鸡SOX6基因真核表达载体构建及其对成肌细胞分化相关基因表达的影响  

Construction of eukaryotic expression vector of chicken SOX6 gene and its effect on gene expression of myoblast differentiation

作　　者：刘书鸣 魏志恒 彭鑫 周博 夏磊 裴军 徐璐 郁建峰 顾志良 LIU Shuming;WEI Zhiheng;PENG Xin;ZHOU Bo;XIA Lei;PEI Jun;XU Lu;YU Jianfeng;GU Zhiliang(School of Biology and Food Engineering,Suzhou University of Technology,Changshu 215500,China;School of Pharmacy,Soochow University,Suzhou 215123,China)

机构地区：[1]苏州工学院生物与食品工程学院,江苏常熟215500 [2]苏州大学药学院,江苏苏州215123

出　　处：《畜牧与兽医》2025年第5期15-23,共9页Animal Husbandry & Veterinary Medicine

基　　金：江苏省自然科学基金项目(BK20191476);江苏省大学生创业创新重点项目(202310333012Z)。

摘　　要：为研究鸡性别决定区域Y盒蛋白转录因子6(SOX6)基因时序表达和在成肌细胞分化中的作用,采用荧光定量PCR方法检测鸡不同组织以及胚胎期(E11~E19)和出壳早期(D0、D3、D6)骨骼肌中SOX6 mRNA的表达;通过RT-PCR扩增并克隆鸡SOX6基因的编码序列区(CDS),经测序分析并利用生物信息学工具预测鸡SOX6蛋白的理化性质和结构功能;构建鸡SOX6基因的真核表达载体并转染至鸡原代成肌细胞,Western blot技术检测重组蛋白的表达;利用荧光定量PCR检测过表达和敲低SOX6基因对成肌分化相关基因表达的影响。结果显示:SOX6 mRNA在鸡12种不同组织中均有表达,其中在胸肌、腿肌较高,而在肌胃和脾脏较低;SOX6 mRNA在胚胎期E11~E19表达量逐渐增加,D6期显著高于其他时期(P<0.05);获得鸡SOX6基因编码区全长2 367 bp,共编码789个氨基酸;生物信息学分析鸡SOX6蛋白为中性、亲水性蛋白,分子量为87.533 kDa,等电点为6.99,不稳定系数为60.95,脂肪系数为62.51;构建SOX6过表达载体并转染至鸡原代成肌细胞中,成功表达出SOX6-MYC融合蛋白;在成肌细胞中过表达SOX6后,肌肉分化标志基因生肌分化因子(MyoD)和肌细胞生成素(MyoG) mRNA表达显著上升(P<0.05),而敲降SOX6后MyoD和MyoG mRNA表达显著下降(P<0.05)。结果表明:SOX6基因对成肌细胞分化发育具有调控作用,为进一步分析SOX6基因功能奠定基础。This study was to investigate the temporal and spatial expression of chicken SOX6 gene and its role in myoblast differentiation.Fluorescence quantitative PCR was used to detect the expression of SOX6 mRNA in different tissues of chickens as well as in skeletal muscles during embryonic stage(E11-E19) and early post-hatching period(D0,D3,D6).The coding sequence region(CDS) of the SOX6 gene was amplified and cloned by RT-PCR.After sequencing,the physicochemical properties and structural functions of the SOX6 protein were predicted by bioinformatics tools.The eukaryotic expression vector of the SOX6 gene was constructed and transfected into the primary myoblasts in the chickens,and the recombinant protein was detected by Western blot.Finally,the effect of the overexpression and knockdown of the SOX6 gene on the expression of myogenic differentiation-related genes was detected by fluorescence quantitative PCR.The results showed that the SOX6 mRNA was expressed in 12 various tissues of the chickens,with higher expression in the pectoral muscle and leg muscle,and lower expression in the stomach and spleen.The expression of SOX6 mRNA gradually increased during the embryonic stage from E11 to E19,and was significantly higher in the D6 stage than in the other stages(P<0.05).The CDS of the chicken SOX6 gene was obtained by RTPCR.The total length of the coding region of the chicken SOX6 gene was 2 367 bp,encoding 789 amino acids.The bioinformatics analysisshowed that the chicken SOX6 protein was a neutral,hydrophilic protein,with a molecular weight of 87.533 kDa,an isoelectric point of6.99,instability coefficient of 60.95,and fat coefficient of 62.51.The SOX6 overexpression vector was constructed and transfected intochicken primary myoblasts,and SOX6-MYC fusion protein was successfully expressed.After SOX6 was overexpressed in the myoblasts,themRNA expressions of the MyoD and MyoG genes increased significantly(P<0.05),while the MyoD and MyoG mRNA expressions were sig-nificantly decreased after knocking down SOX6(P<0.05).The

关 键 词：鸡 SOX6基因 克隆 真核表达 成肌细胞分化 

分 类 号：S831[农业科学—畜牧学]