【作者】 张向阳；

【导师】 李伟辉；

【作者基本信息】 广西大学 ， 生物工程（专业学位）， 2022， 硕士

【摘要】 拟核结合蛋白（Nucleoid-associated proteins,NAPs）是细菌中一类分子量为10-20 k Da的弱碱性小蛋白,它能够凝聚细菌胞内的染色质DNA,并通过结合DNA来广泛调控细菌的多种环境适应能力如抗氧化生长等。结核分枝杆菌是结核病的致病菌,其在宿主胞内生存必须要应对极强的氧化压力,因而进化形成了一系列自己的抗氧化生长调控机制。然而,目前对于这些机制却知之甚少,尤其是拟核结合蛋白是否参与调控分枝杆菌的抗氧化能力还很不清楚。在本论文中,我们以结核分枝杆菌及其模式菌株耻垢分枝杆菌和卡介苗为研究对象,发现了分枝杆菌中新的拟核结合蛋白NapR,并鉴定了NapR下游调控的靶基因,解析了NapR调控分枝杆菌抗氧化生长的具体调控通路与分子机理。（1）利用耻垢分枝杆菌转座插入突变体文库筛选并鉴定到耻垢分枝杆菌napR影响细菌的抗氧化能力。进一步,构建了耻垢分枝杆菌的napR敲除与回补菌株,并检测了相关重组菌株在H₂O₂胁迫下的生长情况,结果发现NapR负调控细菌的抗氧化能力。（2）应用凝胶阻滞迁移实验（EMSA）,DNase I酶切实验,拓扑异构酶解螺旋实验以及蛋白质与染色质DNA荧光共定位实验等发现了NapR具有拟核结合蛋白的特性,即NapR是分枝杆菌中新的拟核结合蛋白。（3）鉴定了NapR下游调控的靶基因ggr（香叶基香叶基还原酶的编码基因）。运用EMSA,实时荧光定量PCR（RT-q PCR）实验和β-半乳糖苷酶活实验证明了NapR直接正调控ggr的表达。进一步,构建了耻垢分枝杆菌的ggr敲除与回补菌株,并检测了相关重组菌株在H₂O₂胁迫下的生长情况,发现ggr增强了细菌对氧化压力的敏感性。（4）构建了耻垢分枝杆菌napR与ggr的交叉重组菌株,并检测与比较了相关重组菌株在H₂O₂胁迫下的生长情况,结果发现NapR负调控细菌的抗氧化能力依赖靶基因ggr,即NapR通过调控ggr的表达进而调控细菌的抗氧化生长。（5）通过分别比对耻垢分枝杆菌与结核分枝杆菌以及卡介苗中NapR的氨基酸序列,发现耻垢分枝杆菌与结核分枝杆菌的NapR一致性为76.6%,结核分枝杆菌与卡介苗的NapR一致性为100%。随后,利用EMSA实验,DNase I酶切实验与拓扑异构酶解螺旋实验等证实了结核分枝杆菌NapR与耻垢分枝杆菌NapR一样也具有拟核结合蛋白的特性。进一步,在BCG中观察了NapR与染色质DNA的荧光共定位情况,结果表明BCG NapR同样具有拟核结合蛋白的特性。以上结果说明,NapR在分枝杆菌中高度同源,且是一个新的拟核结合蛋白。综上,本研究发现了分枝杆菌中新的拟核结合蛋白NapR,鉴定了其下游调控的靶基因ggr,并解析了NapR通过调控ggr的表达来调控分枝杆菌的抗氧化能力的具体分子机理。研究结果为分枝杆菌的拟核结合蛋白家族增添了新成员,为了解分枝杆菌的抗氧化生长机制提供了新证据,为发现新的抗结核药靶提供了思路。更多还原

【Abstract】 Nucleoid-associated proteins（NAPs）are a class of weakly basic small proteins with a molecular weight of 10-20 k Da in bacteria,which can condense the chromatin DNA in bacterial cells and widely regulate various bacterial environmental adaptability such as antioxidant growth by binding to DNA.M.tuberculosis is the causative bacterium of tuberculosis,and it must deal with strong oxidative stress to survive in the host cell,so it has evolved a series of molecular regulatory mechanisms for anti-oxidative growth.However,little is known about these mechanisms,especially whether nucleoid-associated proteins regulate the antioxidant capacity of mycobacteria.In this study,we took Mycobacterium tuberculosis,BCG and its model strains M.smegmatis as research objects,discovered a new nucleoid-associated protein NapR in Mycobacterium,and identified the target genes regulated downstream by NapR.The specific research results obtained are as follows:（1）Using the M.smegmatis transposition insertion mutant library to screen the M.smegmatis gene napR and identify that it affects the antioxidant capacity of bacteria.Furthermore,the knockout and complementation strains of M.smegmatis for napR were constructed,and the growth of related recombinant strains under H₂O₂ stress was detected,and it was found that NapR negatively regulates the antioxidant capacity of bacteria.（2）It was found that NapR has the characteristics of a nucleoid-associated protein by the experiments of Electrophoretic mobility shift assay（EMSA）,DNase I digestion,topoisomerase unwinding and fluorescence co-localization between protein and chromatin DNA.NapR is a novel nucleoid-associated protein in mycobacteria.（3）The target gene ggr（encode geranylgeranyl reductase）regulated downstream by NapR was identified.It is proved that NapR directly positively regulates the expression of ggr by the experiments of EMSA,real-time quantitative PCR（RT-q PCR）andβ-galactosidase activity.Furthermore,the knockout and complementation strains of M.smegmatis ggr were constructed,and the growth of the related recombinant strains under H₂O₂ stress was detected,and it was found that ggr enhanced the sensitivity of bacteria to oxidative stress.（4）The cross-recombinant strains of M.smegmatis napR and ggr were constructed,and the growth of related recombinant strains under H₂O₂ stress was detected and compared.The results showed that the antioxidative ability of NapR by the method of negatively regulating the bacteria depends on the target gene ggr,That is,NapR regulates the antioxidant growth of bacteria by regulating the expression of ggr.（5）By comparing the amino acid sequences of NapR in M.tuberculosis,BCG and M.smegmatis,it was found that the gene sequence of M.tuberculosis was 100%consistent with that of BCG and 85%with that of M.smegmatis.Subsequently,it was confirmed that Mycobacterium tuberculosis NapR and Mycobacterium smegmatis NapR also have the characteristics of nucleoid-associated proteins by the experiments of EMSA,DNase I digestion and topoisomerase helix.Furthermore,the fluorescent co-localization of NapR and chromatin DNA was observed in BCG,and the results showed that BCG NapR also has the characteristics of nucleoid-associated protein.The above results indicate that NapR is highly conserved in mycobacteria and is a new nucleoid-associated protein.In conclusion,this research paper discovered a new nucleoid-associated protein NapR in mycobacteria,identified its downstream target gene ggr,and analyzed the specific mechanism of NapR regulating the antioxidant capacity of mycobacteria by regulating the expression of ggr.The research results add a new member to the mycobacterial nucleoid-associated proteins,provide new evidence for understanding the antioxidant growth mechanism of mycobacteria,and provide ideas for the discovery of new anti-tuberculosis drug targets.更多还原