Identification method of regulatory element for regulating transcriptional activity of FATP1 gene promoter

A technology of regulatory elements and promoters, applied in biochemical equipment and methods, recombinant DNA technology, measurement/inspection of microorganisms, etc., can solve problems such as the transcriptional regulation mechanism of bovine FATP1 gene that has not been reported

Inactive Publication Date: 2018-08-10
GANSU AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although FATP1 gene expression affects the composition of unsaturated fatty acids in bovine skeleta

Method used

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  • Identification method of regulatory element for regulating transcriptional activity of FATP1 gene promoter
  • Identification method of regulatory element for regulating transcriptional activity of FATP1 gene promoter
  • Identification method of regulatory element for regulating transcriptional activity of FATP1 gene promoter

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Embodiment 1

[0028] 1. Analysis of luciferase activity of FATP1 gene promoter

[0029] 1. Design of promoter primers

[0030] According to the 5′UTR sequence of the FATP1 gene in GeneBank, the transcription start site identified by 5′-RACE was +1, and gene-specific primers were designed by Clone Manage software:

[0031] The upstream primer is Forward Primer F1:5′-CTACTGTGGTGGGCACTTG-3′

[0032] The downstream primer is Reverse Primer R1: 5'-TTGTTCCCTGGCTGACCTGGAG-3'.

[0033] Bovine genomic DNA was used as a template for PCR amplification, and the target fragment was 2046bp in total from the promoter sequence -1856 to +190. Add 1.2 μL (100ng) DNA template, 0.4 μL KOD-Plus-Ver 2.0 (1U / μL), 2 μL 10×PCR Buffer, 2 μL dNTP (2 mmol / L), 0.8 μL MgSO in 200 μL PCR tube 4 (25mol / L), 0.6 μL ForwardPrimer F1 (10 μM), 0.6 μL Reverse Primer R1 (10 μM), 12.4 μL ddH 2 O, a total of 20 μL, vortexed and centrifuged, placed in a PCR instrument for reaction, the reaction conditions are as follows: pre-de...

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Abstract

The invention provides an identification method of a regulatory element for regulating the transcriptional activity of an FATP1 gene promoter. The method includes the steps of: cloning an FATP1 gene promoter sequence; constructing a series of deletion FATP1 gene 5' end promoter fragment luciferase reporter vectors; transfecting cells with the luciferase reporter vectors respectively for luciferaseactivity analysis, and determining the activity area of the FATP1 gene core promoter; and analyzing a transcription factor binding site in the activity area, conducting site-directed mutagenesis on the transcription factor binding site, and according to the activity change condition of the FATP1 gene promoter after mutagenesis, determining the regulatory element for regulating the transcriptionalactivity of the FATP1 gene promoter. The method provided by the invention identifies the regulatory element for regulating the transcriptional activity of the FATP1 gene promoter, and finally determines the KLF15 transcription factor binding site as the regulatory element for regulating the transcriptional activity of the FATP1 gene promoter.

Description

technical field [0001] The present invention relates to methods for identifying regulatory elements that regulate the transcriptional activity of the FATP1 gene promoter. Background technique [0002] Fatty acid transport protein 1 (FATP1), as an integral membrane protein that promotes the uptake of long-chain fatty acids, is involved in the regulation of oleic acid synthesis and is one of the target genes for improving beef quality. Oleic acid, as palmitic acid in the main monounsaturated fatty acid, accounts for 33% of beef lipids, and has an inhibitory effect on serum cholesterol concentration. Insulin can induce the ectopic expression of FATP1 around the nucleus and in the cell, increasing the uptake of long-chain fatty acids (LCFA). The FATP1 gene is highly expressed in tissues with high fatty acid metabolism such as heart, skeletal muscle, and fat. The increase of FATP1 gene expression showed the same trend as the increase of oleic acid intake during the differentiat...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/85C12Q1/26
CPCC12N15/52C12N15/85C12Q1/26
Inventor 赵志东昝林森胡江刘秀
Owner GANSU AGRI UNIV
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