Zbed6基因敲除对小鼠骨骼肌生长发育的影响及其分子作用机制研究

doi:10.16431/j.cnki.1671-7236.2023.09.020

摘要/Abstract

摘要： 【目的】研究锌指BED结构域包含蛋白6(ZBED6)基因敲除对青春期小鼠骨骼肌生长发育的影响及其分子作用机制。【方法】以8周龄野生型(WT)和Zbed6基因敲除(Zbed6^-/-)C57BL/6小鼠为试验对象,采集血液,用ELISA法检测血清睾酮含量;分离骨骼肌组织并记录肌肉重、肌肉率。通过骨骼肌组织切片和免疫组化分析检测骨骼肌肌纤维面积及IGF2蛋白表达水平。通过Illumina Novaseq^TM 6000高通量测序对WT和Zbed6^-/-雌、雄小鼠进行转录组测序分析,联合小鼠ZBED6靶基因数据,筛选在Zbed6基因敲除小鼠肌肉组织中发挥作用的下游靶基因,并通过实时荧光定量PCR验证转录组测序结果中差异表达基因的可靠性。【结果】与WT小鼠相比,Zbed6^-/-雌、雄小鼠肌肉重、肌肉率均极显著增加(P＜0.01)。Zbed6基因敲除后,雌、雄小鼠骨骼肌Zbed6基因mRNA表达量显著降低(P＜0.05),IGF2的mRNA及蛋白表达量均显著或极显著增加(P＜0.05;P＜0.01),血清睾酮含量极显著升高(P＜0.01)。转录组测序结果显示,Zbed6^-/-组雄鼠骨骼肌中共筛选到38个差异表达基因,其中22个上调,16个基因下调;雌鼠骨骼肌中筛选到49个差异表达基因,其中19个基因上调,30个基因下调。共有10个差异表达基因在雌、雄小鼠中共同表达,其中Dock3、Lhx2、Brsk2、Caskin1、Gbe1为ZBED6靶基因。实时荧光定量PCR检测发现,4个差异表达基因的表达情况与转录组测序分析结果一致,证实测序结果准确可信。【结论】Zbed6^-/-促进青春期小鼠骨骼肌生长发育,增加血清睾酮含量,筛选出Dock3、Lhx2、Brsk2、Caskin1、Gbe1 5个ZBED6靶基因。研究结果为深入探究ZBED6功能提供了新的基因靶点和研究方向,有助于完善ZBED6功能图谱,为大动物品种改良和畜禽瘦肉性状遗传育种研究提供了重要的理论依据。

关键词: 锌指BED结构域包含蛋白6; 骨骼肌; 生长发育; 转录组测序

Abstract: 【Objective】 The purpose of this study was to investigate the effect of zinc finger BED domain containing protein 6 (ZBED6) gene knockout on skeletal muscle growth and development in adolescent mice and its molecular mechanism.【Method】 Wild type (WT) and Zbed6 gene knockout (Zbed6^-/-) C57BL/6 mice at 8 weeks old were used as experimental subjects.Blood was collected and serum testosterone content was determined by ELISA method.Skeletal muscle tissue was isolated and muscle weight and muscle ratio were recorded.Skeletal muscle fiber area and IGF2 protein expression levels were determined by histological analysis and immunohistochemical analysis.Transcriptome sequencing was performed on WT and Zbed6^-/- male and female mice by Illumina Novaseq^TM 6000 high-throughput sequencing.Combined with ZBED6 target gene data of mice,the downstream target genes that played a role in Zbed6 gene knockout mouse muscle tissue were screened.Real-time quantitative PCR was used to verify the reliability of differentially expressed genes in transcriptome sequencing.【Result】 Compared with WT mice,muscle weight and muscle ratio of Zbed6^-/- male and female mice were extremely significantly increased (P<0.01).After Zbed6 gene knockout,the mRNA expression of Zbed6 gene in skeletal muscle of female and male mice was significantly decreased (P<0.05),and the mRNA and protein expression of IGF2 were significantly or extremely significantly increased (P<0.05 or P<0.01),serum testosterone content was extremely significantly increased (P<0.01).Transcriptome sequencing showed that 38 differentially expressed genes were detected in the skeletal muscle of male mice in Zbed6^-/- group,of which 22 genes were up-regulated and 16 genes were down-regulated.49 differentially expressed genes were detected in skeletal muscle of female mice,among which 19 genes were up-regulated and 30 genes were down-regulated.A total of 10 differentially expressed genes were co-expressed in female and male mice,among which Dock3,Lhx2,Brsk2,Caskin1 and Gbe1 were ZBED6 target genes.Real-time quantitative PCR showed that the expression of the 4 differentially expressed genes was consistent with the transcriptome sequencing analysis,which confirmed the accuracy and reliability of the sequencing results.【Conclusion】 Zbed6^-/- promoted skeletal muscle growth and increased serum testosterone content in adolescent mice.Five ZBED6 target genes including Dock3, Lhx2,Brsk2,Caskin1 and Gbe1 were screened.The results provided new gene targets and research directions for further exploring the function of ZBED6,contribute to improving the functional map of ZBED6,and provided an important theoretical basis for the improvement of large animal varieties and the genetic breeding of lean meat traits of livestock and poultry.

Key words: zinc finger BED domain containing protein 6; skeletal muscle; growth and development; transcriptome sequencing

中图分类号:

刘铃, 王圣楠, 王丹丹, 马月辉, 蒋琳, 崔凯. Zbed6基因敲除对小鼠骨骼肌生长发育的影响及其分子作用机制研究[J]. 中国畜牧兽医, 2023, 50(9): 3641-3651.

LIU Ling, WANG Shengnan, WANG Dandan, MA Yuehui, JIANG Lin, CUI Kai. Effect and Molecular Mechanism of Zbed6 Gene Knockout on the Growth and Development of Skeletal Muscle in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3641-3651.

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