Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (3): 589-601.doi: 10.3864/j.issn.0578-1752.2022.03.013

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

Relationship Between Expression Levels of Guangxi Partridge Chicken m6A Methyltransferase Genes, Myofiber Types and Myogenic Differentiation

SHU JingTing1(),SHAN YanJu1,JI GaiGe1,ZHANG Ming1,TU YunJie1,LIU YiFan1,JU XiaoJun1,SHENG ZhongWei1,TANG YanFei2,LI Hua3,ZOU JianMin1,*()   

  1. 1 Key Laboratory for Poultry Genetics and Breeding of Jiangsu Province, Jiangsu Institute of Poultry Science, Yangzhou 225125, Jiangsu
    2 Guangxi Fufeng Farm Group Co., Ltd. Nanning 530024
    3 Foshan University, Foshan 528225, Guangdong
  • Received:2020-12-16 Accepted:2021-03-30 Online:2022-02-01 Published:2022-02-11
  • Contact: JianMin ZOU E-mail:shujingting@163.com;jqszjm@163.com

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Abstract:

【Objective】 It is widely accepted that the regulation of myofiber type composition and transition is an important source of variation to improve meat quality. Recently, several studies revealed that N6-methyladenosine (m6A) RNA methylation played a important biological role in the regulation of muscle differentiation. In the present study, the myofiber types of different location of skeletal muscles were identified in the native chicken breed-Guangxi partridge chicken, and the expression levels of the m6A methyltransferase genes (METTL3, METTL14, WTAP and KIAA1429) were first studied in different location of skeletal muscle and myogenic differentiation, which would provide a reference for clarifying the regulation mechanism of chicken myofiber type composition and transition.【Method】 Myosin ATPase staining was used to identify the myofiber types and to measure the myofiber size and density of seven location of Guangxi partridge chicken muscles, including the Pectoralis major (XD), pectoralis minor (XX), Sartorius (FJ), Medial head of sciatic pubis (CN), medial head of gastrocnemius (FN), Lateral iliotibial muscle (QW) and Latissimus dorsi (BK). Colorimetric method was used to detect the endogenous m6A methylation levels at different time points before and after myogenic differentiation. Expression of METTL3, METTL14, WTAP and KIAA1429 was quantified by RT-PCR in these seven muscles, as well as at the myoblasts separated on 0 h, 19 h, and cultured in differentiation medium on D0, D2, and D5, and the correlations between the gene expression levels and myofiber characteristics as well as m6A methylation levels were also analyzed by SPSS20.0 software.【Result】 The XD and XX muscles of the breast muscle group were all composed of white myofibers, CN, FN and FJ muscles of the leg muscle group were mainly composed of red myofibers, and QW muscles were mainly composed of white myofibers, while the BK muscles of the back muscle group were mainly composed of red myofibers. The average fiber densities of muscles mainly composed of red myofibers were significantly higher than those mainly composed of white myofibers. The expression patterns of METTL3, METTL14, WTAP and KIAA1429 showed significant differences in tissue and time-specific fashion. Overall, the expression levels of the four genes in muscles mainly composed of red myofibers were higher than those mainly composed of white myofibers, and in differentiated myoblasts were significantly higher than those in proliferative myoblasts. The muscle METTL3, METTL14 and WTAP gene showed similar expression patterns, the highest expression level was appeared in BK muscles, which were significantly higher than that in all the other studied muscles; the expression levels in CN and FN muscles were also significantly higher than those in FJ, XD, XX and QW muscles. The highest expression level of KIAA1429 was appeared in FN muscles, which were significantly higher than that in all the other studied muscles; the expression levels in BK muscles were also significantly higher than those in CN, FJ, XD, XX and QW muscles; the lowest expression level was appeared in XD muscles, but the differences were not significantly when compared to XX and QW. In chicken myoblasts, both METTL3 and METTL14 gene showed continuous increasing and then decreasing expression pattern, the lowest expression level was appeared at 0h newly separated myoblasts, and the highest level was appeared at D2 induced differentiation myoblasts, which were significantly higher than those in proliferative myoblasts and D0 induced differentiation myoblasts. The consistent expression pattern was also found in WTAP and KIAA1429 gene, showed continuous increasing expression pattern; the lowest expression level was appeared at 0h newly separated myoblasts, significantly lower than that in other studied time points, then significantly increased at 19 h, and then slightly increased at D0, then significantly increased from D0 to D5. The endogenous m6A RNA methylation levels increased along with myogenic differentiation, and the levels in differentiation myoblasts also showed significantly higher than those in proliferative myoblasts, the highest methylation level was appeared at D2 induced differentiation myoblasts, then significantly increased from D2 to D5; however, the methylation level at D5 was higher than that at D0. Significant positive relationships were observed for the expression of studied genes in different muscle tissues as well as in myoblasts. Meanwhile, the skeletal muscle expression of these four genes was all showed significant positive relationships with red myofiber ratio, and significant negative relationships with red myofiber ratio. Positive relationships were also observed between the expression of studied genes in myoblasts and m6A methylation levels.【Conclusion】 There were differences in muscle fiber type composition in different parts of the muscle of Guangxi partridge chicken. Differential expression and coordinated developmental regulation of the selected m6A methyltransferase genes in the chicken skeletal muscles and myoblasts, and these genes might play a role in the regulation of the myofiber type composition, maintain, and myogenic differentiation.

Key words: Guangxi partridge chicken, gene expression, m6A methyltransferase genes, myofiber type, myogenic differentiation

Table 1

Gene-specific primer for quantitative RT-PCR"

基因名称
Gene name		基因登录号
Accession number		引物序列
Primer sequence		PCR产物
PCR product (bp)
METTL3 XM_025145967.1 F: GCCATCGATACGCCCGAAAC 148
R: TAGGTGACGATGGTGGGACC
METTL14 NM_001031148.1 F: CTGGATCTTGGCCGAGTGTG 146
R: AGGCAGTGCTCCTTGGTTCT
WTAP XM_025148833 F: TCAGCAGCAGTTGAAGGAGA 166
R: ACCATTGCTTGGTCCGTTAG
KIAA1429 XM_418337 F: GCGCGGAGCAAAGTACCAA 124
R: CCATAAGCCCTGTTGTCGGG
MyoD NM_204214.2 F: GCTACTACACGGAATCACCA 198
R: GGGCTCCACTGTCACTCA
MyoG NM_204184.1 F: AGCGCCATCCAGTACATCG 376
ATCCTTCCAGCATCACCATC
HSP70 AY143693 F: TCTGCTCCTGTTGGATGTC 95
R: TGGGAATGGTGGTGTTACG

Fig. 1

Morphological and fiber-type variations with ATPase staining of different location muscles in Guangxi partridge chickens(A) Morphological variations of different location muscles; (B) Fiber-type variations with ATPase staining of different location muscles. І: slow myofiber; Пa: fast red myofiber; Пb: fast white myofiber. XD: major pectoralis, XX: small pectoralis, QW: iliotibialis lateralis, FN: medial gastrocnemius, CN: medial pubischiofemorales, FJ: Sartorius, BK: latissimus dorsi. The same as below"

Table 2

Fiber-type composition of the different location muscles in Guangxi partridge chickens"

肌肉部位
Location of the muscles		I
(%)		IIa
(%)		I+IIa
(%)		IIb
(%)		平均肌纤维直径
Diameter of myofiber (µm)		平均肌纤维横切面积
Cross-section area of myofiber (µm2)		肌纤维密度（根·mm-2）
Density of myofiber (bundle·mm2)
XD 0 0 0 100 57.28a±4.68 2829.80a±238.40 328.50c±38.50
XX 0 0 0 100 56.66a±5.17 2781.57a±210.32 366.87c±25.56
QW 10.5±0.8 29.1±3.3 39.6±3.4 60.4±4.5 55.87a±5.76 2687.74a±205.58 381.26c±32.25
FN 33.0±2.5 47.9±4.2 80.9±3.6 19.1±1.2 44.57c±5.02 1770.15c±221.05 489.11a±40.85
CN 25.8±2.0 46.1±3.9 71.9±3.3 28.1±2.6 48.82b±4.96 2195.21b±214.21 415.09b±34.52
FJ 6.0±0.9 63.7±5.5 69.7±4.9 30.3±2.8 50.49b±4.65 2266.50b±192.55 407.39b±38.51
BK 16.9±1.2 62.9±5.4 79.8±5.2 20.2±2.1 43.85c±3.58 1682.24c±187.25 505.93a±49.78

Fig. 2

m6A methyltransferase genes expression levels in different location of skeletal muscles of Guangxi partridge chickens(A) METTL3 gene; (B) METTL14 gene; (C) WTAP gene; (D) KIAA1429 gene. Bar diagram values with the same letter are not significantly different among different muscles (P>0.05), and values with different letters are significantly different between different muscles (P<0.05)"

Fig. 3

Expression levels of myogenic differentiation marker genes in chicken primary myoblasts(A) mRNA expression; (B) protein expression. 0 h: the newly separated primary myoblasts; 19 h: primary myoblasts cultured in proliferation medium for 19 h; D0: primary myoblasts cultured in differentiation medium reaching confluence; D2: primary myoblasts cultured in differentiation medium for 2 days; D5: primary myoblasts cultured in differentiation medium for 5 days (the same below); Bar diagram values with the same letter are not significantly different among different stage of primary myoblasts (P>0.05), and values with different letters are significantly different between different stage of primary myoblasts (P<0.05)"

Fig. 4

m6A methyltransferase genes expression levels along with myogenic differentiation of chicken primary myoblasts"

Fig. 5

Endogenous m6A RNA methylation along with myogenic differentiation of chicken primary myoblasts"

Table 3

Correlation between chicken muscle m6A methyltransferase genes and myofiber characteristics"

参数
Index		红肌纤维比例
I+IIa
(%)		白肌纤维比例
IIb
(%)		肌纤维直径
Diameter of myofiber (µm)		肌纤维密度（根·mm-2）
Density of myofiber (bundle·mm-2）		METTL14WTAPKIAA1429
METTL3 0.609** -0.502** -0.573** 0.451** 0.910** 0.854** 0.942**
METTL14 0.563** -0.532** -0.614** 0.459** 1 0.952** 0.950**
WTAP 0.570** -0.498** -0.613** 0.440* 1 0.923**
KIAA1429 0.551** -0.563** -0.594** 0.421* 1

Table 4

Correlation between chicken primary myoblasts m6A methyltransferase genes and m6A RNA methylation levels"

参数
Index		METTL3METTL14WTAPKIAA1429MyoGMyoDm6A含量
m6A (%)
METTL3 1 0.979** 0.950** 0.971** 0.478* 0.453* 0.614**
METTL14 1 0.976** 0.975** 0.425* 0.362 0.506**
WTAP 1 0.931** 0.855** 0.385 0.342
KIAA1429 1 0.532** 0.338 0.403*
MyoG 1 0.651** 0.436*
MyoD 1 0.473*
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