裂殖酵母中人DJ-1同源蛋白亚细胞定位及SpDJ-1在胁迫反应中作用的研究
发布时间：2018-11-20 05:56
【摘要】：人DJ-1 (HsDJ-1)是一种与早期帕金森疾病和很多癌症相关的蛋白。粟酒裂殖酵母中有1个与其同源性很高的SpDJ-1蛋白和5个同源性较远的Hsp31蛋白(Hsp3101, Hsp3102, Hsp3103, Hsp3104, Hsp3105)。已有报道,DJ-1及其同源蛋白在很多的物种中参与抗氧化刺激,但在裂殖酵母中DJ-1蛋白是否参与抗氧化刺激并未报道且这种保护机制并不清楚。本论文主要研究粟酒裂殖酵母细胞中SpDJ-1和Hsp3101-3105蛋白在不同强度启动子调控下的蛋白亚细胞定位、蛋白在不同生长时期的表达水平以及SpDJ-1蛋白抗氧化刺激的作用,并对其这种作用机制做出初步的解释。我们首先通过GFP荧光标记研究发现,在中等强度nmt1启动子调控下,SpDJ-1、Hsp3101、Hsp3105既存在于细胞核又存在于细胞质中,Hsp3102仅存在于细胞质中,Hsp3104仅存在于细胞质中且呈胞质小点状,而在基因自身启动子调控下,SpDJ-1和Hsp3101既定位于细胞核中也定位于细胞质中,Hsp3102只存在于细胞质中,结果表明这些同源蛋白定位于不同的亚细胞器中,预示着功能或者功能发挥位置的不一；继而Western blot检测发现SpDJ-1、Hsp3101、Hsp3102、 Hsp3105蛋白对数期至稳态期有一个明显的递增趋势,而且在稳态期趋于平稳,说明蛋白主要是在稳态期表达并积累,预示着蛋白可能是在稳态期发挥作用。最后我们通过检测敲除SpDJ-1基因和高表达SpDJ-1基因的菌种对氧化刺激物TBHP、H2O2和温度刺激时的应激反应,证实了裂殖酵母中的SpDJ-1蛋白对氧化压力和热激有明显保护细胞的作用。
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R742.5;Q936
本文编号：2343979
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R742.5;Q936
【相似文献】
相关硕士学位论文 前2条
1 陈彩平;裂殖酵母中人DJ-1同源蛋白亚细胞定位及SpDJ-1在胁迫反应中作用的研究[D];南京师范大学;2015年
2 黄霖霆;粟酒裂殖酵母中SpDJ-1，，Hsp3101-Hsp3105的转录水平及其调控途径的研究[D];南京师范大学;2015年
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