本文選題：TET3 + O-GlcNAc糖基化��； 參考：《西北農(nóng)林科技大學》2015年碩士論文

【摘要】：O-GlcNAc(O-linked N-acetylglucosamine,O-GlcNAc)糖基化修飾是存在于細胞質(zhì)和細胞核蛋白質(zhì)絲氨酸或蘇氨酸殘基的羥基基團上的一種翻譯后單糖修飾。O-GlcNAc糖基化修飾通過調(diào)控蛋白質(zhì)細胞定位、蛋白活性、蛋白穩(wěn)定等參與多種生命過程。O-GlcNAc糖基化修飾由兩個酶催化,氧連氮乙酰葡萄糖胺轉(zhuǎn)移酶(O-linked N-acetylglucosamine transferase,OGT)和氧連氮乙酰葡萄胺糖苷酶(O-GlcNAcase,OGA)。其中OGT負責O-GlcNAc的添加,OGA負責O-GlcNAc的移除。TET3蛋白是TET(ten-eleven translocation)蛋白家族成員之一,屬于α-酮戊二酸和Fe2+依賴的雙加氧酶。精卵發(fā)生受精后,TET3蛋白能特異性介導父源基因組發(fā)生快速的主動去甲基化,在胚胎早期發(fā)育和重編程過程中發(fā)揮重要作用。近期研究發(fā)現(xiàn),人OGT不僅和TET3蛋白之間存在相互作用而且能夠調(diào)控TET3蛋白的亞細胞定位和酶活性。這些研究結果提示我們,OGT可能通過調(diào)控TET3蛋白的亞細胞定位和功能來影響早期胚胎的DNA去甲基化過程,進而對胚胎發(fā)育和重編程產(chǎn)生影響。本實驗以小鼠為模型,較系統(tǒng)地研究了小鼠OGT對TET3蛋白亞細胞定位的調(diào)控作用,比較了小鼠、人、牛、羊和豬的TET3和OGT基因的同源性,并檢測了牛體細胞核移植胚胎和體外受精胚胎早期發(fā)育過程中TET3和與O-GlcNAc糖基化修飾相關基因的表達情況。本實驗的主要研究內(nèi)容和結果如下:1.以小鼠為模型,分別構建小鼠TET3蛋白和OGT蛋白過表達載體。當在NIH3T3細胞中過表達TET3蛋白時,免疫熒光顯示TET3蛋白主要定位在細胞核;而在NIH3T3細胞中同時過表達TET3蛋白和OGT蛋白時,大部分TET3蛋白定位于細胞質(zhì)。實驗結果表明OGT蛋白具有調(diào)控TET3蛋白亞細胞定位的作用。2.為了研究OGT是否依賴其O-GlcNAc轉(zhuǎn)移酶活性的調(diào)控TET3蛋白的亞細胞定位,我們使用OGT抑制劑Alloxan對過表達TET3蛋白和OGT蛋白的NIH3T3細胞進行處理,結果發(fā)現(xiàn)處理后TET3蛋白又定位于細胞核。這表明OGT依賴其O-GlcNAc轉(zhuǎn)移酶活性來調(diào)控TET3的核定位。通過使用OGA的抑制劑PUGNAc處理過表達TET3蛋白的NIH3T3細胞,我們發(fā)現(xiàn)PUGNAc處理促進了TET3蛋白的出核;通過改變過表達TET3蛋白的NIH3T3細胞培養(yǎng)液中的葡萄糖濃度,我們發(fā)現(xiàn)低葡萄糖培養(yǎng)液同樣抑制TET3蛋白的出核,降低OGT對TET3蛋白亞細胞定位的調(diào)控作用。這些結果證明OGT通過影響TET3蛋白的O-GlcNAc糖基化水平來調(diào)控TET3蛋白的細胞定位。3.為了進一步探究OGT調(diào)控TET3蛋白亞細胞定位的具體機制,我們構建TET3蛋白核定位信號(nuclear localization signal,NLS)缺失的表達載體;結果顯示無NLS的TET3蛋白主要定位于細胞質(zhì),這提示我們O-GlcNAc糖基化修飾可能通過影響TET3蛋白的NLS來調(diào)控TET3蛋白的亞細胞定位。4.用生物信息學軟件比對了小鼠、人、牛、山羊和豬的TET3和OGT基因序列,結果顯示TET3和OGT基因在這些物種間具有較高的同源性。將小鼠TET3蛋白轉(zhuǎn)入牛成纖維細胞可以正常發(fā)揮其羥甲基化功能,增強牛成纖維細胞的5hmc水平。對牛體細胞核移植胚胎和體外受精胚胎早期發(fā)育過程各時期的TET3和OGT基因進行qRT-PCR檢測,結果發(fā)現(xiàn)TET3基因在胚胎8-細胞期前高表達,桑椹胚和囊胚幾乎不表達;OGT基因也在早期胚胎中表達較高。對牛核移植胚胎和體外受精胚胎發(fā)育過程中甲基化水平進行檢測發(fā)現(xiàn),牛核移植和體外受精胚胎在胚胎基因組激活前都發(fā)生了DNA去甲基化過程,說明TET3蛋白在早期胚胎發(fā)育的DNA去甲基化過程中發(fā)揮重要作用。以上結果表明,OGT對TET3蛋白亞細胞定位的調(diào)控作用可以用于改善牛核移植胚胎去甲基化不完全的研究。綜合以上結果,本實驗發(fā)現(xiàn)小鼠OGT通過改變蛋白的O-GlcNAc糖基化水平調(diào)控TET3蛋白的亞細胞定位;小鼠、人、牛、羊和豬的TET3和OGT基因具有較高的同源性;TET3基因和OGT基因都在早期胚胎中表達較高。這些結果提示我們OGT對TET3蛋白亞細胞定位的調(diào)控作用也可以用于改善牛核移植胚胎去甲基化不完全的研究,為提高牛核移植重編程效率的研究提供了新的思路和理論依據(jù)。
[Abstract]:O-GlcNAc (O-linked N-acetylglucosamine, O-GlcNAc) glycosylated modification is a kind of translated monosaccharide modified.O-GlcNAc glycosylation modification that exists on the cytoplasm and nuclear protein serine or threonine residues. The glycosylation modification of.O-GlcNAc is involved in a variety of life processes by regulating protein cell location, protein viability, protein stability and so on. Glycosylated modification is catalyzed by two enzymes, O-linked N-acetylglucosamine transferase, OGT, and O-GlcNAcase, OGA. OGT is responsible for the addition of O-GlcNAc. OGA responsible for the removal of O-GlcNAc is one of the members of the TET protein family, It belongs to alpha ketopamyl diacid and Fe2+ dependent dioxygenase. After fertilized sperm, TET3 protein can specifically mediate the active demethylation of the parent genome, and play an important role in early embryonic development and reprogramming. Recent studies have found that human OGT not only has interaction with TET3 protein, but also regulates TET. The subcellular localization and enzyme activity of the 3 protein suggest that OGT may affect the DNA demethylation process of early embryos by regulating the subcellular localization and function of TET3 protein, and then influence embryo development and reprogramming. In this experiment, a mouse model was used to systematically study the TET3 protein subcells of mouse OGT. The homology of TET3 and OGT genes in mice, human, cattle, sheep and pigs was compared, and the expression of TET3 and O-GlcNAc glycosylation related genes during the early development of bovine somatic cell nuclear transfer embryos and in vitro fertilized embryos were detected. The main contents and results of this experiment were as follows: 1. the model of mice was divided into mice. TET3 protein and OGT protein overexpression vector were not constructed. When the TET3 protein was overexpressed in NIH3T3 cells, the immunofluorescence showed that the TET3 protein was mainly located in the nucleus; and when the TET3 protein and OGT protein were overexpressed in NIH3T3 cells, most of the TET3 protein was located in the cytoplasm. The experimental results showed that the OGT protein had the regulation of TET3 protein. The role of subcellular localization.2. in order to study whether OGT depends on the subcellular localization of the TET3 protein that is dependent on its O-GlcNAc transferase activity, we use OGT inhibitor Alloxan to process NIH3T3 cells expressing the TET3 protein and OGT protein, and the results show that the TET3 protein is located in the nucleus after treatment. This indicates that OGT depends on its O-GlcNAc transfer. Enzyme activity was used to regulate the nuclear location of TET3. By using the OGA inhibitor PUGNAc to treat NIH3T3 cells expressing TET3 protein, we found that PUGNAc treatment promoted the nucleation of TET3 protein; by changing the glucose concentration in the NIH3T3 cell culture medium that overexpressed the TET3 protein, we found that the low glucose culture also inhibited the TET3 protein. Nucleation, reducing the regulatory role of OGT on the subcellular localization of TET3 protein. These results show that OGT regulates the cell localization of TET3 protein by affecting the O-GlcNAc glycosylation level of TET3 protein to further explore the specific mechanism of OGT regulating TET3 protein subcellular localization. We construct TET3 protein nuclear localization signal (nuclear localization) Nal, NLS) missing expression vector; the results show that the TET3 protein without NLS is mainly located in the cytoplasm, which suggests that the O-GlcNAc glycosylation modification may regulate the subcellular localization of TET3 protein by the NLS of the TET3 protein to regulate the.4. using bioinformatics software to compare the TET3 and OGT gene sequences of mice, cattle, goats and pigs. The results show TET The 3 and OGT genes have high homology among these species. The transfer of mouse TET3 protein into bovine fibroblast cells can normally play its hydroxyl methylation and enhance the 5hmc level of bovine fibroblasts. QRT-PCR detection of TET3 and OGT genes in the early development period of bovine and in vitro fertilized embryos. It was found that the TET3 gene was highly expressed before the 8- cell stage of the embryo, and the morula and blastocysts were almost not expressed, and the OGT gene was also highly expressed in the early embryos. The detection of the methylation level in the bovine embryo and the development of in vitro fertilized embryos found that the bovine and in vitro fertilized embryos were all DNA before the activation of the embryo genome. The methylation process indicates that TET3 protein plays an important role in the DNA demethylation of early embryonic development. The above results show that the regulatory role of OGT on the subcellular localization of TET3 protein can be used to improve the study of the incomplete demethylation of bovine nuclear transplants. The results of the above results show that the mouse OGT can be used to change the O-Gl of the protein in the mouse. The level of cNAc glycosylation regulates the subcellular localization of TET3 protein; the TET3 and OGT genes of mice, human, cattle, sheep and pigs have high homology; both the TET3 gene and the OGT gene are highly expressed in the early embryos. These results suggest that the regulation of OGT on the subcellular localization of TET3 protein can also be used to improve the demethylation of bovine nuclear transplantation embryos. Incomplete research provides a new idea and theoretical basis for improving the reprogramming efficiency of bovine nuclear transfer.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.23

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