鹅p21基因克隆、启动子分析及转录调控研究  

Cloning,Promoter Analysis and Transcriptional Regulation of p21 Gene in Goose

作　　者：陈哲[1,2] 杨鹏霞 雷明明 陈佳彬[3] 闫乐艳 CHEN Zhe;YANG Pengxia;LEI Mingming;CHEN Jiabin;YAN Leyan(Institute of Animal Science,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Key Laboratory of Protected Agriculture Engineering in the Middle and Lower Reaches of Yangtze River,Ministry of Agriculture and Rural Areas,Nanjing 210014,China;Taizhou City Jinpeng Geese Professional Cooperative,Taizhou 225319,China)

机构地区：[1]江苏省农业科学院畜牧研究所,南京210014 [2]农业农村部长江中下游设施农业工程重点实验室,南京210014 [3]泰州市金鹏鹅业专业合作社,泰州225319

出　　处：《中国畜牧兽医》2022年第2期405-415,共11页China Animal Husbandry & Veterinary Medicine

基　　金：泰州市科技支撑计划(TN202017);农业农村部长江中下游设施农业工程重点实验室开放性课题(KF2018)。

摘　　要：【目的】分析鹅p21基因的结构和启动子活性,探讨p21基因的转录调控机制。【方法】以泰州鹅为试验对象,通过同源克隆、RACE和生物信息学分析等方法获得鹅p21基因全长序列和5′-侧翼区序列特征;构建6个不同缺失片段的启动子区双荧光素酶报告载体并分析其荧光素酶活性,进而确定p21基因核心启动子区;对核心启动子区转录因子结合位点生肌决定因子(MyoD)(+25~+36 bp)进行定点突变,并构建突变报告基因载体,在C2C12细胞系内初步鉴定鹅p21基因核心转录调控因子。【结果】鹅p21基因cDNA全长1943 bp,CDS区大小为453 bp,编码151个氨基酸,蛋白序列包含高度保守的CDI家族结合位点。系统进化树分析表明,鹅p21基因与鸭亲缘关系最近,与鸡和火鸡有较强的进化关系。鹅p21基因5′-侧翼区包含启动子元件,-35~+37 bp是核心启动子区,发挥正向调控作用,结合定点突变技术初步鉴定MyoD是鹅p21基因核心转录调控元件。【结论】本研究获得了鹅p21基因完整的cDNA序列和启动子区域,MyoD是p21基因核心转录调控因子,为探究p21基因在鹅胚胎期肌肉发育过程中的调控机制提供理论依据。【Objective】The aim of this study was to analyze the structure and promoter activity of p21 gene in goose,and investigate the transcriptional regulation mechanism.【Method】The Taizhou goose was taken as the research object,the sequence characteristics of the full-length and 5′-flanking region of p21 gene were obtained using homologous amplification,RACE and biological analysis.The dual luciferase reporter vectors with 6 different deletion fragments of the promoter were constructed,its luciferase activities were analyzed,and the core promoter region of p21 gene was identified.Site-directed mutagenesis of the transcription factor binding sites MyoD(+25 to+36 bp)in the core promoter region was carried out,and the key transcription factors of p21 gene was preliminary identified in C2C12 cell line.【Result】The full cDNA sequence of p21 gene in goose was 1943 bp,containing 453 bp coding regions(CDS)which encoded 151 amino acids.p21 protein sequence had highly conserved CDI family binding sites.Phylogenetic tree analysis showed that p21 gene in goose was closely related to Anas platyrhynchos,and had a particularly strong evolutionary relationship with Gallus gallus and Meleagris gallopavo.The 5′-flanking region of p21 gene contained promoter elements,the core promoter was found to be located at-35 to+37 bp,which played a positive regulatory effect.Combined with site-directed mutation demonstrated that MyoD was the key transcription factor of p21 gene in goose.【Conclusion】The complete cDNA sequence and promoter region of p21 gene in goose were obtained,and MyoD was the key transcription factor.The results provided a theoretical basis for exploring molecular regulation mechanism of p21 gene in embryonic muscle development of goose.

关 键 词：鹅 P21基因 克隆 启动子活性 转录调控元件 

分 类 号：Q78[生物学—分子生物学]