本文選題：修飾基因 + 角膜混濁��； 參考：《揚州大學(xué)》2017年碩士論文

【摘要】：角膜病是當(dāng)前致盲的主要眼病之一,在我國是繼白內(nèi)障疾病之后的第二大致盲眼病,約占15.4%。目前絕大多數(shù)嚴重威脅人類健康的疾病如糖尿病、高血壓、冠狀動脈疾病和精神分裂癥,還包括兒童先天性缺陷如唇裂、鄂裂及許多先天性心臟病也都受到多基因的調(diào)控。本實驗室采用ENU化學(xué)誘變的方法獲得一種角膜混濁表型的小鼠模型,將其主基因鑒定為RAX并將該小鼠命名為RAX突變小鼠。本文是對RAX突變小鼠角膜特點及其修飾基因進行研究,主要分為以下三個方面:1、RAX無義突變小鼠角膜特點的研究本文以RAX無義突變小鼠為研究對象,將該小鼠與正常B6小鼠繁殖,篩選具有角膜混濁表型的雜合子小鼠。對角膜混濁小鼠眼球進行組織學(xué)分析發(fā)現(xiàn),突變小鼠角膜基質(zhì)層及角膜層明顯變薄,且角膜表層細胞呈水泡樣。再采用免疫組織化學(xué)的方法對該8W以上小鼠進行蛋白分子分析,結(jié)果顯示突變小鼠角膜蛋白Cytokeratin 12(K12)、Cytokeratin 14(K14)、Cytokeratin13(K13)及 PAX6 均發(fā)生了變化。2、角膜混濁修飾基因的定位采用將B6背景角膜混濁雜合子小鼠與D2小鼠配種獲得F1代小鼠,F1代小鼠繼續(xù)回交B6獲得[(B6×D2)B6]N2代的方案獲得角膜混濁雜合子。篩選突變雜合子N2代小鼠1782只,其中180只小鼠表現(xiàn)有角膜混濁表型,通過基因連鎖分析結(jié)果修飾基因與微衛(wèi)星D1Mit145連鎖(LOD值5.97),初步將該基因定位于小鼠第1號染色體上。為對該修飾基因進行精確定位,在1號染色體上增加微衛(wèi)星密度,最終將該修飾基因定位于1號染色體 68.38cM-74.68cM 之內(nèi)。3、角膜混濁修飾基因的鑒定根據(jù)定位結(jié)果,通過MGI數(shù)據(jù)庫查詢該小鼠修飾基因定位區(qū)域附近相關(guān)基因,并對相關(guān)基因進行基因功能分析,最終初步將候選基因確定為:Pou2f1。繼而對候選目的基因設(shè)計引物,分別PCR擴增候選目的基因,切膠回收后測序,將以上測序結(jié)果與本實驗中心野生型B6小鼠、D2小鼠基因序列進行對比,篩查突變位點。Pou2f1啟動子部分三段引物分別為 Pou2f1717、Pou2f1736、Pou2f1703;Pou2f1 編碼區(qū)部分三段引物分別為 Pou2f1910、Pou2f11029、Pou2f1556。雙向測序結(jié)果顯示突變小鼠Pou2f1在基因編碼區(qū)未發(fā)現(xiàn)重疊峰而在啟動子區(qū)域發(fā)現(xiàn)多個單核苷酸多態(tài)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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R772.2

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