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Regulation factor of targeted FAS (fatty acid synthetase) and application thereof

An adipose tissue and molecular marker technology, applied in the field of non-coding nucleic acids, can solve the problems of low lean muscle mass, low intramuscular fat content, and poor meat quality.

Active Publication Date: 2014-12-24
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dorset sheep (Dorset) is a meat sheep in Australia and New Zealand. It has the characteristics of fast growth and development, high lean meat rate, and low intramuscular fat content, but the meat quality is not good.

Method used

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  • Regulation factor of targeted FAS (fatty acid synthetase) and application thereof
  • Regulation factor of targeted FAS (fatty acid synthetase) and application thereof
  • Regulation factor of targeted FAS (fatty acid synthetase) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 High-throughput transcriptome sequencing to find microRNAs with different expression profiles in adipose tissue of different breeds of sheep

[0057] Abdominal fat tissues were collected from 5 cases of Dorset sheep and 5 cases of HanSheep sheep. After the tissue samples were taken out, they were divided into small pieces and quickly frozen in liquid nitrogen for 30 seconds, and then stored in a -70°C refrigerator. Then it was sent to a sequencing company at low temperature for small RNA sequencing.

[0058] Use Fast-QC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) software to evaluate the overall quality of sequencing data, including base quality value distribution, quality value position distribution, and GC content , PCRduplication content, kmer frequency, etc.

[0059] The counts of the mature small RNA analysis results are added to the deletion of less than 10, and then the difference is screened using the internationally recognized algorit...

Embodiment 2

[0060] Example 2 Detection of miR-4749-5 expression in sheep adipose tissue by Real-time PCR

[0061] Reagent:

[0062] Reverse transcription kit: SG One-Step miRNA RT Kit, #33-30120, SinoGene

[0063] PCR Mix: 2×SG PCR MasterMix, #33-10201, SinoGene

[0064] qPCR reagents: 2×SG Green qPCR Mix(with ROX), #22-10102, SinoGene

[0065] Removal of RNase for related experimental items:

[0066] ① Rinse and soak all glassware with DEPC before use, 120°C for 20 minutes under high pressure, and 180°C for more than 2 hours.

[0067] ②Plastic utensils (such as: EP tubes / tips) need to be soaked in 0.1% DEPC water overnight before use, and then the liquid is controlled to dry, 120°C and high pressure for 20 minutes, and dried in an oven for later use.

[0068] 45 cases of Dorsetp sheep fat tissue and 45 cases of HanSheep sheep fat tissue were selected, and RNA was randomly selected after encoding.

[0069] miRNA extraction:

[0070] (1) Take out the frozen sheep fat tissues from liq...

Embodiment 3

[0098] Embodiment 3miRNA suppresses FAS gene expression

[0099] 1. Plasmid construction and miRNA synthesis

[0100] The carrier plasmid is pcDNA3.1, GFP is connected to the carrier through EcoRI and NotI, and then the 3'UTR of the FAS gene is connected to the carrier through restriction sites XhoI and XbaI to form the vector pcDNA3.1-GFP-3'UTR. The 3'UTR sequence refers to the sequence from the first base after the stop codon of the FAS gene to the last base of the mRNA. Send the company to synthesize miR-4749-5, and dilute the miRNAmimics synthesized by the company for use.

[0101] 2. GFP reporter gene experiment

[0102] The day before transfection, approximately 2.5 × 10 per well 5 Mouse fibroblasts were inoculated in 24-well plates at a density of cells / mL, and cultured in DMEM containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultivate the cells overnight at saturated humidity until 70%-80% confluence, aspirate the cell culture medium, wash the cells twice with PB...

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Abstract

The invention relates to a regulator gene expressed non-coding nucleic acid, in particular to a regulation factor of targeted FAS (fatty acid synthetase) and an application thereof. A targeted FAS gene expressed microRNA (ribonucleic acid) is screened from adipose tissue of sheep and named MiR-4749-5. Further experimental results of the molecular biology show that the MiR-4749-5 can suppress expression of the FAS gene. The invention further provides a detection kit for detecting the microRNA. The regulation factor and the detection kit have important practical application value in molecular marker assisted breeding of sheep.

Description

technical field [0001] The invention relates to a non-coding nucleic acid for regulating gene expression, in particular to a regulatory factor targeting fatty acid synthase and application thereof. Background technique [0002] microRNA (miRNA) is one of the hotspots of biological researchers. miRNA is a type of endogenous non-coding small molecule single-stranded RNA with a length of 19-24 nucleotides (nt), which is highly conserved during evolution and can cause specific base pairing with target gene mRNA. The degradation of target gene mRNA or inhibition of its translation broadly negatively regulates the expression of target genes. Although tens of thousands of miRNAs have been discovered in animals, plants, and viruses since they were reported in 1993, the functions of many miRNAs are still unclear because the number of miRNA target genes that have been identified is very small, and miRNA target genes It is very difficult to predict and identify miRNAs, so it is of gr...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12Q1/68
CPCC12Q1/6876C12N15/113C12N2310/141C12Q2600/124C12Q2600/158C12Q2600/178
Inventor 苗向阳
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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