痢疾志贺菌A1型IroN、ShuA单、双突变体的构建及功能分析
发布时间：2018-08-24 09:02
【摘要】：本文以基因敲除方法研究痢疾杆菌的功能基因，以RecA重组系统为基础，采用低拷贝自杀质粒pCVD442作为本敲除体系的重组载体，为了克服该质粒在进行分子克隆操作中的困难，将Gateway技术应用在敲除前期的重组质粒构建过程中，成功构建了痢疾志贺菌A1型SD51197株运铁相关基因IroN、ShuA部分缺失和插入的单、双突变体MTS-1、MTS-2、MTS，从而建立了在痢疾杆菌中进行定位插入或缺失突变的敲除技术平台。 对各突变株分别在培养基、细胞和动物三个水平进行了功能检测。在丰富培养基中各突变株与野生株生长没有显著差异；在添加150μM铁鳌合剂DIP的条件下，各突变体的生长水平都从4h开始明显低于野生株，在培养基中补加铁离子可以使各突变株的生长回复到丰富培养基条件下的水平；在HeLa细胞和U937细胞的胞内存活增殖能力和胞间扩散能力以及豚鼠角膜结膜炎实验中，各突变株均没有明显的毒力变化，但在HeLa细胞侵袭过程中添加65μM的DIP，突变株MTS-1、MTS-2、MTS的胞内存活增殖能力与野生株相比下降了1／4～1／2。上述结果提示痢疾杆菌中IroN、ShuA基因与细菌的铁转运相关，可能由于细菌中其它铁转运相关系统的存在代偿了敲除基因的功能缺陷，因此突变体在毒力水平没有特别显著的变化。 利用SD51197全基因组芯片进行了铁丰富和铁限制条件下突变株和野生株的表达谱比较分析，结果表明：铁丰富条件下各突变体上调基因数目多于下调基因，双突变体中变化基因的数目及幅度高于任意单突变体。铁限制条件下，整体上各突变体对缺铁的变化比野生株更敏感，各突变体下调基因数目及幅度明显高于上调基因，，双突变体中变化基因的数目及幅度高于任意单突变体，均高于野生株；上调的基因主要涉及转录、辅酶代谢、氨基酸代谢和功能未分类基因，而下调基因中参与能量代谢和碳水化合物代谢的较多，还有许多功能未分类的基因；一些已知的转铁相关基因普遍上调，且各突变株中上调基因的数目及幅度均高于野生株。此结果表明运铁相关基因IroN、ShuA对志贺氏菌的生长具有一定的影响。 利用Gateway技术与自杀质粒相结合的敲除体系具有高效高通量的特点。本研究建立了一种对痢疾杆菌功能基因组研究有效的基因敲除技术平台，并且提供了对痢疾杆菌中未知功能基因研究值得参考的新思路。
[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.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378
本文编号：2200295
[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.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378
【参考文献】
相关期刊论文 前3条
1 刘红,杨帆,张笑冰,张继瑜,杨国威,董杰,薛颖,侯云德,袁正宏,闻玉梅,徐建国,陈洪松,马大龙,王宇,杨剑,沈岩,强伯勤,吴洪涛,贺秉坤,吕渭川,金奇;痢疾杆菌全基因组序列及基因组岛的分析[J];中国工程科学;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
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