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Application of MDFI in regulation of pig skeletal muscle growth and development

A technique for growth and development and skeletal muscle, applied in the field of molecular genetics, which can solve the problems of few research reports

Inactive Publication Date: 2015-03-04
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]In recent years, studies have shown that many miRNAs are involved in the regulation of proliferation and differentiation of skeletal muscle, among which miR-27b is a regulator that plays a role in the process of cell proliferation and differentiation A new miRNA, miR-27b is specifically expressed in different tissues of pigs, and is involved in the growth and development of mammalian muscle, fat metabolism and proliferation and differentiation of osteoblast stem cells, MDFI (MyoD Family Inhibitor) gene, also known as I-mfa (Inhibitor of MyoD family a) gene, is a negative transcriptional regulator, I-mfa affects skeletal muscle growth and development, Trophoblast and chondrogenic differentiation play an important regulatory role; there are very few reports on porcine MDFI gene in the prior art, and its function and molecular mechanism regulating porcine skeletal myogenesis have not been reported

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  • Application of MDFI in regulation of pig skeletal muscle growth and development
  • Application of MDFI in regulation of pig skeletal muscle growth and development
  • Application of MDFI in regulation of pig skeletal muscle growth and development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Culture and Purification of Pig Skeletal Muscle Satellite Cells

[0042] (1) Take 1-day-old healthy purebred Changbai boars, kill them by bleeding from the jugular artery and vein, disinfect the sampling sites with 75% alcohol, and quickly transfer them to the ultra-clean workbench;

[0043] (2) Under sterile conditions, cut the skeletal muscle sample from the gastrocnemius muscle of the hindlimb, put it in a petri dish, wash it with 1% double-antibody PBS three times, cut it into pieces, and remove the visible blood vessels, fascia, tendon and connective tissue. cut into l mm 3 the size of the tissue block;

[0044] (3) Move the tissue block into 75 cm 2 Add 2 times the volume of 0.1% collagenase II digestion solution to a glass Karl Fischer bottle, place it in a constant temperature shaking water bath at 37°C for digestion for 2-2.5 h, and oscillate at 70-100 r / min;

[0045] (4) Add the same amount of stop solution as collagenase II to stop the digesti...

Embodiment 2

[0052] Example 2 Fluorescent quantitative PCR detection of relative gene expression in porcine skeletal muscle satellite cells

[0053] The relative expression levels of MDFI, MyoD, myogenin, Myf5, Myf6, CCNB1, CCND1, CCND2, and P21 genes in porcine skeletal muscle satellite cells were amplified with a Real-Time PCR instrument. The primer sequences are shown in Table 1. Experiments were repeated three times. The reaction system (20 μL) is as follows: upstream primer: 0.4 μL; downstream primer: 0.4 μL; SsoFast EvaGreen super mix with low ROX: 10 μL; RNase / DNase-free ddH 2 O: 5.1 μL; cDNA template: 2 μL. The reaction program is: 95°C / 30s→95°C / 5s→58°C / 30s→65°C / 5s, 40 cycles. The relative expression of genes was analyzed by the method of comparative Ct value.

[0054] △Ct = Ct (target gene) - Ct (internal reference gene)

[0055] △△Ct=△Ct (experimental group)-△Ct (control group)

[0056] Relative expression % = 2 -△△ Ct

[0057] Table ...

Embodiment 3

[0059] Example 3 MDFI As miR-27b regulates the target gene in porcine skeletal muscle growth and development

[0060] (1) Bioinformatics predicts that MDFI is the target gene of miR-27b

[0061] Using the miRNA information provided in the mirBase database, compare the homology of miR-27b, analyze molecular bioinformatics, and predict target genes through online http: / / www.microrna.org / microrna / home.do. Homology comparison of genes is carried out, and miRNAs acting on target genes are predicted through online http: / / www.microrna.org / microrna / home.do. Through bioinformatics prediction, it was found that MDFI may be the target gene of miR-27b ( figure 1 ).

[0062] (2) Dual-luciferase reporter carrier verifies that MDFI is the target gene of miR-27b

[0063] ①Suspension transfection: The constructed MDFI dual reporter vectors pmirGLO-MDFI-3'UTR and pmirGLO-MDFI-3'UT-mut were transfected with the miR-27b fragment into 293FT cells, which are semi-adherent cells , i...

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Abstract

The present invention relates to the technical field of molecular genetics, and specifically discloses an application of the pig MDFI gene in regulation of pig skeletal muscle growth and development. According to the present invention, MDFI is overexpressed and interfered in pig skeletal muscle satellite cells, results of cell count and EdU fluorescence detection show that the MDFI can significantly promote proliferation of the pig skeletal muscle satellite cells, and the results of detection on the expression levels of MyoD, myogenin and MyHC expression show that the MDFI can inhibit differentiation of the pig skeletal muscle satellite cells; and with qRT-PCR and Western Blot methods, the regulation effect on the MRFs and the associated cyclin is detected after MDFI is overexpressed and interfered, and the regulation mechanism of the MDFI on the proliferation and differentiation effect of the pig skeletal muscle satellite cells is primarily explored so as to provide the theoretical basis for further research of the MDFI on the pig skeletal muscle growth and development at the body level and provide the reference for improvement of the pork production breeding work.

Description

technical field [0001] The present invention relates to the technical field of molecular genetics, more specifically, to MDFI Application in regulating the growth and development of porcine skeletal muscle. Background technique [0002] my country is a big pig-producing country, and pork consumption accounts for a large proportion of meat consumption. Improving the amount and quality of pig meat and meeting the national demand for pork is an important goal and arduous task for pig genetics and breeding workers to strive for for a long time. [0003] Earlier studies have shown that muscle fibers are the main building blocks of muscle, and that an animal's muscle mass is largely determined by the number and size of muscle fibers in the muscle. The number and size of muscle fibers in the muscles of meat-producing animals also have important effects on meat quality. A large number of studies have shown that meat with a high number of muscle fibers and a medium diameter in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 王翀徐建段军莉顾婷潘志成焦义仁周晓嘉覃立立徐路路陈叶
Owner SOUTH CHINA AGRI UNIV
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