Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx moil

Class B scavenger receptors （SR-Bs） are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagoeytosis of xenobiotics, and signaling. But little information is available about silkworm SR-Bs; it is necessary to study these SR-Bs for revealing their function. In this study, we cloned the full-length coding sequence of BrnSCRBQ4, a SR-B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA （mRNA） was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.

Identification of a PP2A gene in Bombyx mori with antiviral function against B.mori nucleopolyhedrovirus

Ser/Thr protein phosphatase 2A(PP2A)is one of the type 2 protein phosphatases,which is required for many intracellular physiological processes and pathogen infection.However,the function of PP2A is unclear in silkworm,Bombyx mori.Here,we cloned and identified BmPP2A,a PP2A gene from B.mori,which has two HEAT domains and a high similarity to PP2A from other organisms.Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut,and that Bombyx mori nucleopolyhedrovirus(BmNPV)infection induces down-regulation of BmPP2A expression.Furthermore,up-regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication.In contrast,down-regulation of BmPP2A via RNA interference and okadaic acid(a PP2A inhibitor)treatment allowed robust BmNPV replication.This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A,a potential anti-BmNPV mechanism,and a possible target for the breeding of silkworm-resistant strains.

Evolutionary and functional analyses of the interaction between the Bombyx mori inhibitor of apoptosis(IAP)and nucleopolyhedrovirus IAPs

As an important insect immune response,apoptosis plays a critical role in the interaction between baculoviruses and insect hosts.Previous reports have identified inhibitor of apoptosis(IAP)proteins in both insects and baculoviruses,but the relationship between these proteins is still not clearly understood.Here,we found that insect IAP proteins were clustered with baculovirus IAP3,suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or Diptera.We demonstrated that Bombyx mori inhibitor of apoptosis(Bmiap)gene had an inhibitory effect on apoptosis in silkworm cells.Further analysis of the effects of Bmiap genes on the proliferation of B.mori nucleopolyhedrovirus(BmNPV)showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm cells.Our results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells,implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV infection.Taken together,our results provide important insights into the functional relationships of iap genes,and improve our knowledge of apoptosis in baculoviruses and insect hosts.

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

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, cdtl 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.

E2F4 regulates the cell cycle and DNA replication in the silkworm,Bombyx mori

The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions.Although E2Fs have been widely studied in mammals,there have been few studies performed in insects.Here,we determined the function of E2F4 in the silkworm,Bombyx mori.We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals.Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase,whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase.These results indicate that BmE2F4 is important in cell cycle regulation.We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland.Furthermore,we identified a protein called Bm14-3-3ζthat can interact with BmE2F4 and allow it to localize in the nucleus.Overexpression of the Bm14-3-3ζgene led to cell cycle arrest in the G1 phase,while knocking down the gene increased the proportion of cells in S phase.These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.