Method for screening target gene of miR-29

A mir-29, target gene technology, applied in the field of molecular biology

Inactive Publication Date: 2018-07-20
河南省种牛遗传性能测定中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) miRNA perfectly matches and complements the target gene mRNA, degrades the target gene, thereby inhibiting gene expression

Method used

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  • Method for screening target gene of miR-29
  • Method for screening target gene of miR-29
  • Method for screening target gene of miR-29

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Experimental program
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Embodiment Construction

[0067] This embodiment provides a method for screening chi-miR-29 target genes using biological techniques, including the following steps:

[0068] 1) Using goat ovary tissue cDNA as template, in Taq DNA polymerase, buffer environment, Mg 2+ 1. In the presence of dNTPs, use the primer PTX3 to amplify under PCR conditions, and determine that the obtained PCR product is the sequence of the 3'UTR region of the PTX3 gene;

[0069] Described primer PTX3 is as follows (the underline is the restriction site of XhoI and NotI endonuclease):

[0070] Upstream primer F: 5'-CG CTCGAG TCCTGTCACATCCGGG-3';

[0071] Downstream primer R: 5'-AT GCGGCCGC TAGTTTTATAAAATATTTTTATTTACAGTAGAGCTTT-3'.

[0072] The condition of described PCR amplification is:

[0073] 15 μL reaction system, including 0.5 μL of DNA template, 7.5 μL of MasterMix, 0.5 μL of upstream primer F and downstream primer R of primer PTX3, add sterilized water to 15 μL;

[0074] The PCR reaction program using primer PTX3 i...

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Abstract

The invention discloses a method for screening a target gene of miR-29. The method uses a goat ovary DNA sequence as a template to amplify the 3' untranslated region (UTR) of a PTX3 gene under the condition of polymerase chain reaction (PCR) by using a PTX3 primer in a buffer environment in presence of Taq DNA polymerase, Mg<2+> and dNTPs; a dual-luciferase report system is constructed for detecting luciferase activity; the results show that the miR-29 reduces the luciferase activity, and the PTX3 is initially identified as the target gene of the miR-29; RT-qPCR and Western blot methods are used for verifying the impact of the miR-29 on the expression of the PTX3 gene at mRNA and protein levels. The results show that the miR-29 can inhibit the expression of the PTX3 gene at the mRNA and protein levels, so that the miR-29 is further determined to be bonded to PTX3'UTR, and the PTX3 is the target gene of the miR-29; therefore, a foundation is laid for study on the regulation of the miR-29 on ovary development and lambing traits of dairy goats.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to molecular cloning, RT-qPCR and Western blot detection technology, in particular to a method for screening miR-29 target gene, the method is mainly by constructing a dual-luciferase reporter carrier, using modern molecular biology The technology explores and verifies the target genes of miR-29. Background technique [0002] miRNA is a short (21-23 nucleotides), evolutionarily conserved, non-coding RNA molecule that has the function of post-transcriptional regulation of gene expression. It was first discovered in Caenorhabditis elegans, and it can Binding of the 3' non-coding (UTR) of the target gene inhibits translation of mRNA into protein. The regulation of miRNA is quite complex, each miRNA can regulate multiple target genes through different signaling pathways, and each target gene may be regulated by several miRNAs simultaneously. As an important regulatory factor, miRNA widely e...

Claims

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Application Information

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IPC IPC(8): C12Q1/66C12Q1/6851G01N33/68
CPCC12Q1/66C12Q1/6851G01N33/68C12Q2545/101C12Q2531/113C12Q2563/107
Inventor白跃宇谭旭信魏金销金喜新韩明鹏陈美凯郭利亚曹斌云王沛捷
Owner河南省种牛遗传性能测定中心