巴马小型猪OCA2基因序列、组织表达分析及载体构建研究  

Analysis of Sequence,Tissue Expression and Vector Construction of OCA2 Gene in Bama Miniature Pigs

作　　者：洪潜龙 张颖[1] 曹春伟[1] 王宪龙[1] 赵建国[1] 

机构地区：[1]中国科学院动物研究所干细胞与生殖生物学国家重点实验室,北京100101 [2]安徽大学生命科学学院,合肥230601

出　　处：《畜牧兽医学报》2016年第3期439-448,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：973国家科技重大专项(2011CBA01005);国家高技术研究发展计划(2012AA020602)

摘　　要：旨在为猪的毛色调控机制和模拟与OCA2基因相关的人类疾病模型的研究建立基础。本研究将GenBank公布的猪OCA2基因序列（NC_010457.4）所缺失的5个外显子（20~24号外显子）进行了克隆和划分,在基因组水平和cDNA水平上采用PCR扩增和Sanger测序方法对划分结果进行了验证;并对OCA2基因进行生物信息学分析,同时在广西巴马小型猪中对OCA2基因外显子的多态位点进行了筛选。采用实时荧光定量PCR技术（Q-PCR）分析了OCA2基因在各组织中的表达规律,并以皮肤全长cDNA为模板构建重组表达质粒,并利用XhoⅠ和SacⅡ限制性内切酶进行双酶切验证。在已公布的猪OCA2基因中成功划分和克隆了所缺失的外显子,并在OCA2基因所有外显子中共发现了10个SNPs位点,其中7个为同义突变,3个为错义突变（R124H、G509D、R573H）;猪OCA2基因在多种组织中广泛表达,其中,肺、大脑、小脑相对高表达,脾、胃、盲肠和皮肤中度表达,而在心和肝中表达量较低;OCA2基因重组表达质粒经酶切和测序鉴定证明构建成功。本研究初步探讨了猪OCA2基因的序列信息及组织表达规律,并构建了OCA2基因重组表达质粒,为该基因下一步的功能学研究及疾病模型的研究奠定了基础。In order to establish scientific basis for farm animal models associated with coat color mechanism and human diseases in OCA2 gene,we carried out this experiment.The exons 20-24 of porcine OCA2gene（NC_010457.4）in absence in GenBank were cloned and mapped.The mapping result was confirmed at the genomic and cDNA levels using PCR amplification and Sanger sequencing simultaneously.Bioinformatics analysis was performed for OCA2 gene.Polymorphic loci of all exons for OCA2 gene were screened in Guangxi Bama Miniature pigs.Meanwhile,the mRNA expression in different tissues was detected by real-time fluorescence quantitative PCR（Q-PCR）.A full-length skin cDNA was used as a template to construct recombinant expression plasmid,and then XhoⅠand SacⅡrestriction enzyme double digestion was carried out.We mapped and cloned the porcine OCA2 gene in GenBank successfully.A total of 10 SNP loci were found in all exons,including 7synonymous mutations and 3 missense mutations（R124H,G509 D,R573H）.PorcineOCA2 gene was widely expressed in various tissues.The expression in lung,brain and cerebellum was at relatively higher level compared to that in spleen,stomach,caecum and skin,which exhibited moderate expression level,and the expression level in heart and liver was lower.pEGFP-N1-OCA2 recombinant plasmid was constructed successfully after confirmation of restriction enzyme double digestion and sequencing.This study explored porcine OCA2 gene sequence information and mRNA expression preliminarily,and recombinant expression plasmid of OCA2 gene was constructed,which provide the foundation for the study of disease model and gene biological function in future.

关 键 词：猪 OCA2基因 荧光定量PCR 重组表达质粒 单核苷酸多态性 

分 类 号：S828[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]