民猪FoxN1基因启动子核心区的鉴定及对冷应激的应答研究

发布时间：2018-01-26 04:11

  本文关键词： 民猪 FoxN1基因 转录因子 冷应激　出处：《东北农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：应激反应通常是在动物受到较大频率、较长时间或较短时间但变化剧烈的刺激时产生的,这种应激反应会改变动物机体内环境稳定性、生理和行为等。冷应激是最常见的一种应激反应,会造成机体免疫力下降,生长缓慢,严重的可导致死亡。胸腺是动物机体一个重要的免疫器官,是T淋巴细胞的发生场所。叉头蛋白N1(Forkhead box protein N1,FoxN1)作为翼状螺旋/叉头型转录因子家族的一员,它的突变或缺失都会引起胸腺发育出现严重障碍或缺失的现象,因此该转录因子在维持胸腺细胞、支持T细胞发育中扮演重要角色。本研究针对猪的Foxn N1基因,采用分子生物学方法,对该基因的转录调控以及冷应激对该基因的调控机理进行了研究。主要内容包括:克隆了民猪FoxN1基因5’侧翼的1 001bp序列;通过生物信息学方法预测猪FoxN1基因5’侧翼区1 001bp内潜在转录因子的结合位点;构建不同长度缺失片段猪FoxN1基因5’侧翼序列调控的报告基因荧光表达载体,利用双荧光素酶报告系统分析猪FoxN1基因核心启动子区域位置,利用过表达技术对筛选出的转录因子进行验证。采用定量PCR检测民猪和大白猪的FoxN1基因冷应激后在转录水平上的变化;采用Western-blot检测民猪和大白猪的FoxN1基因冷应激后在翻译水平上的变化。主要结果如下:(1)克隆了猪FoxN1基因5’侧翼的1 001bp序列,经测序及序列比对,该基因与Gen Bank中猪FoxN1序列(NC_010454)同源性达到93.63%。(2)启动子活性检测过程中,第一轮构建了3个系列缺失载体,将起始密码子ATG中的A定义为+1,分别命名为p A(-973/+28)-FoxN1、pB(-620/+28)-FoxN1和pC(-224/+28)-FoxN1。第二轮构建3个质粒,分别命名为p D(-332/+28)-FoxN1、p E(-438/+28)-FoxN1和p F(-530/+28)-FoxN1。以上各质粒经Nhe I和HindⅢ双酶切鉴定并测序后证明构建成功。(3)克隆了猪GATA-1基因完整编码区1 239bp,经测序及序列比对,该基因与Gen Bank中猪GATA-1序列(NC_010461)同源性达到99%。克隆了猪SP1基因完整编码区2 361bp,经测序及序列比对,该基因与Gen Bank中猪SP1序列(NC_010447)同源性达到99%。上述两个基因分别连入pcDNA3.1(+)载体,经Eco R I和Xba I双酶切鉴定及测序,成功构建了GATA-1和SP1的真核表达载体。(4)经双荧光素酶检测发现ATG上游-298bp~-291bp处的GATA-1及SP1转录元件是影响FoxN1基因启动子活性的关键区域,分别单独缺失该转录元件结合区域会显著影响FoxN1基因的启动子活性。过表达SP1转录因子可以显著促进FoxN1基因的表达,证实SP1转录因子是FoxN1基因转录的关键因子。(5)Real-time PCR和Western-blot结果均表明,冷应激后,民猪胸腺中FoxN1基因的表达量显著上升(p0.05),而大白猪胸腺中FoxN1基因的表达呈下降趋势。
[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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