Identification method of pig fat deposition related PNPLA2 mRNA m6A methylation single locus and function application

An identification method and fat deposition technology, applied in the field of molecular biology, can solve problems such as poor pork quality, affecting consumers' health, susceptibility to diseases and reproductive performance, etc.

Active Publication Date: 2017-11-24
ZHEJIANG QINGLIAN FOOD
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AI Technical Summary

Problems solved by technology

Excessive pig fat deposition not only reduces nutrient utilization efficiency, but also excessive body fat deposition will directly affect the health of consumers, including diabetes and cardiovascular and other metabolic diseases that are easily caused by obesity; while too little pig body fat deposition will cause A series of problems such as poor pork quality, low immunity, susceptibility to diseases and reduced reproductive performance

Method used

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  • Identification method of pig fat deposition related PNPLA2 mRNA m6A methylation single locus and function application
  • Identification method of pig fat deposition related PNPLA2 mRNA m6A methylation single locus and function application
  • Identification method of pig fat deposition related PNPLA2 mRNA m6A methylation single locus and function application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Landrace pig and Jinhua pig PNPLA2 mRNA m 6 A difference in methylation levels

[0040]The RNA used in the experiment came from the adipose tissue of Landrace pigs and Jinhua pigs, and the specific operations were as follows:

[0041] 1. Obtain tissue total RNA

[0042] Get pig adipose tissue sample, utilize Trizol method to extract total RNA (conventional method), specific method is:

[0043] 1) Take out the muscle tissue sample frozen in liquid nitrogen, take 50-100mg and grind it into powder in liquid nitrogen, and place it in an RNase-free 1.5ml eppendorf tube;

[0044] 2) Add 1ml Trizol and shake vigorously to a homogeneous solution;

[0045] 3) After adding 200ul chloroform, shake vigorously to a homogeneous solution, centrifuge at 4C, 12000g for 15min;

[0046] 4) Take the supernatant to another RNase-free 1.5ml eppendorf tube, add the same volume of isopropanol, mix gently, incubate at room temperature for 10min, 4C, centrifuge at 12000g for 10...

Embodiment 2

[0126] Embodiment 2: Change the PNPLA2 mRNA m of pig 6 A single point mutation in methylation level

[0127]

[0128]

[0129] (Explanation: Because the A site is located at the second position of the codon, in order not to change the amino acid sequence, only the third position of the codon can be mutated to achieve synonymous mutation; and m 6 The A of A is located in the conserved sequence GGACU. The C behind A is very important for the formation of methylation on A. The mutation of C will change the methylation efficiency of A, resulting in a decrease in methylation, thereby changing the methylation level. the goal of. )

[0130] Cloning the sequence of porcine PNPLA2 gene (NM_001098605.1) and the mutation sequence of C756 mutation to T756 and C768 mutation to T768, adding FLAG sequence (5'-GACTACAAGGACGATGATGACAAG-3') at the N-terminus, and cloning it into Pcdna3.1(+) expression plasmid The HindIII and BamHI sites were used to obtain PNPLA2-WT and PNPLA2-MUT plas...

Embodiment 3

[0131] Example 3: PNPLA2 mRNA m after point mutation 6 A change in methylation level

[0132] 1. Primer design:

[0133] Fluorescent quantitative PCR primers were designed upstream and downstream of the mutation site, and were synthesized by Shanghai Sangon Biological Co., Ltd., as follows:

[0134]

[0135] 2. Isolation and Culture of Porcine Preadipocytes

[0136] The acquisition of porcine preadipocytes refers to the method of Ding et al. Soak and rinse with resistant PBS to remove visible blood vessels and muscles. Use scissors to cut into tissue pieces of uniform size, place them in a sterile bottle, digest with the digestive solution in a 37°C water-bath shaking pot for 1.0h, neutralize the digestive solution with an equal volume of complete medium after digestion, and pass the digest once 200-mesh and 300-mesh nylon sieves, collect the filtrate, and centrifuge at 1500rpm / min for 10min. Discard the supernatant, add red blood cell lysate, pipette evenly, let stand...

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Abstract

The invention provides an identification method of pig fat deposition related PNPLA2 mRNA m6A methylation single locus. According to m6A-seq sequencing, m6A methylation locus exists in a fragment of about 100nt, and method is characterized in that confirmation of a m6A single locus position of a PNPLA2 gene is carried out according to a RRACH(R=G,A;H=A,C,T) sequence with highly conserved methylation. Bioinformatics analysis is used, and according to a conserved sequence of m6A, the concrete locus of m6A is identified; the invention provides a primer sequence and a concrete method for identification of methylation level of PNPLA2 mRNA m6A in pigs; gene single locus mutation changes PNPLA2 m6A level proves that the PNPLA2 m6A locus has function for influencing fat deposition.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a single gene mRNA m 6 A methylation site identification, function and application, specifically related to PNPLA2 mRNA m6A methylation site identification, function and application. Background technique [0002] The rational deposition and regulation of pig fat has always been the focus of animal husbandry. Excessive pig fat deposition not only reduces nutrient utilization efficiency, but also excessive body fat deposition will directly affect the health of consumers, including diabetes and cardiovascular and other metabolic diseases that are easily caused by obesity; while too little pig body fat deposition will cause There are a series of problems such as poor pork quality, low immunity, susceptibility to diseases and decreased reproductive performance. Most local pig breeds in China are high-fat, called fat pigs; imported pig breeds have a high lean meat rate, called lean meat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/85G01N33/68
CPCC12N15/85C12Q1/6851C12Q1/6869G01N33/68C12Q2535/122C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 汪以真王新霞江芹吴睿帆姚永曦蔡旻刘卿
Owner ZHEJIANG QINGLIAN FOOD
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