RAX无义突变引起小鼠角膜混浊及其修饰基因的研究

发布时间：2018-06-14 09:29

本文选题：修饰基因 + 角膜混浊　；参考：《扬州大学》2017年硕士论文

【摘要】：角膜病是当前致盲的主要眼病之一,在我国是继白内障疾病之后的第二大致盲眼病,约占15.4%。目前绝大多数严重威胁人类健康的疾病如糖尿病、高血压、冠状动脉疾病和精神分裂症,还包括儿童先天性缺陷如唇裂、鄂裂及许多先天性心脏病也都受到多基因的调控。本实验室采用ENU化学诱变的方法获得一种角膜混浊表型的小鼠模型,将其主基因鉴定为RAX并将该小鼠命名为RAX突变小鼠。本文是对RAX突变小鼠角膜特点及其修饰基因进行研究,主要分为以下三个方面:1、RAX无义突变小鼠角膜特点的研究本文以RAX无义突变小鼠为研究对象,将该小鼠与正常B6小鼠繁殖,筛选具有角膜混浊表型的杂合子小鼠。对角膜混浊小鼠眼球进行组织学分析发现,突变小鼠角膜基质层及角膜层明显变薄,且角膜表层细胞呈水泡样。再采用免疫组织化学的方法对该8W以上小鼠进行蛋白分子分析,结果显示突变小鼠角膜蛋白Cytokeratin 12(K12)、Cytokeratin 14(K14)、Cytokeratin13(K13)及 PAX6 均发生了变化。2、角膜混浊修饰基因的定位采用将B6背景角膜混浊杂合子小鼠与D2小鼠配种获得F1代小鼠,F1代小鼠继续回交B6获得[(B6×D2)B6]N2代的方案获得角膜混浊杂合子。筛选突变杂合子N2代小鼠1782只,其中180只小鼠表现有角膜混浊表型,通过基因连锁分析结果修饰基因与微卫星D1Mit145连锁(LOD值5.97),初步将该基因定位于小鼠第1号染色体上。为对该修饰基因进行精确定位,在1号染色体上增加微卫星密度,最终将该修饰基因定位于1号染色体 68.38cM-74.68cM 之内。3、角膜混浊修饰基因的鉴定根据定位结果,通过MGI数据库查询该小鼠修饰基因定位区域附近相关基因,并对相关基因进行基因功能分析,最终初步将候选基因确定为:Pou2f1。继而对候选目的基因设计引物,分别PCR扩增候选目的基因,切胶回收后测序,将以上测序结果与本实验中心野生型B6小鼠、D2小鼠基因序列进行对比,筛查突变位点。Pou2f1启动子部分三段引物分别为 Pou2f1717、Pou2f1736、Pou2f1703;Pou2f1 编码区部分三段引物分别为 Pou2f1910、Pou2f11029、Pou2f1556。双向测序结果显示突变小鼠Pou2f1在基因编码区未发现重叠峰而在启动子区域发现多个单核苷酸多态性。
[Abstract]:Corneal disease is one of the main blinding eye diseases at present, and it is the second leading cause of blindness after cataract disease in China, accounting for 15.4%. The vast majority of diseases that pose a serious threat to human health, such as diabetes, hypertension, coronary artery disease and schizophrenia, as well as congenital defects in children such as cleft lip, laceration and many congenital heart diseases, are also regulated by multiple genes. A mouse model of corneal opacity phenotype was obtained by ENU chemical mutagenesis. Its main gene was identified as rax and named as rax mutant mouse. In this paper, the cornea characteristics and its modified genes of rax mutant mice were studied, which were divided into the following three aspects: 1. The cornea characteristics of rax nonsense mutant mice were studied in this paper. The mice were bred with normal B6 mice. To screen heterozygous mice with corneal opacity phenotype. The corneal stroma and corneal layer of the mutant mice were obviously thinned and the corneal surface cells were blisters by histological analysis of the eyes of mice with corneal opacity. The protein molecules of the above 8W mice were analyzed by immunohistochemical method. The results showed that the corneal protein Cytokeratin 12 (K12) and cytokeratin 14K14 (Cytokeratin 13 K13) and PAX6 were changed. The location of corneal opacity modification gene was obtained by mating B6 background corneal opacity heterozygous mice with D2 mice to obtain F1 generation mice. B _ 6 脳 D _ 2 B _ 6 N _ 2 generation was used to obtain corneal opacity heterozygote. 1782 N 2 generation mice with mutated heterozygotes were screened. Among them, 180 mice showed corneal opacity phenotype. The gene was modified by gene linkage analysis and the LOD value of microsatellite D1Mit145 linkage was 5.97. The gene was preliminarily located on chromosome 1 of mice. In order to locate the modified gene accurately, the microsatellite density was increased on chromosome 1. Finally, the modified gene was located within 68.38cM-74.68cM. The MGI database was used to search the related genes near the locational region of the mouse modified gene, and the functional analysis of the related genes was carried out. Finally, the candidate gene was initially identified as: Pou2f1. Then the candidate genes were amplified by PCR and sequenced by PCR. The above sequencing results were compared with those of wild type B6 mice and D 2 mice. The three-piece primer of the promoter was Pou2f1717, Pou2f1736 and Pou2f1703Pou2f1, respectively. The three primers were Pou2f191010, Pou2f11029 and Pou2f1556. the three primer was Pou2f1717, Pou2f1736, Pou2f1703 and Pou2f1, and the three primer was Pou2f191010, Pou2f11029 and Pou2f1556. Bidirectional sequencing showed that no overlapping peaks were found in the gene coding region of the mutant mouse Pou2f1, but multiple single nucleotide polymorphisms were found in the promoter region.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R772.2

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