本文選題：豬 + ANK1基因　； 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：我國(guó)是世界第一大豬肉生產(chǎn)和消費(fèi)國(guó)。隨著中國(guó)經(jīng)濟(jì)的快速發(fā)展,人們對(duì)豬肉的肉質(zhì)要求越來(lái)越高。改良豬肉肉質(zhì)性狀將是育種工作的一個(gè)重點(diǎn)。ANK1(Ankyrinl)屬于錨蛋白家族(Ankyrins)中的一員,其編碼的蛋白 AnkR(Ankyrin-R)由膜結(jié)合域(membrane-binding domain)和血影蛋白結(jié)合域(spectrin-binding domain)及調(diào)控域(regulatory domain)三部分構(gòu)成。該基因不但與豬和牛的部分肉質(zhì)性狀有一定的相關(guān),而且也與2型糖尿病的易感性、遺傳性球形紅細(xì)胞增多癥、冠心病和阿爾茨海默癥等有關(guān)。在基因的表達(dá)調(diào)控過(guò)程中,基因的轉(zhuǎn)錄調(diào)控是一個(gè)非常重要的過(guò)程。基因的轉(zhuǎn)錄過(guò)程受到關(guān)鍵元件啟動(dòng)子的調(diào)控,啟動(dòng)子是一段能與RNA聚合酶和轉(zhuǎn)錄結(jié)合因子結(jié)合的DNA序列,它與相應(yīng)轉(zhuǎn)錄因子相互作用來(lái)調(diào)控基因的表達(dá)。為了解豬ANK1基因的轉(zhuǎn)錄調(diào)控機(jī)制,本課題以廣西巴馬小型豬和長(zhǎng)白豬作為研究對(duì)象,克隆了 ANK1基因的啟動(dòng)子區(qū)域,對(duì)其核心區(qū)域進(jìn)行研究。同時(shí)檢測(cè)了廣西巴馬小型豬、德寶豬、陸川豬和長(zhǎng)白豬ANK1基因啟動(dòng)子區(qū)域的SNP,研究結(jié)果如下:1.成功克隆了廣西巴馬小型豬、長(zhǎng)白豬ANK1基因啟動(dòng)子區(qū)約2074bp序列。從Ensembl截取豬ANK1基因5'端上游調(diào)控序列,利用在線軟件PromoterScan、NNPP、Promoter2.0、Gene-regulation、MethPrimer 等進(jìn)行分析,發(fā)現(xiàn)克隆的2074bp片段存在很多的調(diào)控元件,例如TATAbox、GATA-1、SP1、myogenin、AP-2alpha、CEP-bind等,同時(shí)預(yù)測(cè)表明該區(qū)域存在兩個(gè)CpG島和兩個(gè)核心啟動(dòng)子區(qū)域。2.分別構(gòu)建了長(zhǎng)白豬和廣西巴馬小型豬ANK1啟動(dòng)子缺失片段雙熒光素酶報(bào)告基因載體各8個(gè),并轉(zhuǎn)染C2C12細(xì)胞,檢測(cè)各載體的熒光素酶活性。發(fā)現(xiàn)長(zhǎng)白豬ANK1啟動(dòng)子活性最高的片段為PGL3-P3,巴馬小型豬活性最高片段為PGL3-P2,推測(cè)ANK1基因啟動(dòng)子核心區(qū)域定位為-1080～-1991bp范圍內(nèi)。3.檢測(cè)發(fā)現(xiàn)廣西巴馬小型豬、長(zhǎng)白豬ANK1基因啟動(dòng)子核心區(qū)域存在16個(gè)單核苷酸多態(tài)性位點(diǎn),分別是-19、-133、-147、-187、-228、-246、-322、-360、-369、-468、-549、-550、-615、-696、-725、-798。其中-798、-468、-369位點(diǎn)SNP在廣西巴馬小型豬分別為C、C和G,在長(zhǎng)白豬中分別為T(mén)、G和A。檢測(cè)德保豬和陸川豬這三個(gè)特異性位點(diǎn)時(shí)發(fā)現(xiàn),-468位點(diǎn)在廣西巴馬小型豬、陸川豬、德保豬中均為C,長(zhǎng)白豬均為G。4.ANK1基因核心啟動(dòng)子分析預(yù)測(cè)發(fā)現(xiàn)-468位點(diǎn)存在SP1、GCN4、C/EBP alph轉(zhuǎn)錄因子結(jié)合位點(diǎn)。通過(guò)點(diǎn)突變實(shí)驗(yàn)構(gòu)建野生型和突變型雙熒光素酶報(bào)告基因載體,檢測(cè)發(fā)現(xiàn)突變型和野生型雙熒光素酶報(bào)告基因載體的熒光素酶活性具有極顯著差異(P0.01);構(gòu)建了 SP1的真核表達(dá)載體,與野生型和突變型ANK1雙熒光素酶報(bào)告基因載體進(jìn)行共轉(zhuǎn)時(shí),發(fā)現(xiàn)熒光素酶活性顯著降低(P0.05)。5.利用熒光定量PCR技術(shù)檢測(cè)了巴馬小型豬的ANK1基因在豬11個(gè)組織中的表達(dá)情況,結(jié)果揭示這11個(gè)組織中ANK1基因均有差異性表達(dá),其中在胰腺中表達(dá)量最高,脂肪中表達(dá)量最低。成功構(gòu)建了 ANK1基因CDS區(qū)真核表達(dá)載體,轉(zhuǎn)染細(xì)胞并于轉(zhuǎn)染后48h觀察到細(xì)胞發(fā)出綠色熒光。
[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.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Analysis of gene expression profile of pancreatic carcinoma using cDNA microarray[J];World Journal of Gastroenterology;2003年04期

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本文編號(hào)：1787004