ABSTRACT
Glucose regulatory protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family, that plays an important role in secreted protein folding. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. Previous studies showed that HSP90 members promote BmNPV replication in silkworm, but the function of BmGRP94 in BmNPV infection and proliferation is still not understood. In this study, we investigated the interplay between BmGRP94 and BmNPV infection in silkworm. We first identified a single gene of BmGRP94 in the Bombyx mori genome, which encodes a polypeptide with 810 amino acids in length. Spatio-temporal expression profiles showed that BmGRP94 was highly expressed in hemocytes and midgut, and was significantly induced by BmNPV infection. Furthermore, overexpression of BmGRP94 facilitates viral proliferation, while BmGRP94 inhibition evidently decreased BmNPV proliferation in BmN cells and in silkworm midgut. Mechanistically, BmGRP94 inhibition triggers ER stress, as judged by increased expression of PERK/ATF4/ERO1, H2O2 production, and ER calcium efflux, which promotes cell apoptosis to restrict BmNPV replication in silkworm. These results suggest that BmGRP94 plays an important role in facilitating BmNPV proliferation, and provides a potential molecular target for BmNPV prevention.
Subject(s)
Bombyx , Nucleopolyhedroviruses , Animals , Nucleopolyhedroviruses/physiology , Hydrogen Peroxide/metabolism , HSP90 Heat-Shock Proteins/metabolism , Bombyx/metabolism , Apoptosis/genetics , Cell Proliferation , Insect Proteins/genetics , Insect Proteins/metabolismABSTRACT
N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification that plays an important role in regulating many biological processes, especially embryonic development. However, regulation of m6A methylation during silkworm embryonic development and diapause remains to be investigated. In this study, we analyzed the phylogeny of subunits of methyltransferases BmMettl3 and BmMettl14, and detected the expression patterns of BmMettl3 and BmMettl14 in different tissues and at different developmental stages in silkworm. To investigate the function of m6A on the development of silkworm embryo, we analyzed the m6A/A ratio in diapause and diapause termination eggs. The results showed that BmMettl3 and BmMettl14 were highly expressed in gonads and eggs. Moreover, the expression of BmMettl3 and BmMettl14 and the m6A/A ratio were significantly increased in diapause termination eggs compared with diapause eggs in the early stage of silkworm embryonic development. Furthermore, in BmN cell cycle experiments, the percentage of cells in the S phase increased when lacking BmMettl3 or BmMettl14. This work contributes to understanding the role of m6A methylation during insect embryogenesis and gametogenesis. It also provides a research orientation to further analyze the role of m6A methylation in diapause initiation and termination during insect embryonic development.
Subject(s)
Bombyx , Methyltransferases , Animals , Methyltransferases/genetics , Methyltransferases/metabolism , Bombyx/metabolism , RNA/metabolism , Epigenesis, Genetic , Embryonic Development/genetics , Gene Expression Regulation, Developmental , Ovum/metabolismABSTRACT
De-etiolation during seedling development is antagonistically regulated by blue light (BL) and gibberellins (GAs). The crosstalk between blue light (BL) and GA metabolism and signaling remains unclear. Using the mutant har1 which is specifically hypersensitive to BL in de-etiolation, the involvement possibility of the GA metabolism, GA signaling in the inhibition of mesocotyl elongation of the sorghum (Sorghum bicolor L. var. R111) seeding under BL was investigated. The inhibition of mesocotyl and cell elongation by BL was restored by application of exogenous GA(3) in har1. The endogenous GA(3) level correspondingly decreased in har1 mesocotyl especially from 1 to 4 h after BL irradiation. Putative genes of GA metabolism enzymes SbGA20ox, SbGA3ox and SbGA2ox were detected by Real-Time PCR and the results showed that one of the SbGA2ox homologs appeared significantly higher transcript level in har1 than in R111 at 2 h after BL irradiation. Putative homologous genes of DELLAs increased after BL irradiation and were higher in har1 among the three homologs. Remarkable increase of the DELLA expression was observed responding to exogenous paclobutrazol (PAC). Our research provided evidence in monocot sorghum, that the changes of a set of the GA metabolism and signaling genes might be involved in BL-induced inhibition of cell elongation.
Subject(s)
Gibberellins/metabolism , Light , Seedlings/growth & development , Seedlings/radiation effects , Signal Transduction , Sorghum/growth & development , Sorghum/radiation effects , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Genes, Plant/genetics , Genetic Association Studies , Gibberellins/pharmacology , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reference Standards , Seedlings/cytology , Signal Transduction/drug effects , Sorghum/cytology , Sorghum/genetics , Transcription, Genetic/drug effects , Triazoles/pharmacologyABSTRACT
CRY2 is the apoprotein of cryptochrome 2 and CRY2 cDNA has been cloned from Sorghum bicolor in this study. The results of sequence analysis shows that it contains a complete open reading frame encoding a predicted protein of 690 amino acids sharing 87% identity with the CRY2 of rice, but only 45.5% with that of Arabidopsis and 57% with that of tomato. The DNA sequence of sorghum CRY2 contains three introns and four exons. RT-PCR results show that CRY2 has been transcribed in root, leaf and mesocotyl in sorghum seedlings. CRY2 protein has been shown by Western blot analysis to be present in mesocotyl, leaf and root, and to be accumulated in the dark and to be degraded under blue light.
