新核开关“aac”的发现及外源性硫化氢对裂殖酵母全基因组表达谱的调控

发布时间：2018-06-13 17:45

本文选题：诱导性 + 报告基因载体　；参考：《复旦大学》2011年博士论文

【摘要】：1944年以来,氨基糖苷类抗生素临床应用走过了漫长的道路,细菌也产生了更强的耐药性。氨基糖苷类抗生素耐药机制包括：抗生素乙酰化,腺苷化或磷酸化；细菌外膜通透性改变,使胞内抗生素浓度降低,减少内膜运输,主动外排,药物诱捕；30S核糖体亚基作用位点突变；抗生素结合位点甲基化等。而诱导抗性基因的表达成为当前抗生素耐药的主要问题。 AAC(6')-IIb位于荧光假单胞菌的染色体上,该基因表达使细菌对庆大霉素、妥布霉素、奈替米星、地贝卡星和西索米星产生抗性。我们命名其5'-UTR非翻译区序列为aac, aac可翻译成短肽,具有ermC、fexA一样的性质。vienna RNA结构在线分析显示,aac在不同温度下呈现不同结构,说明其不同结构可能会影响AAC(6’)-IIb的表达。为研究aac的功能,构建报告基因pGEX-aac-lacZa表达载体,选用具有代表性的氨基糖苷类结构模型kanamycin B, lacZ酶活显示加入kanamycin B组酶活升高,证实其能诱导lacZa的表达。而荧光定量PCR发现lacZa转录水平上,加抗生素组比对照组有稍微上调,推测可能是mycin B与aac转录产物作用后,稳定aac的结构进而导致lacZa的稍微上调。不同浓度kanamycin B不引起lacZa转录产物的变化,推测kanamycin B组与对照组aac-lacZa实际上没有转录产物表达变化,只有aac转录产物二级结构的变化。neomycin与gentamycin进一步验证lacZ酶活与lacZa转录产物,结果与kanamycinB相同,推测氨基糖苷类抗生素能与aac结合,进而诱导lacZa的表达。平板诱导实验直观展示lacZa表达情况,除spectinomycin外所用13种氨基糖苷类抗生素均能诱导报告基因表达,推测spectinomycin结构中没有氨基或者其结构未与aac结合,进而导致lacZa表达无变化。SPR实验验证aac RNA结合,并获得结合常数。SPR实验需要aac RNA3’末端标记生物素,经验证标记过程RNA无明显降解,标记效率高,产量多。进一步体外SPR实验证明,aac RNA能与除spectinomycin外氨基糖苷类抗生素结合,而结合常数与抗生素结构中氨基位置和结构有关。RNA与小分子结合调控相关基因表达为“核开关”的定义,而ermC、fexA翻译后停靠调控具体机制仍未知,因为在体外前导肽不能与相对应抗生素结合,因此核开关理论可能适合ermC、fexA(?)(?)aac。构建pGEX-RmtB-aac-lacZa报告载体,16S rRNA甲基化,平板诱导实验显示除spectinomycin外氨基糖苷类抗生素仍能诱导lacZa的翻译,证明aac与氨基糖苷类抗生素直接结合,aac是以氨基糖苷类抗生素为配体的核开关。抗性基因表达菌株诱导环状大小不明显,推测修饰后的核糖体影响了lacZa的翻译,而所需抗生素诱导浓度较高,推测16SrRNA甲基化后氨基糖苷类抗生素结合aac的体内浓度降低,具体机制需进一步研究。 aac作为核开关,其与氨基糖苷类抗生素结合前后应有结构改变。SHAPE实验证实,氨基糖苷类抗生素与aac RNA结合后使翻译起始SD-2及AUG-2结构打开,而spectinomycin不与之结合。通过vienna在线预测RNA结构及CLUSTALW同源性比对分析发现,aac结构中存在一段保守序列AGUC与16SrRNA相同,推测结合位点可能发生在相关位置。经SHAPE数据分析发现,靠近AGUC位置的“CCC"序列信号下降,证明其可能与氨基糖苷类抗生素结合,因此推测其为氨基糖苷类抗生素的结合位点。通过数据分析,预测出aac加抗生素前后的结构,而参与结合位点的反应基团还有待进一步研究。综上所述,本课题发现一个新核开关aac,配体为氨基糖苷类抗生素,这也是首次报导抗生素为配体的核开关,为抗性基因诱导表达的机理研究开辟了一个新的领域。硫化氢是第三大气体信号分子,参与血管张力,心肌收缩,神经传递和胰岛素的分泌调节。当体内缺少硫化氢时,观察到动脉和肺动脉高压,阿尔茨海默病,胃粘膜损伤和各种动物模型肝硬化。外源硫化氢可改善心肌功能障碍与缺血相关／再灌注损伤,降低胃粘膜损伤。另一方面,对其过度表达可能导致炎症性疾病,感染性休克,脑中风,唐氏综合征患者心理障碍的发病,其表达的减少可能对这些疾病的治疗有潜在价值。目前对硫化氢的研究还处于初步阶段,对硫化氢作用的分子机制及直接作用的靶分子至今不明。为了阐明硫化氢细胞效应的分子机制与调控网络,并进一步通过硫化氢途径干预重大疾病寻找新的靶点,我们运用裂殖酵母生物芯片的方法,选用覆盖全基因组编码序列的裂殖酵母的生物芯片,对比在细胞内有硫化氢和没有硫化氢的条件下全基因组表达谱的差异,为分离出参与细胞周期,细胞增殖和凋亡过程中信号通路中的重要蛋白因子做铺垫。经过相关实验验证和分析,采用50μM硫氢化钠为硫化氢的供体,处理后的裂殖酵母进行全基因组RNA提取,反转录后基因芯片杂交。信号采用SAS分析系统标准化,2-fold, t-test分析筛选到83个差异表达基因,1.5-fo1d筛选到280个差异基因。经cluster分析发现,三个生物重复实验具有重复性,实验结论可靠。而采取的1.2-fold变化基因的实时荧光定量PCR分析发现,基因芯片数据可信度高,所以我们采用1.5-fold为主要分析所用基因。采用DAVID在线软件对差异表达基因进行功能分类,默认设置(counts2, EASE score＜0.1),2-fold基因GO生物过程分类显示,有20个下调基因,63个基因上调。1.5—fold的基因中,有156个基因表达上调,124个基因表达下调。其中2-fold中31个基因与应激反应相关,18个基因编码预测的或已知的运输蛋白,11个基因编码与细胞周期/减数分裂相关的蛋白质,10个基因编码的蛋白质参与氧化还原反应。与应激相关基因的比较发现,硫化氢诱导基因中46%的基因与过氧化氢相同,43%的基因与镉相同,49%的基因与热相同。然而,只有12%和24%硫化氢诱导基因与山梨醇和甲基甲烷磺盐相同。与过氧化氢相同基因可能说明硫化氢可以保护细胞免受氧化应激,参与MAPK信号通路中的一些基因的表达变化可能是硫化氢的靶基因。在全基因组蛋白定位研究中,4954个基因中的480个基因定位在线粒体(9.6%基因定位在线粒体)。大于1.5倍基因中124个基因下调,而其中定位在线粒体的基因有23个受硫化氢处理下调(18.5%基因定位于线粒体)。表明硫化氢引起许多线粒体基因表达下调,可能对线粒体功能的有影响。通过检测裂殖酵母的呼吸中的耗氧量和线粒体膜电位来验证硫化氢对线粒体功能的影响,实验结果表明,硫化氢处理后的裂殖酵母耗氧量降低,线粒体膜电位下降,证明线粒体功能损伤,这充分印证基因芯片数据分析结果,硫化氢引起线粒体基因表达下调。硫化氢引起的一些基因差异表达可为人类同源蛋白质研究和分子靶点提供线索,为进一步探索与硫化氢相关的人类疾病机理和治疗提供基础。
[Abstract]:Since 1944, the clinical application of aminoglycoside antibiotics has come a long way, and bacteria have also produced stronger resistance. The mechanisms of aminoglycoside resistance include antibiotic acetylation, adenosine or phosphorylation, bacterial membrane permeability change, reduction of intracellular antibiotic concentration, reduction of endometrium transport, active efflux, drugs Trapping, 30S ribosome subunit mutation and methylation of antibiotic binding sites, and the expression of inducible resistance genes are the main problems of antibiotic resistance at present.
AAC (6') -IIb is located on the chromosomes of Pseudomonas fluorescens, which makes bacteria resistant to gentamicin, tobramycin, netilmicin, netilmicin, and sisomicin. We named its 5'-UTR untranslated region sequence as AAC, AAC can be translated into short peptide, ermC, fexA like properties of.Vienna RNA structure online analysis show that AAC is in AAC. Different structures show different structures, indicating that different structures may affect the expression of AAC (6 ') -IIb. In order to study the function of AAC, the pGEX-aac-lacZa expression vector of the reporter gene is constructed, the representative amino glycoside structure model kanamycin B is selected, and the activity of lacZ enzyme activity is added into the B group of kanamycin B, which proves that it can induce lacZa. The fluorescence quantitative PCR found that at lacZa transcriptional level, the antibiotic group was slightly up-regulated than the control group. It was presumed that after the action of mycin B and AAC transcripts, the structure of the AAC was stable and the lacZa was slightly up-regulated. The kanamycin B of different concentrations did not cause the change of the lacZa transcript, and the kanamycin B group and the control group were presumed to be true. There is no change in the expression of transcriptional products. Only the changes in the two grade structure of the AAC transcriptional products.Neomycin and gentamycin further verify the lacZ enzyme activity and the lacZa transcript, and the results are the same as kanamycinB. It is presumed that the aminoglycoside antibiotics can bind to AAC and then induce the expression of lacZa.
The flat plate induction test shows the expression of lacZa intuitively. 13 aminoglycoside antibiotics except spectinomycin can induce the expression of the reporter gene. It is speculated that there is no amino group in the structure of spectinomycin or its structure is not combined with AAC, which leads to the AAC RNA binding without change of.SPR in lacZa expression, and the.SPR experiment of binding constant is obtained. It is necessary to mark biotin at the end of AAC RNA3 ', which has no obvious degradation, and the labeling efficiency is high and the yield is high. Further in vitro SPR experiment has proved that AAC RNA can combine with spectinomycin amidoside antibiotics, and the binding constant is associated with the binding of the amino position and structure of the amino group and structure in the structure of the antibiotic and the binding of.RNA to small molecules in the structure of the amino group and the structure of the antibiotic. Because the expression is the definition of "nuclear switch", the specific mechanism of ermC, fexA after translation is still unknown, because the leading peptide in vitro can not be combined with relative antibiotics, so the theory of nuclear switch may be suitable for ermC, fexA (?) aac.
The pGEX-RmtB-aac-lacZa report carrier, 16S rRNA methylation, and the plate induction test showed that the spectinomycin external aminoglycoside antibiotics still could induce the translation of lacZa, which proved that AAC was directly combined with aminoglycoside antibiotics, and AAC was a nuclear switch with aminoglycoside antibiotics as the ligand. It is conjectured that the modified ribosome affects the translation of lacZa, and the concentration of antibiotics required is high. It is speculated that the concentration of aminoglycoside antibiotics combined with AAC after 16SrRNA methylation is reduced, and the specific mechanism needs to be further studied.
AAC as a nuclear switch, its structure changes.SHAPE experiment with aminoglycoside antibiotics before and after the combination of aminoglycoside antibiotics. The combination of aminoglycoside antibiotics and AAC RNA opens the translation initiation SD-2 and AUG-2 structure, while spectinomycin does not combine with them. The RNA structure and CLUSTALW homology comparison analysis of CLUSTALW are found to be found in AAC structure through Vienna online. The existence of a conservative sequence AGUC is the same as 16SrRNA, and the binding site may occur at the relevant position. The SHAPE data analysis shows that the "CCC" sequence signal near the AGUC position decreases, which proves that it may be combined with aminoglycoside antibiotics, thus speculates that it is the binding site of aminoglycoside antibiotics. By data analysis, the prediction of a The structure of AC plus and without antibiotics, and the reaction sites involved in binding sites need further study.
In summary, we have found a new nuclear switch AAC, a ligand of aminoglycoside antibiotics, which is the first to report the nuclear switch of the antibiotic as a ligand, which opens up a new field for the mechanism of resistant gene expression.
Hydrogen sulfide (H2S) is the third major gas signal molecule involved in vascular tension, myocardial contraction, neurotransmission and insulin secretion. When hydrogen sulfide is lacking in the body, arterial and pulmonary hypertension, Alzheimer's disease, gastric mucosal injury and various animal models of liver cirrhosis are observed. On the other hand, overexpression of it may lead to inflammatory diseases, septic shock, stroke, and mental disorders in Down's syndrome, and the decrease in expression may be of potential value for the treatment of these diseases.
At present, the study of hydrogen sulfide is still in the initial stage. The molecular mechanism of hydrogen sulfide and the target molecules that have direct action are unknown. In order to clarify the molecular mechanism and regulatory network of the hydrogen sulfide cell effect, and to further explore new targets by interfering with the major diseases through hydrogen sulfide pathway, we use the recipe of fission yeast biochip. Method, the biochip of fission yeast, which covers the whole genome encoding sequence, is used to compare the difference of the whole genome expression profiles under the conditions of hydrogen sulfide and hydrogen sulfide in the cells, and paving the important protein factors in the signal pathways involved in cell cycle, cell proliferation and apoptosis.
Through the experimental verification and analysis, 50 M sodium hydrogen sulfide was used as the donor of hydrogen sulfide. The whole genome RNA was extracted by the treated fission yeast, and after the reverse transcriptional gene chip hybridization. The signal was standardized by SAS analysis system, and 83 differentially expressed genes were screened by 2-fold and t-test analysis, and 280 differential genes were screened by 1.5-fo1d. Through cluster The analysis found that the three biological repeat experiments were repeatable and the experimental conclusions were reliable. The real-time quantitative PCR analysis of the 1.2-fold change gene found that the reliability of the gene chip data was high, so we used 1.5-fold as the main analysis of the genes.
DAVID online software was used to classify the differentially expressed genes by default (counts2, EASE score < 0.1). The 2-fold gene GO bioprocess classification showed that there were 20 down-regulation genes and 63 genes up regulation of.1.5 fold, 156 genes were up-regulated and 124 genes were downregulated. Among them, 31 genes in 2-fold and stress response Related, 18 genes encode the predicted or known transporters, 11 genes encode proteins associated with cell cycle / meiosis, and 10 genes encoded proteins participate in redox reaction.
Comparison with stress related genes found that 46% of the gene induced by hydrogen sulfide was the same as hydrogen peroxide, 43% of the gene was the same as cadmium, and 49% of the gene was the same as heat. However, only 12% and 24% hydrogen sulfide induced genes were the same as sorbitol and methyl methanosylsulfonate. Oxidative stress is involved in the expression of some genes in the MAPK signaling pathway, which may be the target gene of hydrogen sulfide.
In the whole genome protein localization study, 480 of the 4954 genes are located in the mitochondria (9.6% genes are located in the mitochondria). More than 1.5 times more than 124 genes are down, 23 of the genes in the mitochondria are down regulated by the hydrogen sulfide treatment (the 18.5% gene is located in the mitochondria). It shows that hydrogen sulfide causes many mitochondrial genes. Down regulation may have an effect on the function of mitochondria.
The effect of hydrogen sulfide on mitochondrial function was verified by detecting oxygen consumption and mitochondrial membrane potential in the respiration of fission yeast. The experimental results showed that the oxygen consumption of fission yeast after hydrogen sulfide treatment was reduced, the mitochondrial membrane potential decreased, which proved mitochondrial function damage, which fully confirmed the results of DNA chip data analysis, hydrogen sulfide caused by hydrogen sulfide. The expression of mitochondrial gene was downregulated.
Some gene differential expression induced by hydrogen sulfide can provide clues for human homologous protein research and molecular targets, providing a basis for further exploration of the mechanism and treatment of human disease related to hydrogen sulfide.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：Q78;R3416

【参考文献】