本文關(guān)鍵詞：大腸桿菌熱休克蛋白DnaK和DnaJ對(duì)細(xì)胞周期的影響　出處：《內(nèi)蒙古大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 大腸桿菌 DnaK DnaJ DNA復(fù)制起始 細(xì)胞周期

【摘要】：細(xì)胞周期的準(zhǔn)確調(diào)控對(duì)于細(xì)胞生命活動(dòng)非常重要,熱休克蛋白對(duì)細(xì)胞周期的影響尚未見深入的研究報(bào)道。本實(shí)驗(yàn)以大腸桿菌熱休克蛋白DnaK和DnaJ為研究對(duì)象,探討熱休克蛋白對(duì)細(xì)胞周期的影響。首先,檢測(cè)△dnaK和△dnaJ突變細(xì)胞的細(xì)胞周期參數(shù),發(fā)現(xiàn)dnaK基因缺失引起了染色體復(fù)制起始的延遲,而dnaJ的缺失引起了染色體復(fù)制起始的提前。而且,△dnaK和△dnaJ突變體的細(xì)胞大小以及細(xì)胞生長(zhǎng)速度與野生型細(xì)胞也有所差異。在缺失突變體中分別恢復(fù)表達(dá)DnaK和DnaJ蛋白能夠使這些表型缺陷得以恢復(fù)。在野生型細(xì)胞中分別過表達(dá)DnaK和DnaJ蛋白使細(xì)胞周期的各個(gè)參數(shù)與缺失突變體的參數(shù)呈相反趨勢(shì)。通過測(cè)定單個(gè)細(xì)胞的總蛋白含量和復(fù)制起始蛋白DnaA的含量,發(fā)現(xiàn)△dnaK突變中的這兩個(gè)含量相對(duì)于野生型細(xì)胞都有所減少;在△dnaK突變細(xì)胞中以質(zhì)粒表達(dá)DnaK蛋白時(shí),總蛋白和DnaA含量與野生型細(xì)胞比差別不大;在野生型細(xì)胞中過表達(dá)DnaK使總蛋白和DnaA含量都有所提高。而△dnaJ突變、△dnaJ/pdnaJ以及過表達(dá)DnaJ后,單細(xì)胞的總蛋白和DnaA含量與野生型細(xì)胞相比無明顯差別。這說明DnaK有可能是通過影響DnaA量來間接影響染色體復(fù)制起始,而DnaJ可能是通過其他途徑影響復(fù)制起始。通過細(xì)菌雙雜交實(shí)驗(yàn),發(fā)現(xiàn)DnaK和DnaJ與核糖體休眠因子YfiA蛋白之間有相互作用。通過對(duì)基因dnaK和dnaJ點(diǎn)突變發(fā)現(xiàn)了 DnaK蛋白的核心氨基酸是位于與核苷酸相互作用功能域上的賴氨酸、組氨酸和精氨酸;DnaJ核心氨基酸為位于與DnaK相互作用功能域上的組氨酸和天冬氨酸。分析△yfiA△dnaK或△yfiA△dnaJ雙突變體,發(fā)現(xiàn)兩者染色體復(fù)制起始非同步化并且染色體復(fù)制不完整,細(xì)胞肥大呈橢圓形并有出芽現(xiàn)象。穩(wěn)定期△yfiA△dnaK或△yfiA△dnaJ細(xì)胞有DNA復(fù)制而且細(xì)胞出芽現(xiàn)象更為明顯。
[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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q51

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 郎明林,張玉秀,柴團(tuán)耀;基因工程改良植物重金屬抗性與富集能力的研究進(jìn)展[J];生物工程學(xué)報(bào);2004年02期

2 張旭輝;熱休克蛋白70與熱耐受的機(jī)制[J];解放軍預(yù)防醫(yī)學(xué)雜志;2001年01期

3 朱大栩;馬更生;;熱休克蛋白的生物學(xué)功能[J];國(guó)外醫(yī)學(xué)(分子生物學(xué)分冊(cè));1993年06期

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