Tai-hang Liu

BmREEPa Is a Novel Gene that Facilitates BmNPV Entry into Silkworm Cells

We previously established two silkworm cell lines, BmN-SWU1 and BmN-SWU2, from Bombyx mori ovaries. BmN-SWU1 cells are susceptible while BmN-SWU2 cells are highly resistant to BmNPV infection. Interestingly, we found that the entry of BmNPV into BmN-SWU2 cells was largely inhibited. To explore the mechanism of this inhibition, in this study we used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative protein expression profiling and identified 629 differentially expressed proteins between the two cell lines. Among them, we identified a new membrane protein termed BmREEPa. The gene encoding BmREEPa transcribes two splice variants; a 573 bp long BmREEPa-L encoding a protein with 190 amino acids and a 501 bp long BmREEPa-S encoding a protein with 166 amino acids. BmREEPa contains a conserved TB2/DP, HVA22 domain and three transmembrane domains. It is localized in the plasma membrane with a cytoplasmic C-terminus and an extracellular N-terminus. We found that limiting the expression of BmREEPa in BmN-SWU1 cells inhibited BmNPV entry, whereas over-expression of BmREEPa in BmN-SWU2 cells promoted BmNPV entry. Our results also indicated that BmREEPa can interact with GP64, which is the key envelope fusion protein for BmNPV entry. Taken together, the findings of our study revealed that BmREEPa is required for BmNPV to gain entry into silkworm cells, and may provide insights for the identification of BmNPV receptors.

A newly discovered member of the Atlastin family, BmAtlastin-n, has an antiviral effect against BmNPV in Bombyx mori.

Atlastin is a member of the dynamin protein superfamily and it can mediate homotypic fusion of endoplasmic reticulum (ER) membranes, which is required for many biological processes. In this study, a new Atlastin homologous protein, BmAtlastin-n, was characterized in silkworms and was found to contain an N-terminal conserved GTPase domain and a coiled-coil middle domain. BmAtlastin-n is localized in the cytoplasm and enriched in silkworm midgut. Results also showed that overexpression of BmAtlastin-n in BmN-SWU1 cells could enhance resistance to BmNPV. To better confirm its antiviral effect, microRNA was used to knock down the expression of BmAtlastin-n in BmE-SWU1 cells with inducing the reproduction of BmNPV. A transgenic expression vector of BmAtlastin-n was constructed and introduced to silkworm embryos by microinjection. The transgenic silkworm also showed considerable antiviral capacity. In conclusion, these findings demonstrate that BmAtlastin-n plays an important role in BmNPV defense. More importantly, the current study may provide a new clue for Atlastin research.

Effects of starvation and hormones on DNA synthesis in silk gland cells of the silkworm, Bombyx mori

Silk gland cells of silkworm larvae undergo multiple cycles of endomitosis for the synthesis of silk proteins during the spinning phase. In this paper, we analyzed the endomitotic DNA synthesis of silk gland cells during larval development, and found that it was a periodic fluctuation, increasing during the vigorous feeding phase and being gradually inhibited in the next molting phase. That means it might be activated by a self‐regulating process after molting. The expression levels of cyclin E, cdt1 and pcna were consistent with these developmental changes. Moreover, we further examined whether these changes in endomitotic DNA synthesis resulted from feeding or hormonal stimulation. The results showed that DNA synthesis could be inhibited by starvation and re‐activated by re‐feeding, and therefore appears to be dependent on nutrition. DNA synthesis was suppressed by in vivo treatment with 20‐hydroxyecdysone (20E). However, there was no effect on DNA synthesis by in vitro 20E treatment or by either in vivo or in vitro juvenile hormone treatment. The levels of Akt and 4E‐BP phosphorylation in the silk glands were also reduced by starvation and in vivo treatment with 20E. These results indicate that the activation of endomitotic DNA synthesis during the intermolt stages is related to feeding and DNA synthesis is inhibited indirectly by 20E.

BmNHR96 participate BV entry of BmN-SWU1 cells via affecting the cellular cholesterol level.

B.mori nucleopolyhedrovirus (BmNPV), which produces BV and ODV two virion phenotypes in its life cycle, caused the amount of economic loss in sericulture. But the mechanism of its infection was still unclear. In this study we characterized B.mori nuclear hormone receptor 96 (BmNHR96) as a NHR96 family member, which was localized in the nucleus. We also found BmNHR96 over-expression could enhance the entry of BV as well as cellular cholesterol level. Furthermore, we validated that BmNHR96 increased membrane fusion mediated by GP64, which could probably promote BV-infection. In summary, our study suggested that BmNHR96 plays an important role in BV infection and this function probably actualized by affecting cellular cholesterol level, and our results provided insights to the mechanisms of BV-infection of B.mori.

Comparative transcriptome profiling of a thermal resistant vs. sensitive silkworm strain in response to high temperature under stressful humidity condition

Thermotolerance is important particularly for poikilotherms such as insects. Understanding the mechanisms by which insects respond to high temperatures can provide insights into their adaptation to the environment. Therefore, in this study, we performed a transcriptome analysis of two silkworm strains with significantly different resistance to heat as well as humidity; the thermo-resistant strain 7532 and the thermos-sensitive strain Knobbed. We identified in total 4,944 differentially expressed genes (DEGs) using RNA-Seq. Among these, 4,390 were annotated and 554 were novel. Gene Ontology (GO) analysis of 747 DEGs identified between RT_48h (Resistant strain with high-temperature Treatment for 48 hours) and ST_48h (Sensitive strain with high-temperature Treatment for 48 hours) showed significant enrichment of 12 GO terms including metabolic process, extracellular region and serine-type peptidase activity. Moreover, we discovered 12 DEGs that may contribute to the heat-humidity stress response in the silkworm. Our data clearly showed that 48h post-exposure may be a critical time point for silkworm to respond to high temperature and humidity. These results provide insights into the genes and biological processes involved in high temperature and humidity tolerance in the silkworm, and advance our understanding of thermal tolerance in insects.

Two Geminin homologs regulate DNA replication in silkworm, Bombyx mori

ABSTRACT DNA replication is rigorously controlled in cells to ensure that the genome duplicates exactly once per cell cycle. Geminin is a small nucleoprotein, which prevents DNA rereplication by directly binding to and inhibiting the DNA replication licensing factor, Cdt1. In this study, we have identified 2 Geminin genes, BmGeminin1 and BmGeminn2, in silkworm, Bombyx mori. These genes contain the Geminin conserved coiled-coil domain and are periodically localized in the nucleus during the S-G2 phase but are degraded at anaphase in mitosis. Both BmGeminin1 and BmGeminin2 are able to homodimerize and interact with BmCdt1 in cells. In addition, BmGeminin1 and BmGeminin2 can interact with each other. Overexpression of BmGeminin1 affects cell cycle progression: cell cycle is arrested in S phase, and RNA interference of BmGeminin1 leads to rereplication. In contrast, overexpression or knockdown of BmGeminin2 with RNAi did not significantly affect cell cycle, while more rereplication occurred when BmGeminin1 and BmGeminin2 together were knocked down in cells than when only BmGeminin1 was knocked down. These data suggest that both BmGeminin1 and BmGeminin2 are involved in the regulation of DNA replication. These findings provide insight into the function of Geminin and contribute to our understanding of the regulation mechanism of cell cycle in silkworm.

Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation

ABSTRACT Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.

The silkworm GSTe4 is sensitive to phoxim and protects HEK293 cells against UV-induced cell apoptosis.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of super enzymes with multiple functions that play a major role in the detoxification of endogenous and xenobiotic compounds. In our previous study, we have predicted 23 putative cytosolic GSTs in the silkworm genome using bioinformatic methods. In this study, we cloned and studied the insect-specific epsilon-class GST gene GSTe4 from the silkworm, Bombyx mori. The recombinant BmGSTe4 (Bac-BmGSTe4) was overexpressed in SF-9 cell lines, and it was found to have effective GST activity. We also found that the expression of BmGSTe4 was especially down-regulated after the silkworms were fumigated with or ingested phoxim. Moreover, BmGSTe4 protected HEK293 cells against UV-induced cell apoptosis. These results demonstrated that BmGSTe4 has GST activity, is sensitive to phoxim, and plays a role in inhibition of UV-induced cell apoptosis.