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Silkworm microsporidia milRNAs and application thereof

A technology for microsporidia and silkworm, which can be applied to DNA/RNA fragments, recombinant DNA technology, medical preparations containing active ingredients, etc. Effect

Active Publication Date: 2021-09-17
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have shown that non-coding RNAs of microsporidia may be involved in host cell proliferation, apoptosis, and immune processes, and play an important role in the interaction between pathogens and hosts, but the regulation mechanism of non-coding RNAs of microsporidia not yet clear

Method used

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  • Silkworm microsporidia milRNAs and application thereof
  • Silkworm microsporidia milRNAs and application thereof
  • Silkworm microsporidia milRNAs and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1, Prediction and Analysis of Bombyx mori Microsporidia milRNAs

[0033]The proliferation characteristics of N. silkworm were analyzed by immunofluorescence and qPCR experiments, and the life history of N. silkworm was observed by immunofluorescence. It was found that when N. silkworm was infected for 24 hours, a small part of the spores had been ejected from the pole silk. The sporoplasm is injected into the host cytoplasm, and a small amount of sporoplasm can be found in the host cell; at 48 hours after infection, the sporoplasm of the spores becomes larger and develops into schizonts through binary or multiple divisions, which can be released in the host cell. The phenomenon of aggregation and expansion of sporoplasm was found; after 72 hours of infection, the mother spores formed by the fusion of schizonts into one formed sporocytes by binary division. At this time, it can also be observed in the host cells sporoplasm; by the time of infection for 96 hours, ...

Embodiment 2

[0083] Example 2, Identification and Functional Analysis of Bombyx mori Microsporidia milRNA-8

[0084] In order to explore the effect of milRNAs on the proliferation of Nos. silkworm, this example inserts target bands containing 10 milRNAs into puro-OpIE2 prm - In the mCherry-PA vector, the recombinant plasmid puro-OpIE2 was obtained by using the restriction enzyme Asc1 prm -mCherry-U6-milRNA( Figure 4 ), the results of further sequencing showed that 7 overexpression vectors were successfully constructed, and all of them were found to be expressed normally by fluorescence microscope observation. The results are as follows Figure 4 shown.

[0085] After the seven overexpression vectors were successfully constructed, they were transfected to BmE-SWU1 cells at the cellular level. After the overexpression vectors were stably expressed for 48 hours, N. silkworm was added. When the host cells were infected by N. silkworm for 48 hours, At this time, No. silkworm is in the stage...

Embodiment 3

[0088] Example 3, Screening and Identification of Bombyx mori Microsporidia milRNA-8 Target Gene

[0089] In order to identify the target gene of N. silkworm milRNA-8, this paper uses the target gene prediction software miRanda to make a preliminary prediction, and the miRanda threshold is set to score ≥ 140. The results showed that milRNA-8 predicted a total of 569 candidate target genes in the host, and selected 64 candidate target genes whose expression level difference between the Control group and the Nb group was ≥ 2, and performed KEGG functional enrichment analysis on these genes , 8 candidate target genes were found to be enriched in metabolism, neuroactive receptor-ligand pathway and peroxisome pathway. Corresponding quantitative primers were designed for 8 candidate target genes, and the sequences were: BGIBMGA002029-F: 5'-aagtatactgaaggccgagatg-3' (SEQ ID No.37);

[0090] BGIBMGA002029-R: 5'-tccatcaacacgtctcataaca (SEQ ID No. 38);

[0091] BGIBMGA009927-F: 5'-ctc...

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Abstract

The invention discloses silkworm microsporidia milRNAs and application thereof, research finds that milRNA-4, milRNA-8, milRNA-10 and milRNA-12 have a promoting effect on the proliferation of microsporidia, milRNA-1, milRNA-3 and milRNA-11 have an inhibiting effect on the proliferation of microsporidia, and the milRNA-8 has the most obvious promoting effect on the proliferation of microsporidia, so that the milRNA-8 can be used for regulating the proliferation of microsporidia. The research also finds that the target gene of miRNAs is BmPEX16, and the copy number of the overexpressed silkworm microsporidia and the protein expression quantity of the NbPTP2 are both reduced, which indicates that the proliferation of the silkworm microsporidia is inhibited after the overexpression of the BmPEX16; after the BmPEX16 is knocked out, the proliferation of the silkworm microsporidia is promoted, so that the proliferation of the silkworm microsporidia can be regulated by regulating the expression of the BmPEX16 gene, and a new thought is provided for analyzing the prevention and control of the silkworm microsporidia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the microsporidia milRNAs of the silkworm, and also relates to the application of the milRNAs. Background technique [0002] miRNA is the most well-studied type of endogenous non-coding small RNA among non-coding RNAs. It is about 22 nt in length and is highly conserved in evolution. So far, about 30,000 miRNAs have been discovered in animals, plants and viruses. miRNA can effectively regulate target genes through its mechanism of action. It is a key regulator of many life activities such as cell growth, differentiation and apoptosis, and is closely related to the pathogenic mechanism of various diseases. However, there are still many important miRNAs that are still unknown, and there is an urgent need for in-depth research on the expression characteristics and mechanisms of miRNAs. With the research on miRNAs, it will not only help to understand the life processes of cell reproduct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7105A61K45/00A61P33/02
CPCC12N15/113A61K31/7105A61K45/00A61P33/02C12N2310/141Y02A50/30
Inventor 董战旗潘敏慧郑宁胡丛武鲁成陈鹏
Owner SOUTHWEST UNIVERSITY
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