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Interaction identification and application of bovine skeletal muscle differentiation related DNA methylase TET1 and m6A methylase METTL3

A TET1-WT, methylation technology, applied in the field of molecular biology, can solve the problem that the relationship between epigenetic modifications is rarely found.

Active Publication Date: 2022-05-13
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, regarding m 6 The function, mechanism and potential target genes of A modification in bovine skeletal myogenesis have not yet been reported. 6 The correlation between A methylation and other epigenetic modifications is also rarely found

Method used

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  • Interaction identification and application of bovine skeletal muscle differentiation related DNA methylase TET1 and m6A methylase METTL3
  • Interaction identification and application of bovine skeletal muscle differentiation related DNA methylase TET1 and m6A methylase METTL3
  • Interaction identification and application of bovine skeletal muscle differentiation related DNA methylase TET1 and m6A methylase METTL3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: TET1 mRNA m during differentiation of bovine skeletal muscle myoblasts 6 A difference in methylation levels

[0070] The test materials were Qinchuan cattle myoblasts before and after differentiation m 6 The data after A-seq sequencing and the remaining m 6 A mRNA samples after immunoprecipitation.

[0071] The specific operation is as follows:

[0072] 1.m 6 Analysis and visualization of A-seq sequencing data

[0073] (1) Further bioinformatics analysis was performed on the sequencing data, and the m 6 A peak map visualization, indicating the m of TET1 mRNA before and after myogenic differentiation 6 A methylation position and m 6 There are differences in the methylation level of A ( figure 1 left picture);

[0074] (2) According to the m of TET1 in the sequencing results 6 A peak sequence, at m 6 A modification site prediction website (http: / / www.cuilab.cn / sramp) to determine the m of TET1 mRNA 6 The A site is located at the 3999th nucleotide of...

Embodiment 2

[0081] Example 2: Changing the m of TET1 mRNA 6 A method of methylation modification, that is, for m 6 A site for synonymous mutation;

[0082] The nucleotide sequence of the coding region of the bovine TET1 gene is as follows:

[0083] atgtctcgat ctcgccacgc aagaccttcc aaattagtca ggagggaaga tctaaacaag 60

[0084] aagaaaaaca accaactgaa gaagggatct aagccagcca acaaaaatgt ggcgactgtc 120

[0085] aagactgtga gccctggaaa attgaagcaa ttcgtgcaag aaagagatgt taagaaaaaa 180

[0086] acagaaccca aactgactgt gccagtcaga agccttctga caagagctgg agcagcacga 240

[0087] atgaatttgg ataggaccga ggttcttttt cagaacccag agtccttaac ttgcaatggg 300

[0088] tttacaatgg ctctgcgaag cacctctctt agcagacgac tctcccaacc cccaatggtc 360

[0089] atagccagac ccaagaaagt tccaccacct aagaatctag gaaagcaaca tgagtgtaat 420

[0090] tataaggtac ttacagacat gggagtaaag cactcagaaa atgattcagt cccaatgcaa 480

[0091] gaccccccag tcccccctga catagagaag ctaattggca tacaaaatgc atctttcatt 540

[0092] aaagacgaga gccaagagac aacccagtct tg...

Embodiment 3

[0191] Example 3: TET1 mRNA m 6 The effect of A methylation site mutation on the stability of TET1 mRNA

[0192] (1) Cloning the coding region sequence of bovine TET1 gene ((ENSBTAT00000086206.1) and the sequence after synonymous point mutation into pcDNA3.1-3xFlag-EGFP-C mammalian expression vector to obtain wild type (MEF2C-WT) and mutant (MEF2C-MUT) expression plasmids;

[0193] (2) When the Qinchuan bovine skeletal muscle myoblasts grow to a density of 80-90%, subculture the cells into a 6-well plate;

[0194] (3) When the myoblast grows to 80% density, use the Lipofectamine 3000 kit (Invitrogen) to transfect the MEF2C-WT and MEF2C-MUT expression plasmids into the myoblast, mainly refer to the instructions of the Lipofectamine 3000 kit, the specific operation as follows:

[0195] 1) For each well of cells, add 5 μl of p3000 reagent and 2500ng of plasmid DNA to 120 μl of OPTI-MEMI medium;

[0196] 2) For each well of cells, add 5 μl Lipofectamine 3000 reagent to 120 μl ...

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Abstract

The invention provides a method for identifying an m6A methylation site on mRNA of DNA demethylase TET1 related to bovine skeletal muscle differentiation, provides a primer sequence and a method for identifying the m6A methylation level of TET1, and identifies the change of the m6A level of TET1 in myogenic differentiation according to m6A-seq sequencing and m6A-IP-qPCR; the m6A methylation level of TET1 is changed through synonymous mutation of the m6A site, and it is proved that m6A methylation of TET1 has the function of influencing skeletal muscle differentiation; the invention provides a method for detecting the regulation and control of TET1 by m6A methylation modification. A dual luciferase report system and an RIP experiment prove that the expression of TET1 is regulated and controlled by METTL3-m6A-YTHDF1; a method for detecting the influence of DNA methylation on m6A methylation modification is provided, and the interaction of DNA methylation and RNA methylation in bovine skeletal muscle differentiation is proved. Reference is provided for functional research of m6A methylation modification regulation and control of muscle growth and development, a new interaction mechanism in skeletal muscle development is identified, and a new molecular marker is provided for directional breeding of beef cattle.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to the identification, function and application of the interaction between DNA methylation modification and RNA methylation modification, in particular to bovine skeletal muscle differentiation-related DNA methylation enzymes TET1 and m 6 Interaction identification, function and application of A methyltransferase METTL3. Background technique [0002] Studies have shown that the meat production and meat quality of livestock and poultry are inseparable from the growth and development of skeletal muscle and its genetic characteristics. On the one hand, the number and diameter of skeletal muscle fibers are the key factors that determine the meat production of animals; The intramuscular fat content in the muscle, the type and quantity of protein in the muscle fiber are closely related to the tenderness of the muscle and affect the quality of the meat. In addition, skeletal muscl...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12Q1/6809C12Q1/6869C12N15/10G01N33/68C12Q1/66
CPCC12Q1/6888C12Q1/6858C12Q1/6809C12Q1/6869C12N15/102G01N33/68C12Q1/66C12Q2600/124C12Q2600/154C12Q2531/113C12Q2563/107C12Q2523/125
Inventor 杨昕冉昝林森
Owner NORTHWEST A & F UNIV
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