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  

Effect and Molecular Mechanism of Zbed 6 Gene Knockout on the Growthand Development of Skeletal Muscle in Mice

作　　者：刘铃 王圣楠 王丹丹 马月辉[2] 蒋琳[2] 崔凯[1] LIU Ling;WANG Shengnan;WANG Dandan;MA Yuehui;JIANG Lin;CUI Kai(College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]青岛农业大学动物科技学院,青岛266109 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国畜牧兽医》2023年第9期3641-3651,共11页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金青年科学基金项目(32202621);国家自然科学基金优秀青年科学基金项目(32222079)。

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 NovaseqTM6000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。【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 Zbed 6 gene knockout(Zbed 6^(-/-))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 Zbed 6^(-/-)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 Zbed 6 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 Zbed 6^(-/-)male and female mice were extremely significantly increased(P<0.01).After Zbed 6 gene knockout,the mRNA expression of Zbed 6 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 Zbed 6^(-/-)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 Dock 3,Lhx 2,Brsk 2,Caskin 1 and Gbe

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 

分 类 号：Q78[生物学—分子生物学]