黄色粘球菌DZ2中温度应答ECF-σ因子的研究

发布时间：2018-05-27 09:20

本文选题：信号转导 + 转录调控　；参考：《山东大学》2017年硕士论文

【摘要】：胞质外功能σ因子(ECF-σ)是一类简单而多变的可替代σ因子,参与细胞对环境信号的感知和应答,是原核生物普遍使用的一种基本的信号转导与转录调控机制。以复杂多细胞群体行为及次级代谢产物产生能力而闻名的粘细菌(Myxobacteria)是一类广泛分布的环境微生物,可以耐受包括营养、温度、pH、渗透压、氧化等在内广泛的环境刺激而生长发育,但是对其生态适应机制却研究甚少。前期石美琳硕士以黄色粘球菌(Myxococcus xanthus)DK1622基因组为参考序列,在M.xanthus DZ2菌株中构建了所有未见报道的35个ECF-σ因子和14个anti-σ因子的基因敲除突变株,分析了它们在各种非生物环境信号下的生长发育表型。在此基础上本论文开展了如下研究:一、以其筛选出的专一性温度信号应答ECF-σ因子MXAN_3959和MXAN_7214以及一个全局性信号应答(对所研究的环境信号均产生应答)ECF-σ因子MXAN_4147为靶标,研究它们各自的信号传导途径。首先通过基因回补菌株构建及表型分析,证实突变表型确实是由各基因的缺失所导致的。利用RT-qPCR技术检测了特定环境信号下各基因表达量的变化,结果显示除了全局性信号应答ECF-σ因子MXAN_4147的表达量在Na+胁迫条件下的表达量下降外,其他基因在相应条件下均有不同幅度的上升,这与它们各自突变体的生长表型是一致的。鉴于在没有环境刺激时,绝大多数ECF-σ因子与其同源的anti-σ因子结合而失活,我们又通过共转录实验、细菌双杂交系统分析了上述ECF-σ因子可能的anti-σ因子,结果表明,MXAN_3960、MXAN_7215和MXAN_4148可能分别为MXAN_3959、MXAN_7214和MXAN_4147对应的anti-σ因子。为预测它们可能参与调控的靶基因,我们在确定了它们各自所在的operon后,利用5'RACE技术检测了各自的转录起始位点,获得了 MXAN_3959、MXAN_7214的启动子序列,并以其为探针,预测了它们可能参与调控的靶基因。以上结果初步阐明了 M.xanthus DZ2中MXAN_3959和MXAN_7214参与高温应答的信号转导通路。另外,本文还对上述ECF-σ因子进行了异源表达和纯化,正在通过体内、外实验尝试鉴定各自调控的靶基因。以上结果不仅为阐明粘球菌中ECF-σ因子参与的温度应答调控机制奠定了基础,也为相关研究提供了技术积累。二、利用已有的突变株,筛选参与M.xanthus DZ2生物因素应答的ECF-σ因子,重点分析了各突变株对于次级代谢产物抗生素TA(Myxovirescin)合成的影响。通过指示菌株Acinetobacter baumannii 19606 COLR 和E.coli K12 MG1655 抑菌圈实验筛选到三个影响TA产量的ECF-σ因子,分别是在抗生素TA合成过程中发挥正调控作用的MXAN_0203和MXAN_4148以及发挥负调控作用的MXAN_6173,为解析和改造M.xanthus次级代谢产物生物合成调控机制奠定了基础。
[Abstract]:Extracellular function 蟽 factor (ECF- 蟽) is a kind of simple and changeable alternative 蟽 factor, which is involved in cell perception and response to environmental signal, and is a basic signal transduction and transcription regulation mechanism widely used in prokaryotes. Myxobacteria, known for its complex multicellular population behavior and secondary metabolite production, is a class of widely distributed environmental microbes that can withstand a wide range of environmental stimuli, including nutrition, temperature, pH, osmotic pressure, oxidation, and so on. However, there is little research on its ecological adaptation mechanism. Taking Myxococcus xanthus)DK1622 genome as a reference sequence, Shim Lin constructed all 35 unreported gene knockout mutants of ECF- 蟽 factor and 14 anti- 蟽 factor in M.xanthus DZ2 strain. The phenotypes of their growth and development under various abiotic environmental signals were analyzed. On this basis, the following studies have been carried out in this paper: firstly, the specific temperature signal response (ECF- 蟽 factor MXAN_3959 and MXAN_7214) and a global signal response (ECF- 蟽 factor MXAN_4147) are selected as targets. Their respective signal transduction pathways are studied. Firstly, the mutant phenotype was proved to be caused by the deletion of each gene by the construction and phenotypic analysis of the gene complement strain. RT-qPCR technique was used to detect the changes of gene expression under specific environmental signals. The results showed that the expression of ECF- 蟽 factor MXAN_4147 in the global signal response decreased under Na stress. Other genes increased by different extents under the corresponding conditions, which was consistent with the growth phenotypes of their respective mutants. In view of the inactivation of the majority of ECF- 蟽 factors in the absence of environmental stimulation, the possible anti- 蟽 factors of these ECF- 蟽 factors were analyzed by bacterial two-hybrid system through co-transcription experiments. The results show that MXAN3960MXAN7215 and MXAN_4148 may be the anti- 蟽 factors corresponding to MXAN3959 and MXAN_4147 respectively. In order to predict the target genes that they may be involved in, we determined their respective operon, detected their transcriptional initiation sites by 5'RACE technique, obtained the promoter sequence of MXAN3959 and MXAN7214, and used it as a probe. The target genes that they may be involved in are predicted. The above results indicate that MXAN_3959 and MXAN_7214 are involved in the signal transduction pathway of hyperthermia response in M.xanthus DZ2. In addition, the above ECF- 蟽 factors were expressed and purified from different sources, and the target genes were identified by in vivo and in vitro experiments. The above results not only laid a foundation for elucidating the mechanism of temperature response regulation involved by ECF- 蟽 factor in mucococcus, but also provided a technical accumulation for related research. Secondly, the existing mutants were used to screen the ECF- 蟽 factors involved in the response of M.xanthus DZ2 biological factors, and the effects of the mutants on the synthesis of the secondary metabolite TAX Myxovirescinwere analyzed. Three ECF- 蟽 factors affecting TA yield were screened by the bacteriostasis test of Acinetobacter baumannii 19606 COLR and E.coli K12 MG1655. MXAN_0203 and MXAN_4148, which play a positive role in the synthesis of TA, and MXANS / s 6173, which play a negative role in the synthesis of TA, have laid a foundation for the analysis and transformation of the biosynthetic regulation mechanism of secondary metabolites of M.xanthus.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93

【相似文献】