猪ANK1基因启动子活性与转录调控及多态性分析

发布时间：2018-04-22 11:32

本文选题：猪 + ANK1基因　；参考：《广西大学》2017年硕士论文

【摘要】：我国是世界第一大猪肉生产和消费国。随着中国经济的快速发展,人们对猪肉的肉质要求越来越高。改良猪肉肉质性状将是育种工作的一个重点。ANK1(Ankyrinl)属于锚蛋白家族(Ankyrins)中的一员,其编码的蛋白 AnkR(Ankyrin-R)由膜结合域(membrane-binding domain)和血影蛋白结合域(spectrin-binding domain)及调控域(regulatory domain)三部分构成。该基因不但与猪和牛的部分肉质性状有一定的相关,而且也与2型糖尿病的易感性、遗传性球形红细胞增多症、冠心病和阿尔茨海默症等有关。在基因的表达调控过程中,基因的转录调控是一个非常重要的过程。基因的转录过程受到关键元件启动子的调控,启动子是一段能与RNA聚合酶和转录结合因子结合的DNA序列,它与相应转录因子相互作用来调控基因的表达。为了解猪ANK1基因的转录调控机制,本课题以广西巴马小型猪和长白猪作为研究对象,克隆了 ANK1基因的启动子区域,对其核心区域进行研究。同时检测了广西巴马小型猪、德宝猪、陆川猪和长白猪ANK1基因启动子区域的SNP,研究结果如下:1.成功克隆了广西巴马小型猪、长白猪ANK1基因启动子区约2074bp序列。从Ensembl截取猪ANK1基因5'端上游调控序列,利用在线软件PromoterScan、NNPP、Promoter2.0、Gene-regulation、MethPrimer 等进行分析,发现克隆的2074bp片段存在很多的调控元件,例如TATAbox、GATA-1、SP1、myogenin、AP-2alpha、CEP-bind等,同时预测表明该区域存在两个CpG岛和两个核心启动子区域。2.分别构建了长白猪和广西巴马小型猪ANK1启动子缺失片段双荧光素酶报告基因载体各8个,并转染C2C12细胞,检测各载体的荧光素酶活性。发现长白猪ANK1启动子活性最高的片段为PGL3-P3,巴马小型猪活性最高片段为PGL3-P2,推测ANK1基因启动子核心区域定位为-1080～-1991bp范围内。3.检测发现广西巴马小型猪、长白猪ANK1基因启动子核心区域存在16个单核苷酸多态性位点,分别是-19、-133、-147、-187、-228、-246、-322、-360、-369、-468、-549、-550、-615、-696、-725、-798。其中-798、-468、-369位点SNP在广西巴马小型猪分别为C、C和G,在长白猪中分别为T、G和A。检测德保猪和陆川猪这三个特异性位点时发现,-468位点在广西巴马小型猪、陆川猪、德保猪中均为C,长白猪均为G。4.ANK1基因核心启动子分析预测发现-468位点存在SP1、GCN4、C/EBP alph转录因子结合位点。通过点突变实验构建野生型和突变型双荧光素酶报告基因载体,检测发现突变型和野生型双荧光素酶报告基因载体的荧光素酶活性具有极显著差异(P0.01);构建了 SP1的真核表达载体,与野生型和突变型ANK1双荧光素酶报告基因载体进行共转时,发现荧光素酶活性显著降低(P0.05)。5.利用荧光定量PCR技术检测了巴马小型猪的ANK1基因在猪11个组织中的表达情况,结果揭示这11个组织中ANK1基因均有差异性表达,其中在胰腺中表达量最高,脂肪中表达量最低。成功构建了 ANK1基因CDS区真核表达载体,转染细胞并于转染后48h观察到细胞发出绿色荧光。
[Abstract]:China is the largest producer and consumer of pork in the world. With the rapid development of China's economy, the meat quality of pork is becoming more and more demanding. The modified pork meat quality character will be one of the key.ANK1 (Ankyrinl) in the breeding work (Ankyrins), and its encoded protein AnkR (Ankyrin-R) is a membrane binding domain (membran). E-binding domain) and the three part of the domain (spectrin-binding domain) and the regulatory domain (regulatory domain). This gene is related not only to some meat quality traits of pigs and cattle, but also to the susceptibility to type 2 diabetes, hereditary spherical erythrocytosis, coronary heart disease and Alzheimer's disease. Gene transcription regulation is a very important process in the regulation of gene expression. The transcriptional process of the gene is regulated by the promoter of the key element. The promoter is a DNA sequence that can be combined with RNA polymerase and transcription factor. It is used to regulate gene expression with the corresponding transcription factors. In order to solve the pig ANK1 gene, the gene is used to solve the gene expression. The transcriptional regulation mechanism, this topic uses the Guangxi Bama miniature pig and the Changbai pig as the research object, cloned the promoter region of the ANK1 gene, studied its core region, and detected the SNP of the ANK1 gene promoter region of Guangxi Bama miniature pig, Debao pig, Lu Chuan pig and Changbai pig. The results were as follows: 1. the Guangxi was cloned successfully in Guangxi The ANK1 gene promoter region of Bama miniature pig and long white pig is about 2074bp sequence. The upstream regulation sequence of 5'end of porcine ANK1 gene is intercepted from Ensembl, and the on-line software PromoterScan, NNPP, Promoter2.0, Gene-regulation, MethPrimer, etc. are analyzed. It is found that there are many regulatory elements in the 2074bp fragment of the clone. 2alpha, CEP-bind and so on, at the same time, it was predicted that there were two CpG islands and two core promoter regions in the region,.2. constructed 8 pairs of double luciferase reporter gene carrier of the ANK1 promoter of the long white pig and Guangxi Bama miniature pig, and transfected C2C12 cells to detect the luciferase activity of each carrier, and found the ANK1 promoter of the pig. The highest active fragment was PGL3-P3, and the highest activity fragment of Bama miniature pig was PGL3-P2. It was suggested that the core region of the ANK1 gene promoter was located in the range of -1080 ~ -1991bp by.3. detection in Guangxi Bama miniature pig, and there were 16 single nucleotide polymorphisms in the core region of the ANK1 gene promoter of the long white pig, which were -19, -133, -147, -187, -228, respectively. -322, -360, -369, -468, -549, -550, -615, -696, -725, -798., -798, -468 are found respectively in Guangxi Bama miniature pigs. The NK1 gene core promoter analysis and prediction found that the -468 site had SP1, GCN4, and C/EBP alph transcription factor binding sites. Through the point mutation experiment, the wild type and mutant double luciferase reporter gene carrier was constructed, and the luciferase activity of the mutant and wild type double luciferase reporter gene was found to be extremely significant (P0.01). The eukaryotic expression vector of SP1 was constructed, and when the wild type and mutant ANK1 double luciferase reporter gene was co transferred, the luciferase activity was significantly reduced (P0.05).5. using the fluorescence quantitative PCR technique to detect the expression of ANK1 gene in Bama miniature pigs in 11 pig tissues. The results revealed the ANK1 gene in the 11 tissues. It has the highest expression in the pancreas and the lowest expression in the fat. The eukaryotic expression vector of the ANK1 gene CDS region was successfully constructed, and the cells were transfected and the cells were observed to produce green fluorescence after the transfection of 48h.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

【参考文献】