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Method and application thereof for detecting whether mRNA to be detected contains miRNA target site

A technology of target sites and DNA molecules, applied in the biological field, can solve the problems of not being observed, unable to judge that the interaction between miRNA and target genes is not good, and the fluorescence cannot be observed.

Active Publication Date: 2021-08-24
BEIJING UNIV OF AGRI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, as a negative screening method, this method has many problems
For example, when the infection efficiency is not high, the fluorescence cannot be observed in the same way; in addition, if the fluorescence phenomenon is observed in part of the plant tissue during microscope inspection, while the other part is not observed, it will not be possible to judge the area without fluorescence. The interaction between miRNA and target gene is still not good

Method used

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  • Method and application thereof for detecting whether mRNA to be detected contains miRNA target site
  • Method and application thereof for detecting whether mRNA to be detected contains miRNA target site
  • Method and application thereof for detecting whether mRNA to be detected contains miRNA target site

Examples

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Embodiment 1

[0075] Example 1. Detecting whether the mRNA to be tested contains miRNA target sites

[0076] Next, miR393a derived from Arabidopsis thaliana is used as the target miRNA, and the confirmed target gene At3g26810 (AFB2 gene) of miR393a (GenBank Accession Number NM_113593.3, Update Date is 20-MAR-2017) is used as the target gene to be confirmed The 4CL gene (GenBank AccessionNumber NM_001325625.1, Update Date is 23-APR-2017) which is not a target gene of miR393a is taken as an example of a non-target gene of miR393a to illustrate the present invention. The cleavage mechanism of miR393a and its target gene AFB2 gene can be found in the following literature: Xiu-Jie Wang et al. Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets [J]. Genome Biol. 2004;

[0077] 1. Utilize the method of the present invention to detect whether the mRNA to be tested contains miRNA target sites

[0078] 1. Construction of target miRNA expression vector

[0079] wit...

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Abstract

The invention discloses a method for detecting whether the mRNA to be tested contains a miRNA target site and an application thereof. The method includes: 1) providing the target miRNA expression vector and the mRNA expression vector to be tested; the mRNA expression vector to be tested can transcribe the mRNA molecule named as initiation codon region-tested mRNA-kozak-reporter gene mRNA in biological cells , the mRNA molecule is connected by the start codon region, the segment of the mRNA-kozak to be tested downstream of the start codon region and the reporter gene mRNA located at the downstream of the mRNA-kozak to be tested; 2) constructing and introducing the target miRNA expression 3) detecting the expression of the reporter gene in the recombinant biological cells, and determining whether the mRNA to be tested contains the target site of the miRNA of interest. The present invention can be used for screening miRNA target genes.

Description

technical field [0001] The invention relates to a method for detecting whether an mRNA to be tested contains a miRNA target site and an application thereof in the field of biotechnology. Background technique [0002] microRNA (miRNA) is a kind of non-coding small molecule RNA with a length of 19-24nt. As a post-transcriptional regulator, it plays a very important role in the regulation of gene expression in eukaryotes. At the post-transcriptional level, miRNA recognizes target genes by complementing the sequence of mRNAs, and causes the degradation of the target gene or inhibits its translation, and finally achieves the purpose of inhibiting the expression of specific genes. Most of the miRNAs in plants are completely or almost completely complementary to the target gene mRNA, and cleavage occurs between the 10th and 11th nucleotides of the complementary site to regulate the expression of the target gene. [0003] In view of the important role of miRNA, a variety of experim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/65C12N15/63C12N15/11
Inventor 杨明峰何炫程马兰青吕鹤书刘灿马伯宁项晨韦航涛
Owner BEIJING UNIV OF AGRI
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