【摘要】：人DJ-1 (HsDJ-1)是一種與早期帕金森疾病和很多癌癥相關(guān)的蛋白。粟酒裂殖酵母中有1個(gè)與其同源性很高的SpDJ-1蛋白和5個(gè)同源性較遠(yuǎn)的Hsp31蛋白(Hsp3101, Hsp3102, Hsp3103, Hsp3104, Hsp3105)。已有報(bào)道,DJ-1及其同源蛋白在很多的物種中參與抗氧化刺激,但在裂殖酵母中DJ-1蛋白是否參與抗氧化刺激并未報(bào)道且這種保護(hù)機(jī)制并不清楚。本論文主要研究粟酒裂殖酵母細(xì)胞中SpDJ-1和Hsp3101-3105蛋白在不同強(qiáng)度啟動(dòng)子調(diào)控下的蛋白亞細(xì)胞定位、蛋白在不同生長時(shí)期的表達(dá)水平以及SpDJ-1蛋白抗氧化刺激的作用,并對其這種作用機(jī)制做出初步的解釋。我們首先通過GFP熒光標(biāo)記研究發(fā)現(xiàn),在中等強(qiáng)度nmt1啟動(dòng)子調(diào)控下,SpDJ-1、Hsp3101、Hsp3105既存在于細(xì)胞核又存在于細(xì)胞質(zhì)中,Hsp3102僅存在于細(xì)胞質(zhì)中,Hsp3104僅存在于細(xì)胞質(zhì)中且呈胞質(zhì)小點(diǎn)狀,而在基因自身啟動(dòng)子調(diào)控下,SpDJ-1和Hsp3101既定位于細(xì)胞核中也定位于細(xì)胞質(zhì)中,Hsp3102只存在于細(xì)胞質(zhì)中,結(jié)果表明這些同源蛋白定位于不同的亞細(xì)胞器中,預(yù)示著功能或者功能發(fā)揮位置的不一；繼而Western blot檢測發(fā)現(xiàn)SpDJ-1、Hsp3101、Hsp3102、 Hsp3105蛋白對數(shù)期至穩(wěn)態(tài)期有一個(gè)明顯的遞增趨勢,而且在穩(wěn)態(tài)期趨于平穩(wěn),說明蛋白主要是在穩(wěn)態(tài)期表達(dá)并積累,預(yù)示著蛋白可能是在穩(wěn)態(tài)期發(fā)揮作用。最后我們通過檢測敲除SpDJ-1基因和高表達(dá)SpDJ-1基因的菌種對氧化刺激物TBHP、H2O2和溫度刺激時(shí)的應(yīng)激反應(yīng),證實(shí)了裂殖酵母中的SpDJ-1蛋白對氧化壓力和熱激有明顯保護(hù)細(xì)胞的作用。
[Abstract]:Human DJ-1 (HsDJ-1) is a protein associated with early Parkinson's disease and many cancers. There were one SpDJ-1 protein with high homology and five Hsp31 proteins (Hsp3101, Hsp3102, Hsp3103, Hsp3104, Hsp3105) with high homology in Schizoderma cerevisiae. It has been reported that DJ-1 and its homologous proteins are involved in antioxidant stimulation in many species, but whether DJ-1 protein is involved in antioxidant stimulation has not been reported and this protective mechanism is not clear. In this paper, we studied the subcellular localization of SpDJ-1 and Hsp3101-3105 proteins under the control of different intensities of promoters, the expression level of proteins in different growth stages and the role of SpDJ-1 protein in antioxidant stimulation. And to its this kind of action mechanism makes the preliminary explanation. We first found that under the regulation of moderate intensity nmt1 promoter, SpDJ-1,Hsp3101,Hsp3105 existed in both nucleus and cytoplasm, and Hsp3102 only existed in cytoplasm. Hsp3104 existed only in the cytoplasm and showed a small cytoplasmic dot. Under the regulation of gene itself promoter, SpDJ-1 and Hsp3101 were located in the cytoplasm, and Hsp3102 only existed in the cytoplasm. The results showed that these homologous proteins were located in different suborganelles, indicating different functional or functional roles. Western blot analysis showed that there was an obvious increasing trend from logarithmic phase to steady-state phase of SpDJ-1,Hsp3101,Hsp3102, Hsp3105 protein, and it tended to be stable in steady-state phase, indicating that protein was expressed and accumulated mainly in steady-state phase. This indicates that proteins may play a role in the steady-state period. Finally, we tested the stress response of knockout SpDJ-1 gene and high expression SpDJ-1 gene strain to oxidative stimulator TBHP,H2O2 and temperature stimulation. It was confirmed that the SpDJ-1 protein in the merozoite yeast had obvious protective effect on oxidative pressure and heat shock.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R742.5;Q936

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1 陳彩平;裂殖酵母中人DJ-1同源蛋白亞細(xì)胞定位及SpDJ-1在脅迫反應(yīng)中作用的研究[D];南京師范大學(xué);2015年

2 黃霖霆;粟酒裂殖酵母中SpDJ-1，，Hsp3101-Hsp3105的轉(zhuǎn)錄水平及其調(diào)控途徑的研究[D];南京師范大學(xué);2015年

本文編號(hào)：2343979