中国农业科学 ›› 2023, Vol. 56 ›› Issue (1): 165-178.doi: 10.3864/j.issn.0578-1752.2023.01.013
收稿日期:
2021-09-30
接受日期:
2022-04-12
出版日期:
2023-01-01
发布日期:
2023-01-17
通讯作者:
昝林森
作者简介:
杨昕冉,E-mail:基金资助:
YANG XinRan1(),MA XinHao1,DU JiaWei1,ZAN LinSen1,2()
Received:
2021-09-30
Accepted:
2022-04-12
Online:
2023-01-01
Published:
2023-01-17
Contact:
LinSen ZAN
摘要:
【目的】近年来,RNA m6A甲基化修饰在肌肉发育中的作用不断被发现,通过探究m6A甲基化酶相关基因,包括METTL3,METTL14,WTAP,FTO和ALKBH5,在牛肌肉组织以及骨骼肌卫星细胞(skeletal muscle satellite cells, SMSCs)增殖和分化过程中的表达,同时分析体外成肌分化过程中m6A甲基化水平的变化,为阐明m6A修饰在骨骼肌发育中的作用及机制提供参考。【方法】使用实时荧光定量PCR(RT-qPCR)技术检测m6A甲基化酶相关基因在新生和成年牛不同部位骨骼肌中的表达。随后在秦川牛肉用新品系背最长肌中分离SMSCs,通过生长曲线、免疫荧光和RT-qPCR技术验证SMSCs的增殖和成肌分化功能,使用RT-qPCR检测m6A甲基化酶相关基因在SMSCs增殖期24、36、48、60、72 h和分化第0、2、4、6、8天中的时序表达谱。最后利用LC-MS/MS和Dot blot技术检测SMSCs分化过程中m6A甲基化水平的变化。【结果】METTL3、METTL14和WTAP等m6A甲基化转移酶基因在成年牛背最长肌、前腿肌和后腿肌中的表达量均显著低于新生牛(P<0.01)。FTO和ALKBH5等m6A去甲基化酶基因在成年牛后腿肌中的表达更高(P<0.01),ALKBH5在成年牛背最长肌中的表达也较高(P<0.01)。分离的SMSCs具有良好的生长状态且可正常成肌分化。在SMSCs增殖期,METTL3表达逐渐下降,但在增殖后期时明显提高。METTL14在增殖期的表达变化差异不明显,WTAP则在增殖48 h后表达逐渐降低。而FTO和ALKBH5在增殖期的时序表达类似,60 h之前变化不显著,但在72 h时显著提高。在SMSCs成肌分化过程中,METTL3、METTL14和WTAP的表达模式基本一致,分化前期上升,随后下降,在分化末期表达增加。而FTO的表达随分化进行逐渐增加,ALKBH5则在分化前4天表达上升,随后不断下降。此外,在SMSCs分化过程中,mRNA的整体m6A水平下降(P<0.01)。【结论】m6A甲基转移酶和去甲基化酶在新生牛和成年牛骨骼肌中的表达变化存在较为明显的差异,表明m6A修饰可能对秦川牛骨骼肌的发育具有重要作用。同时,这些m6A相关甲基化酶可能参与调控牛骨骼肌卫星细胞的增殖和分化。这一发现为研究m6A甲基化修饰调控骨骼肌生成的作用及机制提供理论依据。
杨昕冉,马鑫浩,杜嘉伟,昝林森. m6A甲基化酶相关基因在牛骨骼肌生成中的表达[J]. 中国农业科学, 2023, 56(1): 165-178.
YANG XinRan,MA XinHao,DU JiaWei,ZAN LinSen. Expression Pattern of m6A Methylase-Related Genes in Bovine Skeletal Muscle Myogenesis[J]. Scientia Agricultura Sinica, 2023, 56(1): 165-178.
表1
RT-qPCR所用引物"
基因名称 Gene name | 引物序列 Primer sequence (5′-3′) | 产物长度 Product length (bp) |
---|---|---|
METTL3 | F: TCGAAAGCTGCACTTCAGAC | 199 |
R: TCCAACGCTCTGTGTAAGGG | ||
METTL14 | F: TGACATCAGAGAACTGACACCC | 198 |
R: AGGTCCAATCCTTCCCCAGA | ||
WTAP | F: GCCTGGAAGTTTACGCCTGA | 176 |
R: TCCTGACTGCTTTTAAGCTCCT | ||
FTO | F: AGCAGCGTACAACGTCACTT | 193 |
R: AGGGTCGTCCTCACTTTCCT | ||
ALKBH5 | F: TACTTCTTCGGCGAGGGCTA | 191 |
R: TGGTAGTCGTTGATGACGGC | ||
MYOD1 | F: AACCCCAACCCGATTTACC | 196 |
R: CACAACAGTTCCTTCGCCTCT | ||
MYOG | F: GGCGTGTAAGGTGTGTAAG | 85 |
R: CTTCTTGAGTCTGCGCTTCT | ||
MYF6 | F: GTGATAACTGCCAAGGAAGGAG | 93 |
R: CGAGGAAATGCTGTCCACGA | ||
MYH3 | F: TGAACGCCCTCTCCAAATCC | 101 |
R: AATGAAGTGCTGTCTCGGCA | ||
GAPDH | F: AGTTCAACGGCACAGTCAAGG | 124 |
R: ACCACATACTCAGCACCAGCA |
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