本文關鍵詞： 民豬 FoxN1基因 轉錄因子 冷應激　出處：《東北農(nóng)業(yè)大學》2016年碩士論文　論文類型：學位論文

【摘要】：應激反應通常是在動物受到較大頻率、較長時間或較短時間但變化劇烈的刺激時產(chǎn)生的,這種應激反應會改變動物機體內環(huán)境穩(wěn)定性、生理和行為等。冷應激是最常見的一種應激反應,會造成機體免疫力下降,生長緩慢,嚴重的可導致死亡。胸腺是動物機體一個重要的免疫器官,是T淋巴細胞的發(fā)生場所。叉頭蛋白N1(Forkhead box protein N1,FoxN1)作為翼狀螺旋/叉頭型轉錄因子家族的一員,它的突變或缺失都會引起胸腺發(fā)育出現(xiàn)嚴重障礙或缺失的現(xiàn)象,因此該轉錄因子在維持胸腺細胞、支持T細胞發(fā)育中扮演重要角色。本研究針對豬的Foxn N1基因,采用分子生物學方法,對該基因的轉錄調控以及冷應激對該基因的調控機理進行了研究。主要內容包括:克隆了民豬FoxN1基因5’側翼的1 001bp序列;通過生物信息學方法預測豬FoxN1基因5’側翼區(qū)1 001bp內潛在轉錄因子的結合位點;構建不同長度缺失片段豬FoxN1基因5’側翼序列調控的報告基因熒光表達載體,利用雙熒光素酶報告系統(tǒng)分析豬FoxN1基因核心啟動子區(qū)域位置,利用過表達技術對篩選出的轉錄因子進行驗證。采用定量PCR檢測民豬和大白豬的FoxN1基因冷應激后在轉錄水平上的變化;采用Western-blot檢測民豬和大白豬的FoxN1基因冷應激后在翻譯水平上的變化。主要結果如下:(1)克隆了豬FoxN1基因5’側翼的1 001bp序列,經(jīng)測序及序列比對,該基因與Gen Bank中豬FoxN1序列(NC_010454)同源性達到93.63%。(2)啟動子活性檢測過程中,第一輪構建了3個系列缺失載體,將起始密碼子ATG中的A定義為+1,分別命名為p A(-973/+28)-FoxN1、pB(-620/+28)-FoxN1和pC(-224/+28)-FoxN1。第二輪構建3個質粒,分別命名為p D(-332/+28)-FoxN1、p E(-438/+28)-FoxN1和p F(-530/+28)-FoxN1。以上各質粒經(jīng)Nhe I和HindⅢ雙酶切鑒定并測序后證明構建成功。(3)克隆了豬GATA-1基因完整編碼區(qū)1 239bp,經(jīng)測序及序列比對,該基因與Gen Bank中豬GATA-1序列(NC_010461)同源性達到99%。克隆了豬SP1基因完整編碼區(qū)2 361bp,經(jīng)測序及序列比對,該基因與Gen Bank中豬SP1序列(NC_010447)同源性達到99%。上述兩個基因分別連入pcDNA3.1(+)載體,經(jīng)Eco R I和Xba I雙酶切鑒定及測序,成功構建了GATA-1和SP1的真核表達載體。(4)經(jīng)雙熒光素酶檢測發(fā)現(xiàn)ATG上游-298bp~-291bp處的GATA-1及SP1轉錄元件是影響FoxN1基因啟動子活性的關鍵區(qū)域,分別單獨缺失該轉錄元件結合區(qū)域會顯著影響FoxN1基因的啟動子活性。過表達SP1轉錄因子可以顯著促進FoxN1基因的表達,證實SP1轉錄因子是FoxN1基因轉錄的關鍵因子。(5)Real-time PCR和Western-blot結果均表明,冷應激后,民豬胸腺中FoxN1基因的表達量顯著上升(p0.05),而大白豬胸腺中FoxN1基因的表達呈下降趨勢。
[Abstract]:Stress reactions usually occur when animals are stimulated with greater frequency, duration, or short time, but change dramatically, which can change the stability of the environment in the animal body. Cold stress is one of the most common stress reactions, which can lead to the decrease of immunity, slow growth and death. Thymus is an important immune organ in animals. Forkhead box protein N1FoxN1) is a member of the pterygoid helix / fork head transcription factor family. Its mutation or deletion can lead to serious thymus development disorders or deletions, so the transcription factor in the maintenance of thymocytes. Supporting T cells play an important role in the development of porcine Foxn N1 gene. The transcriptional regulation of the gene and the regulation mechanism of cold stress on the gene were studied. The main contents were as follows: 1 001bp sequence of 5'flanking of FoxN1 gene was cloned; The binding sites of potential transcription factors in 5'flanking region of porcine FoxN1 gene were predicted by bioinformatics. To construct the reporter gene fluorescent expression vector regulated by 5'flanking sequence of porcine FoxN1 gene with different length deletions, and to analyze the location of the core promoter region of porcine FoxN1 gene by double luciferase reporting system. The transcriptional factors were verified by overexpression technique. Quantitative PCR was used to detect the changes of FoxN1 gene transcription level after cold stress in Min pig and large white pig. Western-blot was used to detect the changes in translation level of FoxN1 gene in Min pig and large white pig after cold stress. The main results were as follows: 1). The 5'flanking sequence of porcine FoxN1 gene was cloned. After sequencing and sequence alignment, the homology of the gene with the porcine FoxN1 sequence NCSCO 10454 in Gen Bank was 93.63. 2) the activity of the promoter was detected. In the first round, three series of deletion vectors were constructed, and the starting codon A in ATG was defined as 1, and named as p Agn-973 / 28C-FoxN1, respectively. Three plasmids were constructed in the second round: pBN-620 / 28-FoxN1 and pCN-224 / 28-FoxN1.Three plasmids were constructed in the second round. They were named as pD- 332 / 28- FoxN1, respectively. P / Ene-438 / 28 / 28 -FoxN1 and p Fang-530 / 28). The above plasmids were identified and sequenced by Nhe I and Hind 鈪，

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