of Baduanjin exercise in coronary heart disease after PCI: A protocol for systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Percutaneous coronary intervention (PCI) is an effective revascularization strategy in patients with coronary heart disease (CHD). However, recent studies had indicated that postPCI patients usually suffer from a low-quality life. Cardiac rehabilitation (CR) has been recommended by numerous guidelines in the clinic for these patients. And Baduanjin exercise can significantly benefit patients with CHD. Regrettably, the effect of Baduanjin exercise on postPCI patients is still not clear. Therefore, this systematic review and meta-analysis protocol is planned to explore the effect of Baduanjin exercise in patients with CHD who have undergone PCI. METHODS: PubMed, Excerpta Medica Database, Cochrane Library, Web of Science, Wanfang Database, SINOMED, China Science and Technology Journal Database, and China National Knowledge Infrastructure will be searched for appropriate articles from respective inceptions until December 1th, 2020. Two reviewers will independently conduct article selection, data collection, and risk of bias evaluation. Disagreements will be resolved first by discussion and then by consulting a third author for arbitration. The primary outcome will include left ventricular ejection fraction. And the change in the scores on the Seattle Angina Questionnaire, SF-36 health survey scale, Zung Self-rating Anxiety scale and self-rating depression scale will be used as the secondary outcomes. RevMan 5.3 will be used for meta-analysis. RESULTS: This systematic review and meta-analysis will explore whether Baduanjin exercise is an effective intervention in postPCI patients. CONCLUSION: This systematic review and meta-analysis will provide convincing evidence of Baduanjin exercise that specifically focuses on CR of Baduanjin exercise on CHD after PCI. REGISTRATION NUMBER: INPLASY202130065.

A transcriptome atlas of silkworm silk glands revealed by PacBio single-molecule long-read sequencing.

The silk gland of the silkworm Bombyx mori is a specialized organ where silk proteins are efficiently synthesized under precise regulation that largely determines the properties of silk fibers. To understand the genes involved in the regulation of silk protein synthesis, considerable research has focused on the transcripts expressed in silk glands; however, the complete transcriptome profile of this organ has yet to be elucidated. Here, we report a full-length silk gland transcriptome obtained by PacBio single-molecule long-read sequencing technology. In total, 11,697 non-redundant transcripts were identified in mixed samples of silk glands dissected from larvae at five developmental stages. When compared with the published reference, the full-length transcripts optimized the structures of 3002 known genes, and a total of 9061 novel transcripts with an average length of 2171 bp were detected. Among these, 1403 (15.5%) novel transcripts were computationally revealed to be lncRNAs, 8135 (89.8%) novel transcripts were annotated to different protein and nucleotide databases, and 5655 (62.4%) novel transcripts were predicted to have complete ORFs. Furthermore, we found 1867 alternative splicing events, 2529 alternative polyadenylation events, 784 fusion events and 6596 SSRs. This study provides a comprehensive set of reference transcripts and greatly revises and expands the available silkworm transcript data. In addition, these data will be very useful for studying the regulatory mechanisms of silk protein synthesis.

The intronic promoter of Actin4 mediates high-level transgene expression mainly in the wing and epidermis of silkworms.

The cytoplasmic actin gene Actin4 (A4) in silkworm (Bombyx mori) was isolated 20 years ago and has a distal promoter upstream of the first exon and a proximal promoter within the first intron; however, how the promoter regulates gene expression has yet to be fully elucidated. Here, we characterized the function and expression of the proximal promoter (named A4IP) by analyzing transgenic Gal4/UAS silkworms, A4IP-Gal4/UAS-EGFP. We demonstrated that A4IP drives the expression of Gal4 and thereby activates UAS-linked EGFP in transgenic silkworms beginning in day-3 embryos through adults. Further detection revealed that EGFP was expressed at a low level in tissues including the trachea, fat body and midgut but was highly expressed in the wing disks/wings and inner epidermis of transgenic silkworms. No EGFP signals were detected in other tissues by western blot assay. Interestingly, EGFP fluorescence had a spot-like distribution on the epidermis of transgenic larvae. These observations are quite different from those in transgenic silkworms driven by the promoter of Actin3 (A3), another cytoplasmic actin gene in B. mori. These findings reveal the expression profiles of the A4IP promoter and provide new insights into the regulatory mechanism of cytoplasmic actin genes in silkworms.

Insights into regulatory characteristics of the promoters of Sericin 1 and Sericin 3 in transgenic silkworms.

Sericin, produced in the middle silk gland (MSG) of silkworms, is a group of glue proteins that coat and cement silk fibers. Several genes are known to encode sericin, but their spatiotemporal regulation has yet to be fully elucidated. Here, we report in detail the expression profiles of the promoters of two major sericin-coding genes, Sericin 1 (Ser1)and Sericin 3 (Ser3), by analyzing Gal4/UAS transgenic silkworms. We found that UAS-linked EGFP fluorescence in transgenic silkworms driven by Ser1-Gal4was detected in only the R3, R4 and R5 regions of MSG starting inday-3 fifth-instar larvae and was continuously expressed until silk gland degradation. In transgenic silkworms driven by Ser3-Gal4, EGFP fluorescence was detected at a low level in the R2 region of MSG since the last day of fifth-instar larvae, and the expression increased during the wandering stages and was continuously detected until silk gland degradation. The molecular detection of EGFP expression in each of the Gal4/UAS transgenic silkworms was consistent with fluorescence observations. These findings reveal clear differences in the regulatory characteristics of the promoters of Ser1and Ser3 and provide new insights into the regulatory mechanism of the expression of sericin-coding genes.

Expression and characterization of recombinant human VEGF165 in the middle silk gland of transgenic silkworms.

Recombinant human vascular endothelial growth factor (rhVEGF) has important applications in therapeutic angiogenesis and inhibition of VEGF-mediated pathological angiogenesis. Previous studies have shown that rhVEGF can be produced in several expression systems, including Escherichia coli, yeasts, insect cells and mammalian cells. However, little is known regarding the effective production of this protein in organs of live organisms. Here, we report for the first time the expression and characterization of rhVEGF165 in the middle silk gland (MSG) of the transgenic silkworm line S1-V165. Our results confirmed that (1) rhVEGF165 was highly expressed in MSG cells and was secreted into the cocoon of S1-V165; (2) the dimeric form of rhVEGF165 could be easily dissolved from S1-V165 cocoons using an alkaline solution; (3) rhVEGF165 extracted from S1-V165 cocoons exhibited slightly better cell proliferative activity than the hVEGF165 standard in cultured human umbilical vein endothelial cells. This study provides an alternative strategy for the production of bioactive rhVEGF165 using the MSG of transgenic silkworms.

Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system.

The silkworm Bombyx mori is a valuable insect that synthesizes bulk amounts of fibroin protein in its posterior silk gland (PSG) and weaves these proteins into silk cocoons. The mechanism by which the fibroin protein is efficiently synthesized and precisely regulated is an important aspect that has yet to be fully elucidated. Here, we describe the regulatory characteristics of the promoters of fibroin protein-encoding genes, namely, fibroin heavy chain (fibH) and fibroin light chain (fibL), using an optimized Gal4/UAS binary system. We found that (1) UAS-linked enhanced green fluorescent protein (EGFP) was effectively activated in the PSGs of Gal4/UAS transgenic silkworms, and fluorescence was continuously detected in the PSGs after complete formation of silk glands. (2) In the PSGs of fourth- and fifth-instar larvae of transgenic silkworms driven by fibL-Gal4 (LG4) or fibH-Gal4 (HG4), EGFP mRNA was detected in only day-3 to day-6 fifth-instar larvae, while the EGFP protein could be detected at each day of both larval stages. (3) High-level expression of Gal4 and UAS-linked EGFP caused a delay in PSG degradation in Gal4/UAS transgenic silkworms. (4) At the early pupal stage, EGFP fluorescence was also detected in fat bodies of Gal4/UAS transgenic silkworms, indicating that the PSG-specific EGFP was transported into fat bodies during PSG degeneration; however, the underlying mechanism needs to be further elucidated. This study provides a modified Gal4/UAS system used for efficient tissue-specific expression of target genes in the PSGs of silkworms and provides new insights into the regulatory characteristics of the promoters of key fibroin protein-encoding genes.

Association of ERAP1 gene polymorphisms with the susceptibility to psoriasis vulgaris: A case-control study.

Psoriasis vulgaris (PsV), also known as plaque psoriasis, is a life-threatening autoimmune skin disease. Inflammatory factors may contribute to the development of PsV. Present study aimed to explore the association of endoplasmic reticulum aminopeptidase 1 (ERAP1) gene polymorphisms (rs26653 and rs27524) with PsV susceptibility in a Chinese Han population. Subgroup analysis was also performed based on the onset of PsV.Present case-control study included 143 patients with PsV and 149 healthy controls. Direct sequencing method was used for genotyping ERAP1 polymorphisms. Chi-squared test was used to estimate the association between ERAP1 polymorphisms and PsV susceptibility. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess association strength.The polymorphism rs26653 was positively correlated with PsV susceptibility (CC vs GG, P = .047, OR = 1.964, 95% CI = 1.006-3.834; C vs G, P = .042, OR = 1.403, 95% CI = 1.011-1.946). Meanwhile, its CC genotype and C allele were positively associated with the early onset of PsV (P = .036, OR = 2.080, 95% CI = 1.044-4.145; P = .034, OR = 1.443, 95% CI = 1.028-2.024) and increased PsV risk in the subgroup with family history (P = .029, OR = 2.149, 95% CI = 1.075-4.296; P = .027, OR = 1.466, 95% CI = 1.044-2.059).ERAP1 gene rs26653 polymorphism may increase the risk of PsV in Chinese Han population.

Genetic association between CD86 polymorphisms and the risk of sepsis in a Chinese Han population.

BACKGROUND: Sepsis is a clinical syndrome that is frequently observed after injury or infection, representing a leading cause of mortality worldwide. CD86 (B7-2) is a co-stimulatory molecule on antigen-presenting cells, and plays critical roles in immune responses. METHODS: A total of 135 sepsis patients and 151 healthy controls were recruited in the current case-control study. Hardy-Weinberg equilibrium (HWE) conformity was examined to assess the representativeness of the study population. CD86 gene polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The relative expression of CD86 mRNA was estimated via quantitative real-time PCR (qRT-PCR). Chi-square test was performed to estimate the associations between CD86 gene polymorphisms and sepsis risk, and the results were presented through odds ratio (OR) and 95% confidence intervals (CI). RESULTS: The genotype distributions of CD86 polymorphisms in the case and control groups conformed to HWE. The GA genotype of the polymorphism rs1129055 was significantly correlated with an increased risk of sepsis (OR = 2.540, 95%CI = 1.288-5.008). The TT genotype of rs1915087 was a risk factor for sepsis (OR = 2.769, 95%CI = 1.292-5.935). High linkage disequilibrium was observed between the two polymorphisms (D' = 1.0, r2 = 0.955). However, no significant association was observed between CD86 polymorphisms and its gene expressions (P > 0.05 for all). CONCLUSION: CD86 gene polymorphisms rs1129055 and rs1915087 may increase the risk of sepsis.

BmYki is transcribed into four functional splicing isoforms in the silk glands of the silkworm Bombyx mori.

Yorkie (Yki), the Drosophila homolog of vertebrate yes-associated protein (YAP), is a key effector of the Hippo pathway, which modulates organ size via the transcriptional regulation of downstream targets involved in cell proliferation and survival. YAP has been shown to be expressed as multiple splicing isoforms in mammals, but thus far, no evidence of alternatively spliced Yki isoforms has been reported in insects. Here, we confirmed that the Yki protein of the silkworm Bombyx mori, BmYki, is transcribed in the silk gland into at least four splicing isoforms, named BmY1329, BmY1314, BmY1188, and BmY1173. Further analysis revealed that BmY1329 and BmY1314 each contain two WW domains, whereas BmY1188 and BmY1173 each contain only one WW domain. Each BmYki isoform functions in regulating expression of Yki target genes in cultured B. mori embryonic cells, and exhibits a few different effects on the expression of Yki targets. Interestingly, the expression of silk fibroin protein genes could also be influenced by each of the BmYki isoforms, suggesting that BmYki is involved in the regulation of silk protein-coding genes. This study provides novel insights into the role of BmYki. The contribution of each BmYki isoform to the modulation of gene expression will be of great interest for further study.

Effects of transgenic overexpression of diapause hormone and diapause hormone receptor genes on non-diapause silkworm.

Diapause is a state of developmental arrest that is most often observed in arthropods, especially insects. The domesticated silkworm, Bombyx mori, is a typical insect that enters diapause at an early embryonic stage. Previous studies have revealed that the diapause hormone (DH) signaling molecules, especially the core members DH and DH receptor 1 (DHR1), are crucial for the determination of embryonic diapause in diapause silkworm strains. However, whether they function in non-diapause silkworm strains remains largely unknown. Here, we generated two transgenic lines overexpressing DH or DHR1 genes in a non-diapause silkworm strain, Nistari. Our results showed that developmental expression patterns of DH and DHR1 are quite similar in transgenic silkworms: both genes are highly expressed in the mid to late stages of pupae and are most highly expressed in day-6 pupae but are expressed at very low levels in other developmental stages. Moreover, the overexpression of DH or DHR1 can affect the expression of diapause-related genes but is not sufficient to induce embryonic diapause in their offspring. This study provides new insights into the function of DH and DHR1 in a non-diapause silkworm strain.

Cloning and expression analysis of BmYki gene in silkworm, Bombyx mori.

The transcriptional coactivator Yorkie(Yki), is a critical downstream effector of the Hippo(Hpo) signaling pathway that controls organ size through the regulation of cell proliferation and apoptosis. During the past ten years the biological function of Yki has been studied extensively in Drosophila and a few other insects, however, little is known about it in the silkworm, Bombyx mori, a major research model of lepidopteran insect. Here, we describe the isolation, characterization and expression of the B. mori Yki ortholog, BmYki. The coding sequence of the BmYki was 1314 bp in length, encoding a protein of 437 amino acids containing two conserved WW domains. BmYki transcripts were ubiquitous but not abundant in all detected tissues and developmental stages. Comparatively, it was expressed at pretty high level in silk glands and at the stage of fifth-instar day-3 larvae. Overexpression of BmYki in cultured B. mori embryonic cells significantly promoted transcription of genes associated with cell proliferation and apoptosis, indicating that BmYki functions in the regulation of organ growth-related biological processes. Interestingly, transcription of silk protein-coding genes and transcription factors regulating the synthesis of silk proteins was downregulated remarkably, suggesting that BmYki was involved in the regulation of silk protein synthesis. This study provides new insights into the role of BmYki in Hpo pathway regulation in silkworm.