Silk Protein Synthesis Research Articles

Deep Insight into the Transcriptome of the Single Silk Gland of Bombyx mori.

The silk gland synthesizes and secretes a large amount of protein and stores liquid silk protein at an extremely high concentration. Interestingly, silk proteins and serine protease inhibitors are orderly arranged in the silk gland lumen and cocoon shells. Silk fiber formation and the spinning mechanism have not been fully elucidated. Therefore, we conducted a comparative transcriptome analysis of seven segments of the single silk gland to characterize internal changes in the silk gland during the 5th instar of mature larvae. In total, 3121 differentially expressed genes were identified in the seven segments. Genes highly expressed in the middle silk gland (MSG) were mainly involved in unsaturated fatty acid biosynthesis, fatty acid metabolism, apoptosis—fly, and lysosome pathways, whereas genes highly expressed in the posterior silk gland (PSG) were mainly involved in ribosome, proteasome, citrate cycle, and glycolysis/gluconeogenesis pathways. Thus, the MSG and PSG differ greatly in energy source use and function. Further, 773 gradually upregulated genes (from PSG to MSG) were involved in energy metabolism, silk protein synthesis, and secretion, suggesting that these genes play an important role in silk fiber formation. Our findings provide insights into the mechanism of silk protein synthesis and transport and silk fiber formation.

Cross-talk between juvenile hormone and ecdysone regulates transcription of fibroin modulator binding protein-1 in Bombyx mori.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are the most important hormones in silkworm and play vital roles in silkworm development, metamorphosis, and silk protein synthesis. Fibroin modulator binding protein-1 (FMBP-1) is a novel transcription factor regulating fibroin heavy chain (fib-H) transcription in Bombyx mori. The roles of JH and 20E on FMBP-1 transcription are less known. Here, we show FMBP-1 transcription is repressed by juvenile hormone analog (JHA) and activated by 20E. We identify two Krüppel homolog 1 (Kr-h1) binding sites (KBS1 and KBS2) and an E74A binding site (EBS) in the promoter of FMBP-1. We demonstrate Kr-h1 directly binds to KBS1 and KBS2 to repress FMBP-1 transcription, and 20E promotes FMBP-1 transcription through E74A. In the presence of JH and 20E, E74A abolishes the repression of Kr-h1 and activates FMBP-1 transcription through direct binding to EBS between KBS1 and KBS2 in FMBP-1 promoter. Further, JHA and 20E treatment and RNA interference confirm the effects of JH and 20E on FMBP-1 transcription in vivo, thus affecting fib-H transcription. Our results reveal the molecular mechanism of FMBP-1 transcription regulated by the cross-talk between JH and 20E in Bombyx mori, and provide novel insights into FMBP-1 transcriptional regulation and silk protein synthesis.

Correlations between some economic larval traits of silkworm

Abstract. Silkworm, Bombyx mori L. is an economic insect that has great importance on cocoon and silk production. The development of sericultural industry depends on quality and yield in terms of qualitative and quantitative silkworm characters. Mature larval weight and silk gland mass are important factors in silkworm breeding. Silk gland mass has significant impact on the synthesis of silk protein in fifth larval stage. The investigation was conducted to evaluate the correlations between silk gland mass and mature larva weight of M and ZF (China), and N and ZB (Japan) lines and their hybrids MxN, ZFxZB. In this research, silkworm lines and hybrids were reared at the laboratory with 600 silkworm larvae for each four lines and their hybrids to determine these silkworm traits. Analysis of variance indicated that the mean values in Chinese lines and hybrids were found to be significant (P<0.01) for mature larva weight. On the other hand, mean value of silk gland mass was determined as important for Chinese, Japanese lines and hybrids (P<0.01). Both mature larva weight and silk gland mass have positive correlation in Chinese lines (r=0.659) and hybrids (r=0.643) (P<0.01). Otherwise, the correlation between these characters were not found to be significant for Japanese lines. As a result, it is necessary to search the correlation between different characters and to protect the genetic diversity in silkworm lines.

Identification of Genes that Control Silk Yield by RNA Sequencing Analysis of Silkworm (Bombyx mori) Strains of Variable Silk Yield.

Silk is an important natural fiber of high economic value, and thus genetic study of the silkworm is a major area of research. Transcriptome analysis can provide guidance for genetic studies of silk yield traits. In this study, we performed a transcriptome comparison using multiple silkworms with different silk yields. A total of 22 common differentially expressed genes (DEGs) were identified in multiple strains and were mainly involved in metabolic pathways. Among these, seven significant common DEGs were verified by quantitative reverse transcription polymerase chain reaction, and the results coincided with the findings generated by RNA sequencing. Association analysis showed that BGIBMGA003330 and BGIBMGA005780 are significantly associated with cocoon shell weight and encode uridine nucleosidase and small heat shock protein, respectively. Functional annotation of these genes suggest that these play a role in silkworm silk gland development or silk protein synthesis. In addition, we performed principal component analysis (PCA) in combination with wild silkworm analysis, which indicates that modern breeding has a stronger selection effect on silk yield traits than domestication, and imply that silkworm breeding induces aggregation of genes related to silk yield.

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.

The silk gland damage and the transcriptional response to detoxifying enzymes-related genes of Bombyx mori under phoxim exposure

Silk gland is a major organ of Bombyx mori for the synthesis and secretion of silk protein. Phoxim exposure can be toxic to B.mori and causes a decrease of fibroin synthesis, finally affecting the silk production in industry. To study the mechanism of metabolism and detoxification of silk gland under phoxim exposure, we measured the residual quantity of phoxim in silk gland and hemolymph after phoxim exposure, and the detoxifying enzymes-related genes and enzyme activity were also investigated. Results indicated that the residual amount of phoxim existed up to 24 h in silk gland compared with that in hemolymph, suggesting that phoxim can accumulate in the silk glands within a certain time course. The transcriptional levels of PI3K/Akt genes, including Akt, Tor1, p70s6k and 4e-bp, were up-regulated by 6.919, 1.358, 10.766 and 7.708-fold, respectively. The expression of two downstream genes (CncC and Keap1) was up-regulated by 1.939 and 3.373-fold, respectively. In addition, the transcriptional levels of detoxification-related genes including CYP6AB, CYP306A, CarE2, GST1 and GSTd1 were up-regulated by 1.731, 1.221, 1.366, 1.376 and 6.591-fold, respectively. The enzymatic activity of CYP450, CarE and GST were increased over time. These results provided possible insights into the injury of silk gland and the transcriptional response to detoxifying enzymes-related genes in silkworm after phoxim exposure.

Titanium nanoparticles influence the Akt/Tor signal pathway in the silkworm, Bombyx mori, silk gland.

Various nanoparticles, such as silver nanoparticles (AgNPs) and titanium nanoparticles (TiO2 NPs) are increasingly used in industrial processes. Because they are released into the environment, research into their influence on the biosphere is necessary. Among its other effects, dietary TiO2 NPs promotes silk protein synthesis in silkworms, which prompted our hypothesis that TiO2 NPs influence protein kinase B (Akt)/Target of rapamycin (Tor) signaling pathway (Akt/Tor) signaling in their silk glands. The Akt/Tor signaling pathway is a principle connector integrating cellular reactions to growth factors, metabolites, nutrients, protein synthesis, and stress. We tested our hypothesis by determining the influence of dietary TiO2 NPs (for 72 h) and, separately, of two Akt/Tor pathway inhibitors (LY294002 and rapamycin) on expression of Akt/Tor signaling pathway genes and proteins in the silk glands. TiO2 NPs treatments led to increased accumulation of mRNAs for Akt, Tor1 and Tor2 by 1.6-, 12.1-, and 4.8-fold. Dietary inhibitors led to 2.6- to 4-fold increases in mRNAs encoding Akt and substantial decreases in mRNAs encoding Tor1 and Tor2. Western blot analysis showed that dietary TiO2 NPs increased the phosphorylation of Akt and its downstream proteins. LY294002 treatments led to inhibition of Akt phosphorylation and its downstream proteins and rapamycin treatments similarly inhibited the phosphorylation of Tor-linked downstream proteins. These findings support our hypothesis that TiO2 NPs influence Akt/Tor signaling in silk glands. The significance of this work is identification of specific sites of TiO2 NPs actions.

Differences in gut microbiota between silkworms (Bombyx mori) reared on fresh mulberry (Morus alba var. multicaulis) leaves or an artificial diet.

Artificial diets for silkworms have many potential applications and they are important in sericulture. However, the challenges of weak larvae and low silk protein synthesis efficiency in silkworms reared on artificial diets have not been resolved. Here, we used high-throughput sequencing to analyse the differences between the gut microbiota of 5th-instar larvae reared on mulberry leaves and larvae reared on an artificial diet. The results showed that at the phylum level, Cyanobacteria, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria are the dominant bacteria in the intestines of silkworm larvae of all the strains. But the abundance of dominant bacteria in the gut microbiota differed between the silkworm strains that were reared on mulberry leaves, as well as between the silkworm strains that were reared on the artificial diet, while the gut microbiota diversity was lower in the silkworm strains that were reared on the artificial diet. Prediction of the functions of the gut microbiota in the hosts indicated that there was no significant difference between the silkworm strains that were reared on mulberry leaves, while there were significant differences between silkworm strains reared on the artificial diet. When the silkworm diet changed from mulberry leaves to the artificial diet, changes in gut microbiota in the silkworms affected host nutrient metabolism and immune resistance. These changes may be related to the adaptation of silkworms to their long evolutionary history of eating mulberry leaves.

QTL analysis of cocoon shell weight identifies BmRPL18 associated with silk protein synthesis in silkworm by pooling sequencing

Mechanisms that regulate silk protein synthesis provide the basis for silkworm variety breeding and silk gland bioreactor optimization. Here, using the pooling sequencing-based methodology, we deciphered the genetic basis for the varied silk production in different silkworm strains. We identified 8 SNPs, with 6 on chromosome 11 and 1 each on chromosomes 22 and 23, that were linked with silk production. After conducting an association analysis between gene expression pattern, silk gland development and cocoon shell weight (CSW), BMGN011620 was found to be regulating silk production. BMGN011620 encodes the 60S ribosomal protein, L18, which is an indispensable component of the 60S ribosomal subunit; therefore we named it BmRPL18. Moreover, the clustering of linked SNPs on chromosome 11 and the analysis of differentially expressed genes reported in previous Omics studies indicated that the genes regulating silk protein synthesis may exhibit a clustering distribution in the silkworm genome. These results collectively advance our understanding of the regulation of silk production, including the role of ribosomal proteins and the clustered distribution of genes involved in silk protein synthesis.

Identification of circular RNA in the Bombyx mori silk gland

Bombyx mori is an economically important holometabolous lepidopteran insect. In B. mori endogenous noncoding RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs play crucial biological functions in metamorphosis and sex determination. In addition, circular RNAs (circRNAs) have been recently identified as noncoding RNAs in most common model organisms and show potential as gene regulators. However, to date, there have been few studies on the circRNAs present in the B. mori genome conducted to date. Here, we identified 3916 circRNAs by deep circular transcriptome sequencing using the silk gland of B. mori. 3155 circRNAs were found to be derived from 1727 parental genes. The circRNAs displayed tissue-specific expression between the middle silk gland (MSG) and posterior silk gland (PSG), with 2532 and 880 being upregulated circRNAs in the MSG and PSG, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that the parental genes from the MSG and PSG were generally annotated to similar categories and pathways. The interaction network of circRNAs and miRNAs showed that circRNAs might act as miRNA sponges or interact with miRNAs in some other way. Overall, the results revealed the complicated patterns of circRNAs in the B. mori silk gland providing a new angle from which to explore the mechanisms of complex gene regulation and efficient silk protein synthesis.

Metabolomics differences between silkworms (Bombyx mori) reared on fresh mulberry (Morus) leaves or artificial diets

Silkworms (Bombyx mori) reared on artificial diets have great potential applications in sericulture. However, the mechanisms underlying the enhancement of metabolic utilization by altering silkworm nutrition are unclear. The aim of this study was to investigate the mechanisms responsible for the poor development and low silk protein synthesis efficiency of silkworms fed artificial diets. After multi-generational selection of the ingestive behavior of silkworms to artificial diets, we obtained two strains, one of which developed well and another in which almost all its larvae starved to death on the artificial diets. Subsequently, we analyzed the metabolomics of larval hemolymph by gas chromatography/liquid chromatography–mass spectrometry, and the results showed that vitamins were in critically short supply, whereas the nitrogen metabolic end product of urea and uric acid were enriched substantially, in the hemolymph of the silkworms reared on the artificial diets. Meanwhile, amino acid metabolic disorders, as well as downregulation of carbohydrate metabolism, energy metabolism, and lipid metabolism, co-occurred. Furthermore, 10 male-dominant metabolites and 27 diet-related metabolites that differed between male and female silkworms were identified. These findings provide important insights into the regulation of silkworm metabolism and silk protein synthesis when silkworms adapt to an artificial diet.

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.

Differentially expressed genes in the silk gland of silkworm (Bombyx mori) treated with TiO2 NPs

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO2 nanoparticles (NPs) promotes silk protein synthesis in silkworms. In this study, digital gene expression (DGE) tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO2 NPs. In total, 5,702,823 and 6,150,719 clean tags, 55,096 and 74,715 distinct tags were detected in TiO2 NPs treated and control groups, respectively. Compared with the control, TiO2 NPs treated silkworms showed 306 differentially expressed genes, including 137 upregulated genes and 169 downregulated genes. Of these differentially expressed genes, 106 genes were related to silk protein synthesis, among which 97 genes were upregulated and 9 genes were downregulated. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 20 pathways were significantly enriched in TiO2 NPs treated silkworms, and the metabolic pathway-related genes were the most significantly enriched. The DGE results were verified by qRT-PCR analysis of eight differentially expressed genes. The DGE and qRT-PCR results were consistent for all three upregulated genes and three of the five downregulated genes, but the expression trends of the remaining two genes were different between qRT-PCR and DGE analysis. This study enhances our understanding of the mechanism of TiO2 NPs promoted silk protein synthesis.

Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori

Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm.

Soluble expression, purification and structural analysis of the bHLH transcription factor Bmsage of Bombyx mori

Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra. The results showed that NusA and MBP could significantly enhance the soluble expression of Bmsage in E. coli, but it was difficult to separate Bmsage from these tags. SUMO could not only increase the soluble expression of Bmsage in E. coli to a certain degree, but also be effectively separated from Bmsage. Other tags did not effectively promote the soluble expression of Bmsage in E. coli. Circular dichroism spectra showed that the purified Bmsage had well-defined α-helix structure in solution, indicating that SUMO may promote the correct folding of Bmsage into native-like structure. These work not only establish a foundation for further study of the property, structure and function of Bmsage, but also provide a reference for the expression and purification of other similar proteins.

Phosphoproteomic analysis of the posterior silk gland of Bombyx mori provides novel insight into phosphorylation regulating the silk production

To understand phosphorylation event regulating silk synthesis in the posterior silk gland of Bombyx mori, phosphoproteome was profiled in a pair of near-isogenic lines, a normally cocooning strain (IC) and a nakedly pupated strain (IN) that the silk production is much lower than IC. In the posterior silk gland of the IC and IN, 714 and 658 phosphosites resided on 554 and 507 phosphopeptides from 431 and 383 phosphoproteins, were identified, respectively. Of all the phosphosites, the single phosphosite was the dominate phosphorylation form, comprising>60% of all the phosphosites in two phenotypic of silk production. All these phosphosites were classified as acidophilic and proline-directed kinase classes, and three motifs were uniquely identified in the IC. The motif S-P-P might be important for regulating phosphorylation network of silk protein synthesis. The dynamically phosphorylated proteins participated in ribosome, protein transport and energy metabolism suggest that phosphorylation may play key roles in regulating silk protein synthesis and secretion. Furthermore, fibroin heavy chain, an important component of silk protein, was specifically phosphorylated in the IC strain, suggesting its role to ensure the normal formation of silk structure and silk secretion. The data gain new understanding of the regulatory processes of silk protein synthesis and offer as starting point for further research on the silk production at phosphoproteome level. Significance of this studyDespite the knowledge on regulation of silk protein synthesis in the posterior silk gland has gained at the gene or protein levels, how phosphorylation event influences the silk yield is largely unknown. To this end, we constructed a pair of silkworm near-isogenic lines that showed different cocooning phenotypes, and the phosphoproteome of the posterior silk gland of two isolines was compared. Here, we reported the first phosphoproteome data on the silkworm and found several key pathways related protein synthesis are regulated by phosphorylation, thereby influencing the silk production. The data provide valuable resources for further functional assay of targeted protein phosphorylation that regulates the silk synthesis in silkworm.

Nuclear hormone receptor BmFTZ-F1 is involved in regulating the fibroin heavy chain gene in the silkworm, Bombyx mori

BackgroundThe synthesis of silk protein is controlled by hormones. The expression of the nuclear hormone Bmftz-f1 in the posterior silk gland (PSG) is induced by 20-hydroxyecdysone in vivo and in vitro. However, whether Bmftz-f1 regulates silk protein expression is unknown. MethodsIn our study, western blotting and quantitative polymerase chain reactions were conducted to detect the expression of FTZ-F1 in the PSG. Electrophoretic mobility shift, chromatin immunoprecipitation, far-western blotting, bimolecular fluorescence complementation, and dual luciferase reporter assays were performed to investigate the effect of FTZ-F1 on the fibH promoter. Results(1) The expression of the hormone receptor BmFTZ-F1 was opposite to that of fibH. It was highly expressed in the PSG during the fourth molting stage and the beginning of the fifth instar, and then its expression decreased gradually until it disappeared at the end of the fifth instar and the wandering stage. (2) We identified a FTZ-F1 response element 390bp upstream of the transcription initiation site of the fibH promoter. (3) BmFTZ-F1 interacted with the basic helix-loop-helix transcription factor Bmdimm. (4) BmFTZ-F1 down-regulated fibH promoter activity and counteracted the effect of Bmdimm on fibH expression. ConclusionsIntegrating these results, we conclude that BmFTZ-F1 regulates the transcription of fibH by binding to the FTZ-F1 response element in the fibH promoter and counteracts the effect of Bmdimm on fibH expression. General significanceThese findings provide new insights into the mechanism of regulation of the silk protein gene.

Effects of TiO2 nanoparticles on nutrition metabolism in silkworm fat body.

ABSTRACTSilkworm (Bombyx mori) is an important economic insect with a fat body that plays a crucial role in the storage and transfer of nutrients. It is also known that TiO2 nanoparticles (NPs) can improve feed efficiency and promote silk protein synthesis in the silkworm. In this study, we profiled gene expression in the silkworm fat body after TiO2 NP treatment, validated the major RNA-seq findings, and determined the contents of trehalose and triglyceride, the activity of lipase, and the amount of total proteins. RNA-seq analysis revealed that TiO2 NP treatment caused significant expression changes in 341 genes (P≤0.01), 138 of which were upregulated while the other 203 were downregulated. The expression levels of two target genes in the insulin signaling pathway and two protein metabolism-related target genes, three lipid metabolism-associated target genes, two carbohydrate metabolism related target genes and expression levels of seven heat shock protein genes were increased, and that of threonine dehydratase gene and fatty acid transport protein gene were decreased. The RNA-seq results of 16 genes were validated by quantitative real-time PCR. The lipase activity, content of trehalose, and amount of total proteins were elevated by 3.86-fold, 1.34-fold, and 1.21-fold, respectively, and the content of triglyceride was decreased by 0.94-fold after TiO2 NP treatment. These results indicated that TiO2 NPs activated the insulin signaling pathway, promoted the metabolism of protein, fat, and carbohydrate, and improved nutrition metabolism. Our study provides new support for the understanding of the beneficial effect of TiO2 NPs on silkworm nutrient metabolism.

Effect of BBX-B8 overexpression on development, body weight, silk protein synthesis and egg diapause of Bombyx mori.

Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5-3.5days. Cocoon shell weight of transgenic silkworms was reduced by 4.79% in females and 7.44% in males, pupal weight of transgenic silkworms was reduced 6.75% in females and 13.83% in males compared to non-transgenic silkworms, and 5.56-14.29% of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm.

Comparative transcriptomic analysis of silkworm Bmovo-1 and wild type silkworm ovary.

The detailed molecular mechanism of Bmovo-1 regulation of ovary size is unclear. To uncover the mechanism of Bmovo-1 regulation of ovarian development and oogenesis using RNA-Seq, we compared the transcriptomes of wild type (WT) and Bmovo-1-overexpressing silkworm (silkworm+Bmovo-1) ovaries. Using a pair-end Illumina Solexa sequencing strategy, 5,296,942 total reads were obtained from silkworm+Bmovo-1 ovaries and 6,306,078 from WT ovaries. The average read length was about 100 bp. Clean read ratios were 98.79% for silkworm+Bmovo-1 and 98.87% for WT silkworm ovaries. Comparative transcriptome analysis showed 123 upregulated and 111 downregulated genes in silkworm+Bmovo-1 ovaries. These differentially expressed genes were enriched in the extracellular and extracellular spaces and involved in metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems. Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development and oogenesis and oocyte proliferation and transport of nutrients to ovaries by altering nutrient partitioning, which would support ovary development. Excessive consumption of nutrients for ovary development alters nutrient partitioning and deters silk protein synthesis.