【摘要】：本文以基因敲除方法研究痢疾桿菌的功能基因，以RecA重組系統(tǒng)為基礎(chǔ)，采用低拷貝自殺質(zhì)粒pCVD442作為本敲除體系的重組載體，為了克服該質(zhì)粒在進(jìn)行分子克隆操作中的困難，將Gateway技術(shù)應(yīng)用在敲除前期的重組質(zhì)粒構(gòu)建過(guò)程中，成功構(gòu)建了痢疾志賀菌A1型SD51197株運(yùn)鐵相關(guān)基因IroN、ShuA部分缺失和插入的單、雙突變體MTS-1、MTS-2、MTS，從而建立了在痢疾桿菌中進(jìn)行定位插入或缺失突變的敲除技術(shù)平臺(tái)。 對(duì)各突變株分別在培養(yǎng)基、細(xì)胞和動(dòng)物三個(gè)水平進(jìn)行了功能檢測(cè)。在豐富培養(yǎng)基中各突變株與野生株生長(zhǎng)沒(méi)有顯著差異；在添加150μM鐵鰲合劑DIP的條件下，各突變體的生長(zhǎng)水平都從4h開(kāi)始明顯低于野生株，在培養(yǎng)基中補(bǔ)加鐵離子可以使各突變株的生長(zhǎng)回復(fù)到豐富培養(yǎng)基條件下的水平；在HeLa細(xì)胞和U937細(xì)胞的胞內(nèi)存活增殖能力和胞間擴(kuò)散能力以及豚鼠角膜結(jié)膜炎實(shí)驗(yàn)中，各突變株均沒(méi)有明顯的毒力變化，但在HeLa細(xì)胞侵襲過(guò)程中添加65μM的DIP，突變株MTS-1、MTS-2、MTS的胞內(nèi)存活增殖能力與野生株相比下降了1／4～1／2。上述結(jié)果提示痢疾桿菌中IroN、ShuA基因與細(xì)菌的鐵轉(zhuǎn)運(yùn)相關(guān)，可能由于細(xì)菌中其它鐵轉(zhuǎn)運(yùn)相關(guān)系統(tǒng)的存在代償了敲除基因的功能缺陷，因此突變體在毒力水平?jīng)]有特別顯著的變化。 利用SD51197全基因組芯片進(jìn)行了鐵豐富和鐵限制條件下突變株和野生株的表達(dá)譜比較分析，結(jié)果表明：鐵豐富條件下各突變體上調(diào)基因數(shù)目多于下調(diào)基因，雙突變體中變化基因的數(shù)目及幅度高于任意單突變體。鐵限制條件下，整體上各突變體對(duì)缺鐵的變化比野生株更敏感，各突變體下調(diào)基因數(shù)目及幅度明顯高于上調(diào)基因，，雙突變體中變化基因的數(shù)目及幅度高于任意單突變體，均高于野生株；上調(diào)的基因主要涉及轉(zhuǎn)錄、輔酶代謝、氨基酸代謝和功能未分類(lèi)基因，而下調(diào)基因中參與能量代謝和碳水化合物代謝的較多，還有許多功能未分類(lèi)的基因；一些已知的轉(zhuǎn)鐵相關(guān)基因普遍上調(diào)，且各突變株中上調(diào)基因的數(shù)目及幅度均高于野生株。此結(jié)果表明運(yùn)鐵相關(guān)基因IroN、ShuA對(duì)志賀氏菌的生長(zhǎng)具有一定的影響。 利用Gateway技術(shù)與自殺質(zhì)粒相結(jié)合的敲除體系具有高效高通量的特點(diǎn)。本研究建立了一種對(duì)痢疾桿菌功能基因組研究有效的基因敲除技術(shù)平臺(tái)，并且提供了對(duì)痢疾桿菌中未知功能基因研究值得參考的新思路。
[Abstract]:In this paper, the functional genes of Shigella dysenteriae were studied by gene knockout method. Based on the RecA recombination system, the low copy suicide plasmid pCVD442 was used as the recombinant vector of the knockout system. The Gateway technique was applied to construct the recombinant plasmid of Shigella dysenteriae type A1 SD51197 strain during the construction of recombinant plasmids. The deletion and insertion of the iron-transport associated gene IroN,ShuA of Shigella dysenteriae A1 strain were successfully constructed. The double mutant MTS-1,MTS-2,MTS, thus established a knockout technology platform for localizing insertion or deletion mutation in Shigella. The function of each mutant was detected at three levels: culture medium, cell and animal. There was no significant difference between the growth of the mutants and the wild plants in the rich medium, and the growth level of the mutants was significantly lower than that of the wild plants from 4 hours after the addition of 150 渭 M Tieao mixture DIP. The addition of iron ions in the medium could restore the growth of the mutant to the level of rich medium, and in the experiment of HeLa cell and U937 cell, the ability of cell survival and proliferation and the ability of intercellular diffusion and corneal conjunctivitis of guinea pig. The virulence of all the mutants was not significantly changed, but the viability and proliferation ability of 65 渭 M DIP, mutant MTS-1,MTS-2,MTS decreased by 1 / 4 / 1 / 2 compared with the wild strain during the invasion of HeLa cells. These results suggest that the IroN,ShuA gene in Shigella is related to the iron transport of the bacteria, and the existence of other iron transport-related systems in the bacteria may compensate for the functional defects of the knockout gene, so the virulence level of the mutant has not changed significantly. The expression profiles of mutant and wild strain under iron enrichment and iron restriction were analyzed by SD51197 genomic microarray. The results showed that the number of up-regulated genes was higher than that of down-regulated gene in iron rich condition. The number and amplitude of mutant genes in double mutants were higher than that in arbitrary single mutants. Under the condition of iron restriction, all the mutants were more sensitive to the changes of iron deficiency than wild plants, the number and amplitude of down-regulated genes were significantly higher than those of up-regulated genes, and the number and amplitude of variation genes in double mutants were higher than those of any single mutants. The up-regulated genes were mainly involved in transcription, coenzyme metabolism, amino acid metabolism and functional unclassified genes, while down-regulated genes involved in energy metabolism and carbohydrate metabolism, and there were many unclassified genes. Some known transferrin related genes were generally up-regulated, and the number and amplitude of up-regulated genes in each mutant were higher than that in wild strains. The results indicated that the IroN,ShuA gene had a certain effect on the growth of Shigella. The knockout system using Gateway and suicide plasmids has the characteristics of high efficiency and high throughput. In this study, an effective gene knockout platform for the study of dysentery bacillus functional genomes was established, and a new idea for the study of unknown functional genes in Shigella was provided.
【學(xué)位授予單位】：沈陽(yáng)藥科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 劉紅,楊帆,張笑冰,張繼瑜,楊國(guó)威,董杰,薛穎,侯云德,袁正宏,聞?dòng)衩?徐建國(guó),陳洪松,馬大龍,王宇,楊劍,沈巖,強(qiáng)伯勤,吳洪濤,賀秉坤,呂渭川,金奇;痢疾桿菌全基因組序列及基因組島的分析[J];中國(guó)工程科學(xué);2002年10期

2 ;Construction, detection and microarray analysis on the Shigella flexneri 2a sitC mutant[J];Science in China(Series C:Life Sciences);2005年03期

3 ;Comparative genomics and phylogenetic analysis of S. dysenteriae subgroup[J];Science in China(Series C:Life Sciences);2005年04期

本文編號(hào)：2200295