本文選題：信號(hào)轉(zhuǎn)導(dǎo) + 轉(zhuǎn)錄調(diào)控��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：胞質(zhì)外功能σ因子(ECF-σ)是一類簡(jiǎn)單而多變的可替代σ因子,參與細(xì)胞對(duì)環(huán)境信號(hào)的感知和應(yīng)答,是原核生物普遍使用的一種基本的信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄調(diào)控機(jī)制。以復(fù)雜多細(xì)胞群體行為及次級(jí)代謝產(chǎn)物產(chǎn)生能力而聞名的粘細(xì)菌(Myxobacteria)是一類廣泛分布的環(huán)境微生物,可以耐受包括營(yíng)養(yǎng)、溫度、pH、滲透壓、氧化等在內(nèi)廣泛的環(huán)境刺激而生長(zhǎng)發(fā)育,但是對(duì)其生態(tài)適應(yīng)機(jī)制卻研究甚少。前期石美琳碩士以黃色粘球菌(Myxococcus xanthus)DK1622基因組為參考序列,在M.xanthus DZ2菌株中構(gòu)建了所有未見報(bào)道的35個(gè)ECF-σ因子和14個(gè)anti-σ因子的基因敲除突變株,分析了它們?cè)诟鞣N非生物環(huán)境信號(hào)下的生長(zhǎng)發(fā)育表型。在此基礎(chǔ)上本論文開展了如下研究:一、以其篩選出的專一性溫度信號(hào)應(yīng)答ECF-σ因子MXAN_3959和MXAN_7214以及一個(gè)全局性信號(hào)應(yīng)答(對(duì)所研究的環(huán)境信號(hào)均產(chǎn)生應(yīng)答)ECF-σ因子MXAN_4147為靶標(biāo),研究它們各自的信號(hào)傳導(dǎo)途徑。首先通過基因回補(bǔ)菌株構(gòu)建及表型分析,證實(shí)突變表型確實(shí)是由各基因的缺失所導(dǎo)致的。利用RT-qPCR技術(shù)檢測(cè)了特定環(huán)境信號(hào)下各基因表達(dá)量的變化,結(jié)果顯示除了全局性信號(hào)應(yīng)答ECF-σ因子MXAN_4147的表達(dá)量在Na+脅迫條件下的表達(dá)量下降外,其他基因在相應(yīng)條件下均有不同幅度的上升,這與它們各自突變體的生長(zhǎng)表型是一致的。鑒于在沒有環(huán)境刺激時(shí),絕大多數(shù)ECF-σ因子與其同源的anti-σ因子結(jié)合而失活,我們又通過共轉(zhuǎn)錄實(shí)驗(yàn)、細(xì)菌雙雜交系統(tǒng)分析了上述ECF-σ因子可能的anti-σ因子,結(jié)果表明,MXAN_3960、MXAN_7215和MXAN_4148可能分別為MXAN_3959、MXAN_7214和MXAN_4147對(duì)應(yīng)的anti-σ因子。為預(yù)測(cè)它們可能參與調(diào)控的靶基因,我們?cè)诖_定了它們各自所在的operon后,利用5'RACE技術(shù)檢測(cè)了各自的轉(zhuǎn)錄起始位點(diǎn),獲得了 MXAN_3959、MXAN_7214的啟動(dòng)子序列,并以其為探針,預(yù)測(cè)了它們可能參與調(diào)控的靶基因。以上結(jié)果初步闡明了 M.xanthus DZ2中MXAN_3959和MXAN_7214參與高溫應(yīng)答的信號(hào)轉(zhuǎn)導(dǎo)通路。另外,本文還對(duì)上述ECF-σ因子進(jìn)行了異源表達(dá)和純化,正在通過體內(nèi)、外實(shí)驗(yàn)嘗試鑒定各自調(diào)控的靶基因。以上結(jié)果不僅為闡明粘球菌中ECF-σ因子參與的溫度應(yīng)答調(diào)控機(jī)制奠定了基礎(chǔ),也為相關(guān)研究提供了技術(shù)積累。二、利用已有的突變株,篩選參與M.xanthus DZ2生物因素應(yīng)答的ECF-σ因子,重點(diǎn)分析了各突變株對(duì)于次級(jí)代謝產(chǎn)物抗生素TA(Myxovirescin)合成的影響。通過指示菌株Acinetobacter baumannii 19606 COLR 和E.coli K12 MG1655 抑菌圈實(shí)驗(yàn)篩選到三個(gè)影響TA產(chǎn)量的ECF-σ因子,分別是在抗生素TA合成過程中發(fā)揮正調(diào)控作用的MXAN_0203和MXAN_4148以及發(fā)揮負(fù)調(diào)控作用的MXAN_6173,為解析和改造M.xanthus次級(jí)代謝產(chǎn)物生物合成調(diào)控機(jī)制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93

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