[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) have emerged as key players in host-pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was and constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV.
Preview · Article · Sep 2015 · Saudi Journal of Biological Sciences
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) regulate expression of genes at post-transcriptional level by binding on complementary sequences of target mRNAs and play multiple roles in biological processes. To investigate the differential expression of miRNAs in posterior silk gland (PSG) of silkworm (Bombyx mori) in different periods and regulation of miRNAs on the expression of fibroin genes, Solexa sequencing technology was used to detect miRNAs in PSGs of fourth-instar day-2 larvae and fifth-instar day-3 larvae, respectively. As a result, 466 previously reported miRNAs, and 35 novel miRNAs were detected, and 499 of these detected miRNAs are predicted to target 13,383 genes by target prediction softwares. Additionally, 29 miRNAs expressed differently between the PSG of fourth-instar day-2 larvae and fifth-instar day-3 larvae were found, and the differential expression of these miRNAs may play an important role in the expression of fibroin genes.
No preview · Article · Aug 2015 · Genes & genomics
[Show abstract][Hide abstract] ABSTRACT: Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.
Preview · Article · Sep 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a class of non-protein coding small RNAs of 18-24 nucleotides in length that regulate expression of genes at post-transcriptional levels and play multiple roles in biological processes. Bm-ase plays an important role in the course of nerve development of the silkworm, Bombyx mori. Bmo-miR-9a is a conservative miRNA. By using target prediction software RNA22 and RNAhybrid, we found a target site of Bmo-miR-9a in the 3'UTR of Bm-ase gene. To verify the regulation function of Bmo-miR-9a on the expression of Bm-ase gene, a Bmo-miR-9a over-expressing vector and Bm-ase 3'UTR fused firefly luciferase gene reporter plasmid were constructed, respectively. Then they were used to co-transfect the BmN cells. The result showed that luciferase activity in the co-transfected cells was suppressed compared with the control. A similar result was obtained when BmN cells were co-transfected with artificial synthetic Bmo-miR-9a mimics and Bm-ase 3'UTR fused luciferase reporter plasmid. These results suggest that Bmo-miR-9a can down regulate the expression of Bm-ase gene.
No preview · Article · Mar 2013 · Bioorganicheskaia khimiia
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that the 3' UTR of BmVMP23 was destroyed by an inserted fragment, and the BmVMP23 expression was downregulated in the lethal egg of "Ming" (l-em). In this study, we found a miRNA (bmo-miR-1a-3p) that matches the 3' UTR sequence of BmVMP23 perfectly. The relationship between bmo-miR-1a-3p and the BmVMP23 expression was examined by the co-transfection in vitro. The luciferase assay result showed that luc expression was strongly repressed. This result inferred that bmo-miR-1a-3p may downregulate BmVMP23 expression via complementary interaction with the target sites at the 3' UTR.
[Show abstract][Hide abstract] ABSTRACT: The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around one hour, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2,287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that a ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation.
[Show abstract][Hide abstract] ABSTRACT: Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977 bp in length with an open reading frame of 885 bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62 kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5-87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon-5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis.
No preview · Article · Feb 2012 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: microRNAs (miRNAs) are 20-24 nucleotide (nt) RNAs that regulate eukaryotic gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). To identify miRNA target genes will help a lot by understanding their biological functions. Sophisticated computational approaches for miRNA target prediction, and effective biological techniques for validating these targets now play a central role in elucidating their functions. Owing to the imperfect complementarity of animal miRNAs with their targets, it is difficult to judge the accuracy of the prediction. Complexity of regulation by miRNA-mediated targets at protein and mRNAs levels has made it more challenging to identify the targets. To date, only a few miRNAs targets are confirmed. In this article, we review the methods of miRNA target prediction and the experimental validation for their corresponding mRNA targets in animals.