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Simple Summary: Bombyx mori is an important economic insect and a model organism of Lepidoptera. Its hemolymph contains rich nutrient storage proteins, which participate in material transportation, immune regulation, and other physiological functions. Additionally, a large number of proteins in hemolymph can be acetylated. Apolipophorin-II is a kind of nutrition storage protein that can bind lipid substances. In the article, we confirm that the Bombyx mori acetyltransferase cyclic adenosine monophosphate response element binding protein (CREB) binding protein (CBP) can interact with the apolipophorin-II protein. Therefore, CBP protein, as an acetyltransferase, can catalyze the acetylation of apolipophorin-II protein and thus affect the stability of apolipophorin-II protein. Acetyltransferase CBP can affect the growth and development of insects. In general, the research on nutrition storage protein and CBP protein can develop a new direction for biological control and pest resistance in the future. Acetylation is an important and reversible post-translational modification (PTM) of protein, which is involved in many cellular physiological processes. In previous studies, lots of nutrient storage proteins were found to be highly acetylated in silkworms, and acetylation can improve the stability of these proteins. However, the related acetyltransferase was not involved. In the present work, a Bombyx mori nutrient storage protein, apolipophorin II (BmApoLp-II), was further confirmed to be acetylated, and the acetylation could improve its protein expression. Furthermore, RNAi and Co-IP showed that the acetyltransferase BmCBP was found to catalyze the acetylation modification of BmApoLp-II, and thus affect its protein expression. Meanwhile, it was proved that acetylation could improve the stability of the BmApoLp-II protein by completing its ubiquitination. These results lay a foundation for further study on the mechanism of regulating nutrition storage and hydrolysis utilization of storage proteins by BmCBP and the acetylation in the silkworm Bombyx mori. [ABSTRACT FROM AUTHOR]

Abstract MicroRNA is an important regulation factor in insect development and metamorphosis. It has been reported that E(spl)m4 is a miRNA-targeted gene, as well as the target of the Notch signaling pathway in Drosophila. The expression of E(spl)m4 can be regulated by microRNA and further affect the neural development of Drosophila. Here, we found that BmEm4 , an ortholog of E(spl)m4 from Bombyx mori , was the target gene of bmo-miR-79 , with target sites containing the Brd and K boxes of the BmEm4 _3′UTR, which was validated by the dual luciferase reporter (DLR) assay. Furthermore, bmo-miR-79 mimics can inhibit the expression of BmEm4 in BmN cells after transfection, and bmo-miR-79 can also inhibit the expression of BmEm4 in different developmental stages of Bombyx mori at a posttranscriptional level, to different degrees. The EMSA test further showed that bmo-miR-79 could bind to BmAGO2, which is the Bombyx mori argonaute2 protein, suggesting that bmo-miR-79 might regulate the expression of BmEm4 by forming miRISC complexes with BmAGO2. Taken together, bmo-miR-79 could regulate the expression of BmEm4 mediated by BmAGO2 and further affect its function in the silkworm Bombyx mori. Highlights • The target sites of bmo-miR-79 located in the 3′UTR of BmEm4. • Bmo-miR-79 inhibits the expression of BmEm4 in vitro. • Bmo-miR-79 might affect the expression profile of BmEm4 in Bombyx mori. • BmAGO2 is the mediator involved in the regulation of BmEm4 by Bmo-miR-79. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) are the family of noncoding single-strand RNA molecules of 21–25 nucleotides in length and play a broad and key regulation role in various physiological and pathological processes including differentiation, apoptosis, proliferation, and tumorigenesis. In Bombyx mori, a total of 487 pre-miRNAs and 562 mature miRNAs were identified by experimental or computational approaches, but their functions remain unknown. To carry out the research of gain-of-function of miRNAs in BmN cells, we firstly identified the endogenous expression of miRNAs in BmN cells by microarray and found that only 73 miRNAs could be detected by miRNA microarray. Then three low abundance or undetected miRNAs, pri-mir-1a, pri-mir-8 and pri-mir-133, were selected to express in BmN cells. The eukaryotic expression vector pIEx-1 harboring baculovirus ie1 promoter and hr5 enhancer was screened and used for expressing miRNA in BmN cells. Three miRNA expression vectors pIEx-1-EGFP-pri-mir-1a/8/133 were constructed, which contained the three corresponding pri-miRNA sequences, respectively. The constructed miRNA vectors were successfully transfected into BmN cells and the qRT-PCR analysis showed that relative abundance of bmo-mir-1a, bmo-mir-8 and bmo-mir-133 in BmN cells transfected with the pIEx-1-EGFP-pri-mir-1a/8/133 is as 32, 4.4 and 904 times as that in BmN cells transfected with the control vector pIEx-1-EGFP, respectively. The present work lays a foundation for the further functional studies of miRNAs in silkworm. [Copyright &y& Elsevier]

Abstract: Background: Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown. Methods: To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae. An His-tagged BmPHB fusion protein was expressed in Escherichia coli Rosetta (DE3) and purified with affinity and reversed-phase chromatography. Purified rBmPHB was used to generate anti-BmPHB polyclonal antibody. The subcellular localization of BmPHB was analysed by immunohistochemistry. Results: BmPHB gene has an ORF of 825bp, encoding a predicted peptide with 274 amino acid residues. Immunostaining indicate that prohibitin is expressed in nucleus and predominately in cytoplasm. Western blot analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine, and head. However, no expression was detected in larva''s silk gland and epidermis. In addition, BmPHB was expressed in the nascent egg, larva and pupa, but not in the moth. Conclusions: The expression of BmPHB gene presents differential characteristic in different stage and tissues. It may play important roles in the development of silkworm. General significance: Studies on prohibitin have been still restricted to a few specific insects and insect cell lines such as Drosophila, Acyrthosiphon pisum and mosquito cell lines, not yet in silkworm. This is a first characterization of prohibitin in silkworm, B. mori. [Copyright &y& Elsevier]

Abstract: 14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands. [Copyright &y& Elsevier]