关岭牛<i>MEF2A</i>启动子甲基化负向调控mRNA转录

doi:10.7668/hbnxb.20195667

摘要/Abstract

摘要：

肌肉生成依赖于各种细胞内外的信号和因子的相互调控,通过组织和细胞水平探究肌细胞增强因子2A(MEF2A)表达量与其启动子甲基化的调控关系,为关岭牛的遗传发育提供理论参考。以关岭牛为试验对象,首先对关岭牛组织MEF2A表达量与其启动子甲基化水平联合分析。其次,将MEF2A基因的干扰和超表达载体转染至关岭牛原代成肌细胞内,分析MEF2A表达量变化对其启动子甲基化水平的影响。结果表明,关岭牛犊牛各组织MEF2A表达量高于成年牛,而犊牛组织MEF2A甲基化率低于成年牛,DNMT1表达量趋势与之一致。此外,MEF2A表达量升高导致其甲基化率降低,降低MEF2A表达量则导致其甲基化率升高。从组织和细胞两方面证实关岭牛MEF2A基因表达水平与其甲基化率呈负相关,为遗传标记辅助牛的遗传改良提供了理论依据。

关键词: 关岭牛, MEF2A, 启动子, DNA甲基化, 成肌细胞

Abstract:

Muscle formation depends on the interplay of various cellular and extracellular signals and factors.This study investigates the regulatory relationship between Myocyte enhancer factor 2A(MEF2A)expression levels and promoter methylation at both tissue and cellular levels,to provide theoretical references for the genetic development of Guanling cattle.Guanling cattle were used as experimental subjects.Initially,the expression levels of the MEF2A in Guanling cattle tissues were analyzed in conjunction with their promoter methylation status.Subsequently,overexpression and interference vectors of the MEF2A gene were successfully transfected into primary myoblasts of Guanling cattle,and the effects of MEF2A expression level changes on promoter methylation levels were analyzed.The results showed that the MEF2A expression levels in various tissues of young Guanling cattle were higher than those in adult cattle,while the methylation rate of MEF2A in young cattle tissues was lower than that in adult cattle,with DNMT1 expression trends consistent with this.Additionally,increased MEF2A expression led to a decrease in its methylation rate,whereas decreased MEF2A expression resulted in an increase in its methylation rate.This study confirms from both tissue and cellular perspectives that the expression level of the MEF2A gene in Guanling cattle is negatively correlated with its methylation rate,providing a theoretical basis for genetic marker-assisted genetic improvement of cattle.

Key words: Cuanling cattle, MEF2A, Promoter, DNA methylation, Myoblasts

中图分类号:

S823

孙金魁, 许厚强. 关岭牛MEF2A启动子甲基化负向调控mRNA转录[J]. 华北农学报, 2025, 40(4): 203-209. doi: 10.7668/hbnxb.20195667.

SUN Jinkui, XU Houqiang. MEF2A Promoter Methylation Negatively Regulates mRNA Transcription in Guanling Cattle[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 203-209. doi: 10.7668/hbnxb.20195667.

http://www.hbnxb.net/CN/Y2025/V40/I4/203

图/表 6

表1 引物序列信息

Tab.1 Primers sequence information

名称 Name	引物序列(5'-3') Primer sequence(5'-3')	长度/bp Length
MEF2A	F: AATGAACCTCACGAAAGCAGAAC	106
	R:TTAGCACATAGGAAGTATCAGGGTC
DNMT1	F: GCACAGTCAAGGCAGAGAA	132
	R: CCAGCCTTCTCCATGGTAGT
GAPDH	F: TTGTGATGGGCGTGAACC	169
	R: GTCTTCTGGGTGGCAGTGAT
CPG-1	F: ATTATTATTAGTAGTAAAGATTAGTAGG	520
	R: ACCTCCCAAAACAACC
CPG-2	F: GGTTGTTTTGGGAGGT	110
	R: CTCCTAAAAAAAATATTAATCA
CPG-3	F: GTGATTAATATTTTTTTTAGGAGAATA	246
	R: TCTTCTTAATAAAACCCAAAACACTTAA

图1 MEF2A CpG岛(A)及在关岭牛不同阶段各组织中的表达量(B) 不同小写字母代表差异显著(P<0.05);不同大写字母表代表极显著(P<0.01)。图2,4同。

Fig.1 Expression of MEF2A CpG island(A)and its expression in Guanling cattle tissues(B) Different lowercase letters represent significant differences(P<0.05);different capital letters represent extremely significant(P<0.01).The same as Fig.2,4.

表2 RNA质检信息

Tab.2 RNA quality information

组织 Tissue	犊牛Calves		成年牛Adult
组织 Tissue	浓度/ (ng/μL) Concentration	OD_260/280	浓度/ (ng/μL) Concentration	OD_260/280
心脏Heart	1 082.3	1.86	1 124.7	1.85
肝脏Liver	985.6	1.91	1 356.9	1.92
肺脏Lung	1 428.5	1.93	1 003.8	1.86
肾脏Kidney	1 126.8	1.83	895.4	1.91
背最长肌	879.2	1.87	1 156.7	1.82
Dorsal muscle
脂肪Fat	685.1	1.81	752.6	1.81

图2 MEF2A在牛心脏与脂肪甲基化率(A)及DNMT1表达量(B)

Fig.2 Effects of MEF2A on heart and adipose methylation rate(A)and DNMT1 expression(B)in cattle

图3 免疫荧光鉴定(200×)

Fig.3 Immunofluorescence identification(200×)

图4 各重组载体对MEF2A启动子甲基化率及DNMT1表达量的影响 A.MEF2A超表达载体效率;B.超表达MEF2A对甲基化率影响;C.超表达MEF2A对DNMT1的影响;D.MEF2A干扰载体效率;E.抑制MEF2A对甲基化率影响;F.抑制MEF2A对DNMT1的影响。oe-NC.超表达阴性载体;oe-MEF2A.超表达载体;sh-NC.干扰阴性载体;sh-MEF2A.干扰载体。

Fig.4 Effects of recombinant vectors on MEF2A promoter methylation rate and DNMT1 expression A. MEF2A overexpression vector efficiency;B.The effect of overexpression of MEF2A on methylation rate;C.The effect of overexpression of MEF2A on DNMT1;D.MEF2A interference carrier efficiency;E.The effect of inhibition of MEF2A on methylation rate;F.The effect of inhibit in of MEF2A on DNMT1.oe-NC.Overexpression negative control vector;oe-MEF2A.Overexpression vector;sh-NC.Interference negative control vector;sh-MEF2A.Interference vector.

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关岭牛MEF2A启动子甲基化负向调控mRNA转录

MEF2A Promoter Methylation Negatively Regulates mRNA Transcription in Guanling Cattle

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