Methods for determining core promoter of miR-27a gene and internal transcription factor Myod binding site of core promoter

A mir-27a and core promoter technology, applied in the field of molecular genetics, can solve the problems of restricting the economic benefits of animal husbandry, low heritability of reproductive traits, long selection cycle, etc., and achieve the effect of high speed, high sensitivity and low cost

Active Publication Date: 2018-05-11
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Reproductive traits have defects such as low heritability, long selection cycle, and slow breeding progress, which have become the main reasons restricting the economic benefits of animal husbandry

Method used

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  • Methods for determining core promoter of miR-27a gene and internal transcription factor Myod binding site of core promoter
  • Methods for determining core promoter of miR-27a gene and internal transcription factor Myod binding site of core promoter
  • Methods for determining core promoter of miR-27a gene and internal transcription factor Myod binding site of core promoter

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 determines the core promoter of miR-27a gene

[0029] 1. Mouse blood DNA extraction and detection

[0030] 1.1 Use Biotech Cell / Tissue Genomic DNA Extraction Kit (spin column type) to extract the whole genomic DNA from mouse blood, and store the extracted DNA at -20°C for future use.

[0031] 1.2 Genomic DNA concentration measurement: Use NanoDrop 2000 DNA / RNA concentration analyzer to measure DNA concentration and OD value, dilute the DNA in the tube according to the measured concentration, aliquot 50ng / μL, and store at -20°C for later use .

[0032] 1.3 Genomic DNA quality detection: Take 3 μL DNA, add 4 μL sample buffer, mix well and spot on 1% agarose gel (EB staining solution), and spot 5 μL standard molecular weight DL 2000 DNA Marker as a reference. Electrophoresis was carried out under the condition of 15V / cm, and then the results were observed in the gel imaging system and photographed for preservation.

[0033] 2. Bioinformatics analysis of miR...

Embodiment 2

[0083] Embodiment 2, the method for determining the transcription factor Myod binding site in the core promoter of miR-27a gene

[0084] 1. Overexpression of transcription factor Myod regulates the fluorescence activity of miR-27a core promoter

[0085] The prepared Myod overexpression vector pc-Myod and the core promoter dual luciferase reporter vector pGL3-D7 of miR-27a were transiently co-transfected into CHO cells, and the dual luciferase activity was detected after 24 hours. The results showed that ( Figure 7 ), the overexpression of Myod can significantly increase the fluorescence activity of the core promoter of miR-27a, and Myod has the ability to promote the activity of miR-27a.

[0086] 2. Activity detection of transcription factor Myod binding site mutant luciferase reporter carrier

[0087] A site-directed mutagenesis technique was used to change the common part of the two Myod binding sites, and the sequences were respectively shown in SEQ ID NO: 21 in the sequenc...

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Abstract

The invention provides a method for determining a core promoter of a miR-27a gene. According to the method, 8 miR-27a promoter deletion fragments are amplified from genomic DNA in mouse blood, recombinant dual-luciferase expression vectors from pGL3-D1 to pGL3-D8 are constructed, after transfer-in of CHO cells, luciferase activity of the 8 deletion fragments is detected, and a core promoter area is obtained; a core area of miR-27a is identified, and a new promoter resource is provided for genetic engineering and molecular breeding. The invention further provides a method for determining an internal transcription factor Myod binding site of the core promoter of the miR-27a gene. By means of overexpression of the transcription factor Myod and site-specific mutagenesis of the transcription factor binding site in the core promoter area, a mutant type fluorescent expression vector is constructed, luciferase activity is detected, and the transcription factor binding site is determined; basisis provided for research of applying expression regulation of miR-27a to high-fertility molecular regulation mechanism of livestock.

Description

technical field [0001] The invention relates to the field of molecular genetics, in particular to a method for determining the core promoter of miR-27a gene and the binding site of transcription factor Myod in the core promoter of miR-27a gene. Background technique [0002] my country is a big country in animal husbandry, and animal husbandry plays an important role in the national economy. Reproduction and growth are the two main economic traits that determine the output of large-scale farming. In animal husbandry, the reproductive benefits of female animals account for a larger proportion of the total benefits. Reproductive traits have defects such as low heritability, long selection cycle, and slow breeding progress, which have become the main reasons for restricting the economic benefits of animal husbandry. Ovulation rate is an important component of litter size traits, and the growth and development of follicles in the ovary determines the number of ovulations. There...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/70C12N15/85C12Q1/66G01N21/64
CPCC12N15/66C12N15/70C12N15/85C12Q1/66G01N21/6428G01N21/6486
Inventor 陶虎陈明新熊琪张凤李晓锋索效军张年杨前平刘洋张鹤山田宏熊军波
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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