本文選題：sRNA　切入點(diǎn)：結(jié)核分枝桿菌　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的在細(xì)菌等原核生物中,sRNA是存在于非編碼區(qū)的一類新型調(diào)節(jié)子,其主要功能是調(diào)控相應(yīng)基因的表達(dá),使細(xì)菌適應(yīng)環(huán)境變化。近年來(lái)研究者發(fā)現(xiàn)sRNA與多種抗生素暴露甚至耐藥性的產(chǎn)生有關(guān),其中就包括氨基糖苷類藥物,而在結(jié)核分枝桿菌中的相關(guān)報(bào)道較少。氨基糖苷類藥物鏈霉素是最早用于抗結(jié)核治療的藥物之一,其同類的卡那霉素作為二線藥物也被用于臨床結(jié)核病治療。本研究的目的是篩選在結(jié)核分枝桿菌中響應(yīng)氨基糖苷類藥物刺激,可能與耐藥性產(chǎn)生相關(guān)的sRNA,檢測(cè)其表達(dá)量變化并進(jìn)行初步的功能學(xué)實(shí)驗(yàn)研究。方法將氨基糖苷類藥物鏈霉素和卡那霉素分別以2倍倍比稀釋濃度作用于結(jié)核分枝桿菌標(biāo)準(zhǔn)菌株H37Rv,根據(jù)各濃度組的生長(zhǎng)情況,選取OD600值較為接近的濃度組菌液,提取總RNA,建立轉(zhuǎn)錄組文庫(kù),通過(guò)高通量測(cè)序分析鏈霉素和卡那霉素作用下轉(zhuǎn)錄組的改變,并預(yù)測(cè)新的sRNA。通過(guò)計(jì)算FPKM值,將sRNA的表達(dá)量進(jìn)行標(biāo)準(zhǔn)化,對(duì)FPKM值進(jìn)行假設(shè)檢驗(yàn)和差異倍數(shù)計(jì)算,篩選上調(diào)或下調(diào)表達(dá)的sRNA。采用Northern blot、qRT-PCR驗(yàn)證差異表達(dá)sRNA的實(shí)際表達(dá)量變化。選擇在鏈霉素和卡那霉素作用后共同下調(diào)的sRNA-ms28進(jìn)行初步功能分析。構(gòu)建過(guò)表達(dá)質(zhì)粒、電轉(zhuǎn)入結(jié)核分枝桿菌H37Rv得到ms28的過(guò)表達(dá)菌株,通過(guò)比較過(guò)表達(dá)菌株與野生株和轉(zhuǎn)入空載體的菌株的生長(zhǎng)速率和基因表達(dá)情況,初步分析ms28的功能。結(jié)果提取鏈霉素組,卡那霉素組及對(duì)照組的總RNA,建立了3個(gè)轉(zhuǎn)錄組測(cè)序文庫(kù)。通過(guò)基因差異分析,得到結(jié)核分枝桿菌標(biāo)準(zhǔn)菌株H37Rv在鏈霉素作用下差異表達(dá)基因287條(7.2%),其中上調(diào)表達(dá)基因184條,下調(diào)表達(dá)基因103條;卡那霉素作用下差異基因230條(5.8%),其中61條上調(diào),169條下調(diào)。GO富集分析顯示,所有差異編碼基因中,細(xì)胞組分、膜蛋白和代謝過(guò)程的差異基因富集程度最高。本研究共預(yù)測(cè)了174條sRNA中,其中88條位于正鏈,分別編碼為ms01-ms88,86條位于負(fù)鏈,分別編碼為ms89-ms174。組間差異表達(dá)分析得到鏈霉素作用后差異表達(dá)sRNA 5條,其中ms03、ms75、ms172上調(diào),ms28、ms88下調(diào);卡那霉素作用后差異表達(dá)sRNA 6條,其中ms113、ms146上調(diào),ms28、ms42、ms127、ms137下調(diào),位于基因組1512822-1513027的ms28是鏈霉素和卡那霉素作用后共同下調(diào)的sRNA。采用Northern blot和q RT-PCR檢測(cè)鏈霉素和卡那霉素作用后sRNA表達(dá)量變化,10條差異表達(dá)的sRNA中的7條檢測(cè)結(jié)果與分析結(jié)果一致。將ms28過(guò)表達(dá)后,前期預(yù)測(cè)的ms28靶基因中有6條上調(diào)表達(dá)。與電轉(zhuǎn)入空載體的菌株相比,ms28過(guò)表達(dá)株生長(zhǎng)曲線左移,生長(zhǎng)對(duì)數(shù)期縮短3-4d。結(jié)論在轉(zhuǎn)錄組水平,結(jié)核分枝桿菌受到氨基糖苷類藥物作用后,部分基因表達(dá)量發(fā)生變化,差異表達(dá)基因在細(xì)胞組分、膜蛋白和代謝過(guò)程高度富集。在鏈霉素作用下,結(jié)核分枝桿菌sRNA—ms03、ms172表達(dá)上調(diào),ms28、ms88表達(dá)下調(diào);卡那霉素作用下ms113表達(dá)上調(diào),ms28、ms42、ms127表達(dá)下調(diào)。結(jié)核分枝桿菌sRNA—ms28的過(guò)表達(dá)使結(jié)核分枝桿菌H37Rv菌株的生長(zhǎng)發(fā)生改變。ms28的潛在靶基因包括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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R52

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相關(guān)博士學(xué)位論文 前1條

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本文編號(hào)：1603365