大肠杆菌热休克蛋白DnaK和DnaJ对细胞周期的影响

发布时间：2017-12-28 20:20

  本文关键词：大肠杆菌热休克蛋白DnaK和DnaJ对细胞周期的影响　出处：《内蒙古大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 大肠杆菌 DnaK DnaJ DNA复制起始 细胞周期

【摘要】：细胞周期的准确调控对于细胞生命活动非常重要,热休克蛋白对细胞周期的影响尚未见深入的研究报道。本实验以大肠杆菌热休克蛋白DnaK和DnaJ为研究对象,探讨热休克蛋白对细胞周期的影响。首先,检测△dnaK和△dnaJ突变细胞的细胞周期参数,发现dnaK基因缺失引起了染色体复制起始的延迟,而dnaJ的缺失引起了染色体复制起始的提前。而且,△dnaK和△dnaJ突变体的细胞大小以及细胞生长速度与野生型细胞也有所差异。在缺失突变体中分别恢复表达DnaK和DnaJ蛋白能够使这些表型缺陷得以恢复。在野生型细胞中分别过表达DnaK和DnaJ蛋白使细胞周期的各个参数与缺失突变体的参数呈相反趋势。通过测定单个细胞的总蛋白含量和复制起始蛋白DnaA的含量,发现△dnaK突变中的这两个含量相对于野生型细胞都有所减少;在△dnaK突变细胞中以质粒表达DnaK蛋白时,总蛋白和DnaA含量与野生型细胞比差别不大;在野生型细胞中过表达DnaK使总蛋白和DnaA含量都有所提高。而△dnaJ突变、△dnaJ/pdnaJ以及过表达DnaJ后,单细胞的总蛋白和DnaA含量与野生型细胞相比无明显差别。这说明DnaK有可能是通过影响DnaA量来间接影响染色体复制起始,而DnaJ可能是通过其他途径影响复制起始。通过细菌双杂交实验,发现DnaK和DnaJ与核糖体休眠因子YfiA蛋白之间有相互作用。通过对基因dnaK和dnaJ点突变发现了 DnaK蛋白的核心氨基酸是位于与核苷酸相互作用功能域上的赖氨酸、组氨酸和精氨酸;DnaJ核心氨基酸为位于与DnaK相互作用功能域上的组氨酸和天冬氨酸。分析△yfiA△dnaK或△yfiA△dnaJ双突变体,发现两者染色体复制起始非同步化并且染色体复制不完整,细胞肥大呈椭圆形并有出芽现象。稳定期△yfiA△dnaK或△yfiA△dnaJ细胞有DNA复制而且细胞出芽现象更为明显。
[Abstract]:The accurate regulation of cell cycle is very important for cell life, and the effect of heat shock protein on cell cycle has not yet been reported. In this experiment, the effect of heat shock protein (HSPs) on the cell cycle of Escherichia coli heat shock protein DnaK and DnaJ was studied. First, detect cell cycle parameters of delta dnaK and delta dnaJ mutant cells, found dnaK gene deletion caused delayed chromosome replication initiation, and the loss of dnaJ caused the early initiation of chromosome replication. Also, Delta dnaK and delta dnaJ mutant cell size and cell growth rate and wild-type cells are also different. The recovery of these phenotypic defects can be made by restoring the expression of DnaK and DnaJ protein in the missing mutants. The overexpression of DnaK and DnaJ protein in wild type cells showed the opposite trend of the parameters of the cell cycle with the parameters of the missing mutant. The total protein content was measured by single cell and replication initiation protein content of DnaA, it was found that the two content of delta dnaK mutations in wild-type cells were decreased compared with dnaK mutant cells; in a plasmid expression of DnaK protein, total protein and DnaA content compared with wild-type cells overexpressing DnaK had no significant difference; in wild type cells in the total protein and DnaA content was increased. DnaJ, Delta dnaJ/pdnaJ and delta mutation and overexpression of DnaJ, the total protein content and DnaA content of single cells compared to wild-type cells had no significant difference. This suggests that DnaK may indirectly affect the replication initiation of chromosomes by affecting the amount of DnaA, and DnaJ may affect replication initiation through other pathways. The interaction between DnaK and DnaJ and the ribosome dormancy factor YfiA protein was found through the double cross test. The point mutation of gene dnaK and dnaJ revealed that the core amino acid of DnaK protein is lysine, histidine and arginine located in the functional domain of nucleotide interaction. DnaJ core amino acid is histidine and aspartate located in the interaction domain of DnaK. Analysis of delta yfiA Delta dnaK delta or delta yfiA dnaJ double mutant, found that the initiation of chromosome replication and non synchronization of chromosome replication is not complete, and oval cell hypertrophy sprouting. Stable yfiA Delta dnaK delta or delta yfiA Delta dnaJ cell DNA replication and cell budding phenomenon is more obvious.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51

【参考文献】

相关期刊论文 前3条

1 郎明林,张玉秀,柴团耀;基因工程改良植物重金属抗性与富集能力的研究进展[J];生物工程学报;2004年02期

2 张旭辉;热休克蛋白70与热耐受的机制[J];解放军预防医学杂志;2001年01期

3 朱大栩;马更生;;热休克蛋白的生物学功能[J];国外医学(分子生物学分册);1993年06期

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