Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (21): 4685-4693.doi: 10.3864/j.issn.0578-1752.2021.21.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Interference in TP53INP2 Gene Inhibits the Differentiation of Bovine Myoblasts

DU JiaWei1(),DU XinZe1,YANG XinRan1,SONG GuiBing1,ZHAO Hui1,ZAN LinSen1,2,WANG HongBao1,2,*()   

  1. 1College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi;
    2National Beef Cattle Improvement Center, Yangling 712100, Shaanxi
  • Received:2020-09-25 Accepted:2021-03-12 Online:2021-11-01 Published:2021-11-09
  • Contact: HongBao WANG E-mail:614502306@qq.com;wanghongbao@nwsuaf.edu.cn

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Abstract:

【Background】Muscle can maintain the motor function of mammals and regulate body metabolism. Its quantity and distribution have an important influence on meat quality. The growth and development of skeletal muscle and its genetic characteristics influence and even determine the meat production and meat quality to a large extent. It is of great significance to study the growth and development of skeletal muscle. The regulatory effect of TP53INP2 on autophagy and the regulation mechanism on the differentiation of preadipocytes have been studied, but whether it affects the differentiation of bovine myoblasts has not been reported. 【Objective】 This study aims to explore the effect of TP53INP2 on the differentiation of Qinchuan bovine myoblasts in order to provide a theoretical basis for the molecular breeding of beef cattle meat traits. 【Method】 The real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the expression characteristics of TP53INP2 in different tissues of Qinchuan cattle at 24 months of age. At the same time, the expression patterns of bovine skeletal myoblasts cultured in vitro at different stages of differentiation were analyzed. TP53INP2 gene siRNA was synthesized and transfected to Qinchuan bovine myoblasts, cells were induced to differentiation 12 hours after transfection, phenotypic changes of myoblasts were observed at different time pionts, and RT-qPCR and Western Blot technologies were performed respectively to detect the expression of differentiation marker genes and proteins on the fourth day of induced differentiation. 【Result】 1. RT-qPCR results showed that the expression level of TP53INP2 was the highest in adult Qinchuan cattle Longissimus dorsi muscle tissue and the lowest in the small intestine tissue. It is higher in adult bovine heart, liver, kidney, reticulum and rumen, and lower in other tissues (compared with longissimus dorsi). 2. With the differentiation of myoblasts, the expression of this gene increased from 0 to 4 days and reached a peak on the 4th day, and then decreased. 3. After interfering with TP53INP2 siRNA in myoblasts, the number and length of myotubes in the test group were significantly lower than those in the control group. 4. RT-qPCR results showed that the expression of myoblast differentiation marker genes myogenin (MYOG) and myosin heavy chain protein 3 (MYH3) was significantly lower than that of the control group. Western Blot results showed that the protein expressions of MYOG, MYH3 and MYOD in the test group were reduced and the differences were extremely significant compared with the control group.【Conclusion】Interfering of TP53INP2 has an inhibitory effect on the differentiation of bovine myoblasts, suggesting that this gene may have an important regulatory effect on the growth and development of Qinchuan cattle muscle tissue, and it can be used for in-depth functional research for molecular breeding of beef cattle practice.

Key words: TP53INP2, differentiation, myoblasts, Qinchuan cattle

Table 1

Real-time PCR primer sequence"

基因名称
Gene name		 GenBank号
GenBank No.		 引物名称
Primer name		 引物序列 (5′—3′）
Primer sequence (5′--3′）		 产物长度
Product length (bp)
TP53INP2 XM_003586843.5 TP53INP2-F GCGGCTGTAGACTCAAAG 130
TP53INP2-R GTTATGAGGCGGAGTGTC
GAPDH NM_001034034.2 GAPDH -F AGTTCAACGGCACAGTCAAGG 124
GAPDH -R ACCACATACTCAGCACCAGCA
MYOD NM_001040478.2 MYOD-F AACCCCAACCCGATTTACC 196
MYOD-R CACAACAGTTCCTTCGCCTCT
MYOG NM_001111325.1 MYOG-F GGCGTGTAAGGTGTGTAAG 85
MYOG-R CTTCTTGAGTCTGCGCTTCT
MYH3 NM_001101835.1 MYH3-F TGAACGCCCTCTCCAAATCC 101
MYH3-R AATGAAGTGCTGTCTCGGCA

Fig. 1

The expression of TP53INP2 gene in different tissues of adult Qinchuan cattle Different lowercase letters indicate significant differences（P<0.05), different uppercase letters indicate extremely significant differences（P<0.01), and the same letters indicate that the difference is not significant"

Fig. 2

Expression of TP53INP2 in muscle cells on different days D0: day 0 of induced differentiation of myoblasts; D2: day 2 of induced differentiation of myoblasts; D4: day 4 of induced differentiation of myoblasts; D6: day 6 of induced differentiation of myoblasts; D8: induction of myoblasts Day 8 of differentiation; D10: Day 10 of differentiation of myoblasts"

Fig. 3

Detection of TP53INP2 interference efficiency **represents extremely significant differences. The same below"

Fig. 4

The phenotype of the interference group and the control group on the fourth day of induced differentiation of myoblasts The red arrow indicates the myotube formed by the fusion of muscle cells"

Fig. 5

Detection of mRNA levels of myoblast differentiation marker genes"

Fig. 6

Western Blot detection of myoblast differentiation marker genes"

Fig. 7

Relative expression of different marker proteins"

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