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Applications of transcription factor CREB in JHDM2A gene expression regulation and pig lipid deposition reduction

A technology of JHDM2A and transcription factors, applied in the field of cell signal transduction and epigenetics, can solve the problems that the functions have not been fully discovered and reported, and achieve the effects of reducing fat deposition, increasing lean meat percentage, and small fat cells

Inactive Publication Date: 2014-10-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the target genes that CREB can regulate and the functions involved have not been fully discovered and reported.

Method used

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  • Applications of transcription factor CREB in JHDM2A gene expression regulation and pig lipid deposition reduction
  • Applications of transcription factor CREB in JHDM2A gene expression regulation and pig lipid deposition reduction
  • Applications of transcription factor CREB in JHDM2A gene expression regulation and pig lipid deposition reduction

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Experimental program
Comparison scheme
Effect test

Embodiment 1W

[0030] Embodiment 1Western Blot analysis

[0031] The nuclear extracts of various porcine tissues and cells were prepared using the nucleus-cytoplasm extraction kit. SDS polyacrylamide gel electrophoresis was used to separate the protein after the protein concentration was determined by the BCA kit, and then the protein was transferred to the PVDF membrane and hybridized with the antibodies of JHDM2a, CREB, p-CREB (phosphorylated CREB) and His respectively, H3 As an internal reference for nucleoprotein.

[0032] Experimental results such as figure 1 Under B, figure 2 Under A, figure 2 B upper right, figure 2 C is shown on the upper right. The specific experimental steps are as follows:

[0033] SDS-PAGE:

[0034] (1) Assemble the electrophoresis device: ①Wash the glass plate with distilled water and dry it, and prepare two clean small beakers; ②Fix the glass plate on the glue-filling bracket.

[0035] (2) Prepare different concentrations of separating gels according...

Embodiment 2

[0058] Embodiment 2 Quantitative PCR (qRT-PCR)

[0059] Total RNA was extracted from porcine tissues and cells using TRIzon kit, and cDNA was synthesized by reverse transcriptase. Quantitative PCR primers were designed. The sequences of quantitative PCR primers are shown in Table 1. ABI7900HT quantitative PCR instrument and SYBR Green were used to detect the mRNA level of specific endogenous genes.

[0060] Table 1 Primers for quantitative PCR of porcine histone modifying enzymes

[0061]

[0062] Experimental results such as figure 1 A. figure 1 on B, figure 2 A is shown on the upper left.

[0063] The specific experimental steps are as follows:

[0064] Extraction of total RNA:

[0065] (1) DEPC-treated experimental equipment, autoclaved or dry-baked at 180°C for 8 hours.

[0066] (2) Take 30-50mg of porcine tissue, put it in 1ml TRlzol, and homogenize it for 1-5min.

[0067] (3) Place on ice for 5 minutes to completely dissolve the nucleoprotein complex, add 0.2...

Embodiment 3

[0078] Example 3 Constructing the recombinant vector

[0079] About 3kb of the pig and human JHDM2a promoter regions were amplified and connected to the multiple cloning site of the luciferase reporter gene vector pGL3-Basic vector (no promoter) to construct recombinant s / hJHDM2a-Luc (s - pig, h-human) luciferase reporter gene vector. Nhe Ⅰ and Xho Ⅰ restriction sites were added to both ends of the promoter region of JHDM2a, and the pGL3-Basic vector was cut with Nhe Ⅰ and Xho Ⅰ respectively, and then ligated into a recombinant vector. The s / hCRE1 and s / hCRE2 mutation primers were used to amplify the corresponding vectors to construct the mutant vectors Δs / hCRE1-Luc and Δs / hCRE2-Luc.

[0080] In order to obtain the pEGFP-CREBα / Δα-His recombinant plasmid, amplify the CREBα / Δα full-length cDNA sequence, add the His tag sequence, and clone it into the pEGFP-N1 vector between the NheI and HindIII endonuclease sites to form pEGFP-CREBα / Δα-His recombinant vector.

[0081] See Ta...

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Abstract

The invention relates to applications of a transcription factor CREB in JHDM2A gene expression regulation and pig lipid deposition reduction. By overexpression of CREB in pigs, or by improvement of the CREB phosphorylation level in pigs, or by promotion of combination of pig CBP protein and the phosphorylated CREB, JHDM2A gene expression in pigs is up-regulated, thus regulating lipid metabolism pathways from the epigenetics and reducing pig lipid deposition. Novel applications of the transcription factor CREB in the epigenetics are provided for the first time and the transcription factor CREB can be used for reducing pig lipid deposition, namely, expression of the histone demethylase JHDM2a is directly activated so that the histone demethylase JHDM2a can regulate the lipid metabolism pathways from the epigenetics, thus minifying lipid cells and increasing the meat factor. A novel candidate gene is provided for genetic breeding, meat quality improvement, and the like in the animal husbandry.

Description

technical field [0001] The invention relates to the field of cell signal transduction and epigenetics, in particular to the application of transcription factor CREB in regulating JHDM2A gene expression and reducing pig fat deposition. Background technique [0002] Clenbuterol (CLB), the chemical name is α-[(tert-butylamino)methyl]-4-amino-3,5-dichlorobenzyl alcohol hydrochloride, originally used as clenbuterol, ammonia asthma It was synthesized and used to prevent and treat diseases such as bronchial asthma, and was later used as a stimulant in competitive sports. The most widely used CLB is as a feed additive "lean meat" in animal husbandry, which can not only increase the growth rate of livestock and poultry, but also significantly reduce fat deposition and increase carcass lean meat rate. Can cause poisoning and is prohibited to use. These functions are all derived from the fact that clenbuterol hydrochloride is a β2-adrenergic receptor agonist, which can bind to adrene...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P3/04
Inventor 胡晓湘李旸李宁
Owner CHINA AGRI UNIV
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