氨基糖苷类药物作用下结核分枝杆菌sRNA分析和初步功能研究

发布时间：2018-03-12 21:30

本文选题：sRNA　切入点：结核分枝杆菌　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的在细菌等原核生物中,sRNA是存在于非编码区的一类新型调节子,其主要功能是调控相应基因的表达,使细菌适应环境变化。近年来研究者发现sRNA与多种抗生素暴露甚至耐药性的产生有关,其中就包括氨基糖苷类药物,而在结核分枝杆菌中的相关报道较少。氨基糖苷类药物链霉素是最早用于抗结核治疗的药物之一,其同类的卡那霉素作为二线药物也被用于临床结核病治疗。本研究的目的是筛选在结核分枝杆菌中响应氨基糖苷类药物刺激,可能与耐药性产生相关的sRNA,检测其表达量变化并进行初步的功能学实验研究。方法将氨基糖苷类药物链霉素和卡那霉素分别以2倍倍比稀释浓度作用于结核分枝杆菌标准菌株H37Rv,根据各浓度组的生长情况,选取OD600值较为接近的浓度组菌液,提取总RNA,建立转录组文库,通过高通量测序分析链霉素和卡那霉素作用下转录组的改变,并预测新的sRNA。通过计算FPKM值,将sRNA的表达量进行标准化,对FPKM值进行假设检验和差异倍数计算,筛选上调或下调表达的sRNA。采用Northern blot、qRT-PCR验证差异表达sRNA的实际表达量变化。选择在链霉素和卡那霉素作用后共同下调的sRNA-ms28进行初步功能分析。构建过表达质粒、电转入结核分枝杆菌H37Rv得到ms28的过表达菌株,通过比较过表达菌株与野生株和转入空载体的菌株的生长速率和基因表达情况,初步分析ms28的功能。结果提取链霉素组,卡那霉素组及对照组的总RNA,建立了3个转录组测序文库。通过基因差异分析,得到结核分枝杆菌标准菌株H37Rv在链霉素作用下差异表达基因287条(7.2%),其中上调表达基因184条,下调表达基因103条;卡那霉素作用下差异基因230条(5.8%),其中61条上调,169条下调。GO富集分析显示,所有差异编码基因中,细胞组分、膜蛋白和代谢过程的差异基因富集程度最高。本研究共预测了174条sRNA中,其中88条位于正链,分别编码为ms01-ms88,86条位于负链,分别编码为ms89-ms174。组间差异表达分析得到链霉素作用后差异表达sRNA 5条,其中ms03、ms75、ms172上调,ms28、ms88下调;卡那霉素作用后差异表达sRNA 6条,其中ms113、ms146上调,ms28、ms42、ms127、ms137下调,位于基因组1512822-1513027的ms28是链霉素和卡那霉素作用后共同下调的sRNA。采用Northern blot和q RT-PCR检测链霉素和卡那霉素作用后sRNA表达量变化,10条差异表达的sRNA中的7条检测结果与分析结果一致。将ms28过表达后,前期预测的ms28靶基因中有6条上调表达。与电转入空载体的菌株相比,ms28过表达株生长曲线左移,生长对数期缩短3-4d。结论在转录组水平,结核分枝杆菌受到氨基糖苷类药物作用后,部分基因表达量发生变化,差异表达基因在细胞组分、膜蛋白和代谢过程高度富集。在链霉素作用下,结核分枝杆菌sRNA—ms03、ms172表达上调,ms28、ms88表达下调;卡那霉素作用下ms113表达上调,ms28、ms42、ms127表达下调。结核分枝杆菌sRNA—ms28的过表达使结核分枝杆菌H37Rv菌株的生长发生改变。ms28的潜在靶基因包括irtA、irt B、mbtK、mbtN、FABG2、lprD。
[Abstract]:In prokaryote, sRNA is a novel regulator in non encoding region, its main function is to regulate the expression of the corresponding gene, allowing bacteria to adapt to the changing environment. In recent years, the researchers found that sRNA and even the emergence of drug resistance related to antibiotic exposure, including the aminoglycosides, and related reported in Mycobacterium tuberculosis less. Aminoglycosides streptomycin is the earliest one of the drugs used in treatment of tuberculosis, the similar kanamycin as second-line drugs are also used in the clinical treatment of tuberculosis. The purpose of this study is to screen in Mycobacterium tuberculosis in response to aminoglycoside drug stimulation, which may be the sRNA and drug resistance, detection the expression and changes of the initial function. Methods the aminoglycosides streptomycin and kanamycin were 2 fold dilution The concentration effect on Mycobacterium tuberculosis standard strain H37Rv, according to the growth status of each concentration group, select the OD600 concentration is close to the group of bacteria, total RNA extracted from the established transcriptome library by high-throughput sequencing analysis of streptomycin and kanamycin under the action of transcriptome changes, and predict the new sRNA. calculated by FPKM the value of sRNA expression were normalized to FPKM values of hypothesis testing and fold difference calculation, screening up-regulated or down regulated expression of sRNA. by Northern blot, the actual change in the expression level of sRNA qRT-PCR expression difference is verified. Choose in streptomycin and kanamycin preliminary functional analysis of down regulated sRNA-ms28 effects after construction. Over expression plasmid of Mycobacterium tuberculosis H37Rv was electroporated into overexpression strain MS28, the growth rate and gene overexpression strains strains and wild strains and into the empty expression vector The situation, preliminary analysis of the function of MS28. The results of extraction of streptomycin, kanamycin and total group RNA control group, established 3 transcriptome sequencing library. Through genetic variance analysis, get the Mycobacterium tuberculosis standard strain H37Rv gene expression differences in streptomycin under article 287 (7.2%), including 184 up-regulated genes the down regulated expression of 103 genes; kanamycin gene was under 230 difference (5.8%), of which 61 were up-regulated, 169 down regulated.GO enrichment analysis showed that all differences encoding genes, cellular components, differences in membrane protein and metabolism gene enrichment level is the highest. This study has predicted that 174 sRNA, of which 88 are located in the chain, respectively located in the negative strand for ms01-ms88,86 encoding, encoding respectively obtained after streptomycin differential expression of sRNA 5, as the difference of ms89-ms174. expression between the MS03, MS75, MS28, upregulation of ms172, downregulation of MS88; kanamycin Rapamycin differentially expressed sRNA 6, including ms113, MS28, ms42, upregulation of ms146, ms127, ms137 down, 1512822-1513027 MS28 is located in the genome of Northern blot and Q RT-PCR detection of streptomycin by down regulated sRNA. streptomycin and kanamycin and effect of kanamycin. After sRNA expression, 7 the detection results of 10 differentially expressed in sRNA and the results are consistent. The expression of MS28, expression of 6 genes with MS28 target gene. Compared with the previous forecast of electricity into the empty vector strains, MS28 overexpression strains growth curve to the left, growing logarithmic period shortened 3-4d. conclusion at transcriptional level Mycobacterium tuberculosis, by aminoglycoside drugs, gene expression changes of differentially expressed genes in cells, membrane proteins and metabolic processes in highly enriched. Under the action of streptomycin, M.tuberculosis sRNA - MS03, MS17 2 the expression of MS28 and downregulation of MS88; kanamycin under the effect of the expression of ms113, MS28, ms42, ms127 expression of Mycobacterium tuberculosis sRNA MS28 overexpression to potential target genes of Mycobacterium tuberculosis H37Rv strain growth change of.Ms28 include irtA, IRT B, mbtK, mbtN, FABG2. LprD.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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