本文選題：恥垢分枝桿菌 + TetR家族轉(zhuǎn)錄調(diào)控因子�。� 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：結(jié)核病是由結(jié)核分枝桿菌(Mycobacterium tuberculosis)引起的一種重要人類(lèi)傳染病,其產(chǎn)生的抗藥性問(wèn)題已經(jīng)引發(fā)了全球范圍的廣泛關(guān)注。然而,致病性的結(jié)核分枝桿菌生長(zhǎng)非常緩慢,感染性極強(qiáng),目前對(duì)其抗藥調(diào)控機(jī)制的認(rèn)識(shí)仍然十分有限。相比而言,恥垢分枝桿菌(M.smegmatis)生長(zhǎng)較快,遺傳操作方法成熟,而且很多基因與結(jié)核分枝桿菌高度同源,因此已經(jīng)成為研究病原性分枝桿菌的基因調(diào)控和生理代謝特點(diǎn)的重要模式菌株。本研究以恥垢分枝桿菌為研究對(duì)象,發(fā)現(xiàn)了兩個(gè)新的TetR家族的轉(zhuǎn)錄因子,分別正調(diào)控恥垢分枝桿菌對(duì)利福平和乙胺丁醇藥物的抗性。具體結(jié)果如下:(1)Ms4022是恥垢分枝桿菌基因組編碼的一個(gè)功能未知的轉(zhuǎn)錄因子,由199個(gè)氨基酸組成,其N(xiāo)端含有一個(gè)TetR家族的HTH結(jié)構(gòu)域。通過(guò)構(gòu)建ms4022敲除和超表達(dá)重組菌株并測(cè)定利福平藥物對(duì)它們的最小抑菌濃度(MIC),結(jié)果發(fā)現(xiàn):ms4022敲除菌株的MIC(3.13μg/ml)比野生型菌株的(6.25μg/ml)低2倍,而超表達(dá)菌株的MIC(25μg/ml)大約是野生型菌株的4倍。因此,ms4022基因的表達(dá)水平顯著影響恥垢分枝桿菌對(duì)利福平的抗性。進(jìn)一步,通過(guò)EMSA和DNA足跡實(shí)驗(yàn)發(fā)現(xiàn),Ms4022在其自身啟動(dòng)子區(qū)域含有有兩個(gè)結(jié)合位點(diǎn):第一個(gè)命名為motif 1,其長(zhǎng)度為19 bp,靠近翻譯起始位點(diǎn);第二個(gè)則包含了兩個(gè)motif,分別命名為motif 2和motif 3,它們距離翻譯起始位點(diǎn)都較遠(yuǎn)。序列比對(duì)分析顯示,這三個(gè)motif的5’端9 bp序列的同源性很高,具有明顯的保守性。利用Ms4022識(shí)別的3個(gè)motif在全基因組范圍內(nèi)搜索恥垢分枝桿菌的啟動(dòng)子區(qū)域,發(fā)現(xiàn)Ms4022可廣泛調(diào)節(jié)約315個(gè)潛在靶基因的表達(dá)。有趣的是,這些潛在靶基因中包括有31個(gè)運(yùn)輸相關(guān)的基因(占9.84%)。隨后,采用EMSA和Ch IP實(shí)驗(yàn)證實(shí)Ms4022在恥垢分枝桿菌體內(nèi)體外能與這些運(yùn)輸相關(guān)基因的靶啟動(dòng)子序列特異性結(jié)合。進(jìn)一步采用qRT-PCR分析發(fā)現(xiàn),與野生型菌株相比,ms4022敲除菌株中絕大部分這些運(yùn)輸相關(guān)靶基因的表達(dá)量都明顯下調(diào);相反,ms4022超表達(dá)菌株中它們的表達(dá)量則顯著上調(diào)。進(jìn)一步的β-半乳糖苷酶活實(shí)驗(yàn)也清楚證明,Ms4022正調(diào)控這些基因的表達(dá)。最后,通過(guò)分別構(gòu)建這些運(yùn)輸相關(guān)靶基因的超表達(dá)菌株并測(cè)定生長(zhǎng)曲線(xiàn),結(jié)果發(fā)現(xiàn)7個(gè)靶基因的超表達(dá)能夠特異性增強(qiáng)恥垢分枝桿菌對(duì)利福平的抗藥性。綜合這些結(jié)果,Ms4022是一個(gè)廣泛調(diào)控因子,發(fā)揮一個(gè)轉(zhuǎn)錄激活子的作用,它通過(guò)正調(diào)控與運(yùn)輸相關(guān)的靶基因的表達(dá),增強(qiáng)恥垢分枝桿菌對(duì)利福平的抗藥性。(2)LerR是恥垢分枝桿菌基因組編碼的另一個(gè)轉(zhuǎn)錄因子,含有Lux R結(jié)構(gòu)域,也屬于TetR家族;它與激酶LerS組成一對(duì)雙組分系統(tǒng),共同調(diào)節(jié)恥垢分枝桿菌對(duì)抗結(jié)核藥物乙胺丁醇的抗性。通過(guò)構(gòu)建lerR超表達(dá)菌株并測(cè)定它在不同抗結(jié)核藥物脅迫下的生長(zhǎng)曲線(xiàn),結(jié)果發(fā)現(xiàn):與野生型菌株相比,超表達(dá)菌株對(duì)乙胺丁醇的抗性能力有顯著提高。隨后,EMSA和Ch IP實(shí)驗(yàn)證實(shí)LerR能夠在體內(nèi)和體外特異性地結(jié)合Ms6242和Ms6239(1,3-丙二醇脫氫酶PDH)的啟動(dòng)子序列。進(jìn)一步的qRT-PCR分析發(fā)現(xiàn),與野生型菌株相比,lerR敲除菌株中PDH以及操縱子Ms6242、Ms6241、Ms6240的表達(dá)量都顯著下降;相反,lerR超表達(dá)菌株中這些靶基因的表達(dá)量則增加。比較轉(zhuǎn)錄組分析發(fā)現(xiàn),與沒(méi)有藥物脅迫相比,乙胺丁醇處理下的恥垢分枝桿菌中l(wèi)erR及其靶基因PDH、Ms6242、Ms6241、Ms6240的表達(dá)量增加了2-4.5倍,說(shuō)明這些基因均能響應(yīng)乙胺丁醇的刺激。最后,通過(guò)分別構(gòu)建LerR靶基因的超表達(dá)菌株并測(cè)定生長(zhǎng)曲線(xiàn),發(fā)現(xiàn)只有超表達(dá)PDH的恥垢分枝桿菌對(duì)乙胺丁醇的抗藥性增加。因此,LerR在恥垢分枝桿菌中也是一個(gè)正調(diào)控蛋白,通過(guò)激活包括PDH在內(nèi)的多個(gè)靶基因的表達(dá),調(diào)節(jié)恥垢分枝桿菌對(duì)乙胺丁醇的抗性。
[Abstract]:Tuberculosis is an important human infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis), and its resistance to drug resistance has attracted worldwide attention. However, the pathogenicity of Mycobacterium tuberculosis is very slow and highly infectious, and the understanding of its anti drug regulation mechanism is still limited. In comparison, Mycobacterium foul Mycobacterium (M.smegmatis) grows rapidly, the genetic manipulation is mature, and many genes are highly homologous with Mycobacterium tuberculosis. Therefore, it has become an important model for the study of gene regulation and physiological metabolism of Mycobacterium tuberculosis. This study was based on the study of Mycobacterium foul Mycobacterium and found two The transcriptional factor of the new TetR family regulates the resistance of Mycobacterium tumefaciens to rifampicin and ethambutol respectively. The specific results are as follows: (1) Ms4022 is a functional unknown transcription factor, composed of 199 amino acids, which contains a HTH domain of the TetR family in the N end of the Mycobacterium tumefaciens genome. Through the construction of ms4022 The recombinant strain was knocked out and overexpressed and the minimal inhibitory concentration (MIC) of rifampin was measured. The results showed that the MIC (3.13 mu g/ml) of the ms4022 knockout strain was 2 times lower than that of the wild type (6.25 mu g/ml), and the MIC (25 mu g/ml) of the overexpression strain was about 4 times that of the wild type. Therefore, the expression level of ms4022 gene significantly affected the disgrace scale. The resistance of Mycobacterium to rifampin. Further, through EMSA and DNA footprints experiments, it is found that Ms4022 has two binding sites in its own promoter region: the first is named motif 1, and its length is 19 BP, close to the translation initiation site, and the second contains two motif, named motif 2 and motif 3 respectively, which are from the beginning of translation. The sequence alignment analysis showed that the 5 'end 9 BP sequences of the three motif were highly homologous and conserved. Using 3 motif identified by Ms4022 to search the promoter region of Mycobacterium scale in the whole genome, it was found that Ms4022 could regulate the expression of about 315 potential target genes. 31 transport related genes were included in the target gene (9.84%). Subsequently, EMSA and Ch IP experiments were used to confirm that Ms4022 can specifically bind to the target promoter sequences of these transport related genes in vitro and in vitro. Further qRT-PCR analysis was used to find that most of the ms4022 knockout strains were compared with the wild type strains. The expression of these transport related target genes was obviously down; on the contrary, the expression of ms4022 overexpressed strains was significantly up-regulated. Further beta galactosidase experiment also clearly demonstrated that Ms4022 was regulating the expression of these genes. Finally, the overexpressed strains of these transport related target genes were constructed and the growth was determined. The results show that the overexpression of 7 target genes can specifically enhance the resistance of Mycobacterium tumefaciens to rifampicin. These results suggest that Ms4022 is a wide regulator and plays a role in a transcriptional activator. It enhances the resistance of Mycobacterium tumefaciens to rifampicin by regulating the expression of target genes associated with transport. (2) (2) LerR is another transcription factor that encodes the genome of Mycobacterium tumefaciens, which contains the Lux R domain and also belongs to the TetR family; it forms a pair of two component systems with kinase LerS to regulate the resistance of Mycobacterium tumefaciens to the anti tuberculosis drug ethambutol. By constructing a lerR superexpression strain and determining it in different anti tuberculosis drug coerced strains. The results showed that the resistance ability of the overexpression strain to ethambutol was significantly improved compared with the wild strain. Subsequently, EMSA and Ch IP experiments confirmed that LerR could specifically combine the promoter sequence of Ms6242 and Ms6239 (1,3- propanediol dehydrogenase PDH) in vivo and in vitro. The expression of PDH, operon Ms6242, Ms6241 and Ms6240 in the lerR knockout strains decreased significantly compared with the strain of the strains; on the contrary, the expression of these target genes increased in the lerR overexpression strain. The comparison of the transcriptional analysis found that the lerR and its target gene PDH, Ms624 in ethambutol under the absence of drug stress, lerR and its target gene PDH, Ms624 2, the expression of Ms6241 and Ms6240 increased by 2-4.5 times, indicating that all the genes could respond to the stimulation of ethambutol. Finally, the growth curve was determined by constructing the overexpressed strains of the LerR target gene and the growth curve was measured. It was found that only the overexpressed PDH was anti drug of ethambutol. Therefore, LerR was also one of the mycobacteria in Mycobacterium foul. A positive regulatory protein regulates the resistance of Mycobacterium tuberculosis to ethambutol by activating multiple target genes including PDH.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R378.91

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 徐志弘;周愛(ài)萍;姚玉峰;;結(jié)核分枝桿菌ABC轉(zhuǎn)運(yùn)蛋白與物質(zhì)的跨膜轉(zhuǎn)運(yùn)[J];微生物學(xué)報(bào);2014年06期

，

本文編號(hào)：1810404