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Bombyx Mori Nuclear Polyhedrosis Virus-encoded miRNA and Its Preliminary Functional Analysis

Author: ChenWei
Tutor: ZhaoQiaoLing
School: Jiangsu University of Science and Technology
Course: Special Economic Animal Feeding
Keywords: Bombyx mori nuclear polyhedrosis virus (BmNPV) miRNA VMir High-throughput sequencing Target genes
CLC: S884.51
Type: Master's thesis
Year: 2010
Downloads: 47
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Abstract


MicroRNAs (miRNAs), length 21-25nt small non-protein-coding RNAs, which can regulate the expression of genes involved in the process to regulate the organism's growth, development and metabolism. miRNAs initially found Caenorhabits elegan nematodes, recently also found in many viral miRNAs in existence. Bombyx mori nuclear polyhedrosis virus (of BmNPV) in order to study miRNAs function of this experiment were used the bioinformatics prediction method and high-throughput sequencing methods the BmNPV genome encoded miRNAs were identified; online software to predict the possible role of the target gene on two known BmNPV miRNAs sequence m1 and m2 functional analysis. The main results are as follows. (1) BmNPV genome encodes miRNAs precursor predicted PCR validation. BmNPV whole genome scan using VMir software, Rating \u0026 hairpin structure resulting sequence analysis, and exclude the sequences in the protein coding region, and 7 may be provided by the BmNPV genomic coding miRNAs candidates precursor sequence finally obtained. Silkworm hybrids S16 · S17 × A1 · A16 larvae of the experimental material, with a concentration of 5 × 10 7 PIB / mL of BmNPV licking infection five instar silkworm hemolymph, 72 h after take to extract total RNA by RT-PCR identified 5 may be encoded by BmNPV genome miRNA precursor molecules. Further analysis of the 5 hairpin structure of the precursor molecules, and miRNA mature sequence verified by RT-PCR amplified a total of six and viral genome sequences consistent mature sequence results in 5 precursor molecules, they are located in different the ORF between two sequences of positive transcriptional sequence, the sequence of four reverse transcription. We believe that these six mature sequence is BmNPV genome encoded miRNAs. Precursor sequence and mature sequence over time incremental expression by quantitative analysis found. (2) of BmNPV genome encodes miRNAs precursor high-throughput sequencing. The method of using the high throughput sequencing detected in the BmNPV genomic 14 miRNAs. But by RT-PCR method only identified seven miRNAs which are located in different ORF, 3 forward transcribed sequences, four sequences for reverse transcription. (3) of BmNPV genomic coding miRNAs target genes in the viral genome on the prediction. Online target gene prediction program common predict the target genes of 13 BmNPV miRNAs in the viral genome, RNA hybrid and RNA 22. Results 28 may be of a target gene, wherein 4 is encoded the capsule membrane protein gene, 3 is the gene encoding the capsid protein, 5 are coded lef-5, LEF-6, of SOD, polyhedrin and GP37 gene 16 non-annotated genes. Thus, we speculate that the BmNPV-encoded miRNAs may be involved in regulating the process of the virus to infect the host. (4) of BmNPV miRNA sequence m1 and m2. Using RNA hybrid software prediction two known the BmNPV miRNA sequence m1 and m2 of the target gene, PCR amplification of m1, m2 of the precursor sequence and the sequence of the 3'-UTR of the predicted target genes were constructed, the expression of miRNA pcDNA recombinant plasmid BmN cells co-transfected with the recombinant plasmid and expression of the target gene 3'-UTR of pGL. Systems analysis by the dual luciferase reporter gene assay, overexpression the m1 pGL-38k 3'-UTR downregulation of 14%; overexpression m2 pGL-Polyhedrin3'-UTR and the pGL-ODV-EC27 3'-UTR of expression down 9.8% and 9.2%, respectively. The results showed 38k and Polyhedrin, ODV-EC27 may m1 and m2 target genes.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Sericulture > Pest and Disease Control of the silkworm > Virus disease ( pus disease) > The nuclear polyhedrosis body disease
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