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Application of microRNAs associated with nuclear polyhedrosis virus infection in silkworm

A nuclear polyhedron and virus infection technology, applied in the field of silkworm microRNA, can solve the problems of increasing the complexity of host-virus interaction

Active Publication Date: 2018-08-31
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, virus-encoded miRNA can also regulate itself and genes related to virus replication or host immunity, which further increases the complexity of host-virus interaction.
[0004] At present, there are few researches on microRNAs related to silkworm nuclear polyhedrosis virus infection. Therefore, it is necessary to identify relevant microRNAs or to study the functions and uses of known microRNAs in this field, so as to provide a basis for the study of the interaction mechanism between silkworm and nuclear polyhedrosis virus. At the same time, it provides a new idea for the detection and control of silkworm nuclear polyhedrosis virus

Method used

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  • Application of microRNAs associated with nuclear polyhedrosis virus infection in silkworm
  • Application of microRNAs associated with nuclear polyhedrosis virus infection in silkworm
  • Application of microRNAs associated with nuclear polyhedrosis virus infection in silkworm

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Effect test

Embodiment 1

[0030] Example 1 The expression level of bmo-miR-277-5p in BmNPV-infected silkworm and normal silkworm was detected by using a stem-loop fluorescent quantitative PCR kit.

[0031] 1 Composition of Stem-loop Fluorescence Quantitative PCR Kit

[0032] The stem-loop fluorescent quantitative PCR kit of the present invention is composed of a reverse transcription system, an amplification system and internal reference primers, specifically as follows:

[0033] (1) The reverse transcription system consists of gDNA Eraser, 5×gDNA Eraser Buffer, PrimeScript RTEnzyme Mix I, 5×PrimeScript Buffer (except stem-loop primers, other reverse transcription reagents were purchased from TaKaRa Company), stem-loop primers (sequences such as shown in SEQ ID NO.3), RNase-free ddH 2 O composition;

[0034] (2) The amplification system consists of 2×SYBR Premix Ex Taq (purchased from TaKaRa Company), bmo-miR-277-5p forward primer (sequence shown in SEQ ID NO.4), bmo-miR-277-5p reverse primer Direct...

Embodiment 2

[0057] Example 2 Target gene biological prediction and laboratory verification of bmo-miR-277-5p

[0058] Step 1, the target gene of bmo-miR-277-5p was predicted by RNAHybrid biological software. Set the complete pairing of the seed region and the minimum free energy of -20kcal / mol as the screening criteria, and predict that the target gene of bmo-miR-277-5p is silkworm DNA cytosine methyltransferase, such as figure 2 shown.

[0059] The genebank accession number of the silkworm DNA cytosine-5methyltransferase gene (Bombyx mori DNA cytosine-5methyltransferase, Dnmt2) is NM_001043469.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0060] Step 2, the dual-luciferase reporter gene system verifies the target gene, and the schematic diagram of vector construction is shown in image 3 , the specific process is as follows:

[0061] (1) chemically synthesize the target gene Dnmt23'UTR and the mutant fragment, the nucleotide sequence of Dnmt23'UTR is shown in SEQ ID NO.12...

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Abstract

The invention discloses an application of microRNA related to silkworm nuclear polyhedrosis virus infection. The application is the application of bmo-miR-277-5p in the preparation of a kit for detecting the infection of silkworm nuclear polyhedrosis virus, or the application of bmo-miR-277-5p in the inhibition of gene expression, The mRNA after gene transcription has a 3'UTR of the sequence shown in SEQ ID NO.12. The present invention opens up the new application of silkworm microRNA, especially bmo-miR-277-5p, which can provide a new research direction for the study of the interaction mechanism between silkworm and nuclear polyhedrosis virus, and at the same time provide a new method for the diagnosis and treatment of silkworm nuclear polyhedrosis virus. Prevention provides new ideas.

Description

technical field [0001] The invention relates to silkworm microRNA, in particular to the application of a microRNA related to silkworm nuclear polyhedrosis virus infection. Background technique [0002] The silkworm nucleopolyhedrovirus (Bombyx mori nucleopolyhedrovirus, BmNPV) belongs to the insect Baculoviridae (Baculoviridae), the nucleopolyhedrovirus (Nucleopolyhedrovirus), the virion is rod-shaped, the genome size is about 128kb, and the code is about more than 100 Gene. The virus is highly contagious, difficult to control, frequent outbreaks, and has caused huge economic losses to the sericulture industry. In host insect cells, the replication of BmNPV DNA and gene expression is a cascade event that occurs in an orderly manner, and its infection process depends on the staged regulation and sequential expression of its genes, that is, the gene products of the previous stage directly or indirectly The trans action acts on the next stage of gene transcription. In the ea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 吴萍郭锡杰李龙陈涛
Owner JIANGSU UNIV OF SCI & TECH
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