【摘要】：目的:非編碼RNA(non-coding RNA,ncRNA)在基因表達(dá)調(diào)節(jié)網(wǎng)絡(luò)中發(fā)揮重要的作用,在很多細(xì)菌中已被廣泛發(fā)現(xiàn)和研究。本研究組通過(guò)對(duì)傷寒沙門菌(S.Typhi)不同應(yīng)激條件下的轉(zhuǎn)錄組RNA-seq的分析,發(fā)現(xiàn)rpo H基因?qū)?cè)反向編碼的RNA,命名AS-RpoH,本論文旨在對(duì)AS-RpoH進(jìn)一步鑒定,并對(duì)其分子表達(dá)特性及在S.Typhi中的功能展開初步研究。方法:1.AS-RpoH分子特性分析:設(shè)計(jì)AS-RpoH特異性引物和雜交探針,用RT-PCR和Northern Blot證實(shí)AS-RpoH在S.Typhi中的表達(dá)存在;用5′RACE實(shí)驗(yàn)和RT-PCR探究AS-RpoH的轉(zhuǎn)錄起始和可能的終止位點(diǎn),分析其分子全長(zhǎng)和基因定位。2.AS-RpoH表達(dá)特性分析:用Northern Blot和實(shí)時(shí)熒光定量PCR(q RT-PCR)技術(shù)分析S.Typhi在不同生長(zhǎng)時(shí)相的AS-RpoH的表達(dá)水平;用q RT-PCR分析對(duì)數(shù)期野生株(OD600 0.4)在酸、氧、高滲及熱應(yīng)激環(huán)境刺激下AS-RpoH的表達(dá)情況。3.分析sigma因子對(duì)AS-RpoH表達(dá)影響:選用實(shí)驗(yàn)室前期已經(jīng)制備的sigma因子的缺失變異株(Δrpo E和Δrpo S),用q RT-PCR分析不同條件下不同缺陷株中AS-RpoH的表達(dá)特點(diǎn)。4.制備AS-RpoH高表達(dá)菌株:在ncRNA AS-RpoH中覆蓋rpo H編碼區(qū)序列處設(shè)計(jì)一對(duì)引物,引物加上NocⅠ和Xho I酶切位點(diǎn)。經(jīng)PCR擴(kuò)增AS-RpoH基因片段,通過(guò)酶切和連接,插入至p BAD/Myc-His A載體,經(jīng)過(guò)酶切、PCR及測(cè)序鑒定后,將重組載體(p BAD-AS-RpoH)和空載體(p BAD/Myc-His A)電轉(zhuǎn)入S.Typhi野生株中,構(gòu)建高表達(dá)菌株(WT-p BAD-AS-RpoH)和空質(zhì)粒對(duì)照株(WT-p BAD)。5.動(dòng)力試驗(yàn):制備0.3%的等滲半固體動(dòng)力培養(yǎng)基,吸取1μL培養(yǎng)至對(duì)數(shù)生長(zhǎng)期的AS-RpoH高表達(dá)菌株和對(duì)照株菌液,分別接種于動(dòng)力板,37℃培養(yǎng)一定時(shí)間后根據(jù)細(xì)菌圈直徑來(lái)比較細(xì)菌的動(dòng)力差異。6.生長(zhǎng)曲線測(cè)定:以時(shí)間為橫坐標(biāo),OD600為縱坐標(biāo)描制曲線,觀測(cè)AS-RpoH高表達(dá)菌株和對(duì)照株在不同條件下的生長(zhǎng)情況。7.He La上皮細(xì)胞侵襲實(shí)驗(yàn):將AS-RpoH高表達(dá)菌株和對(duì)照株培養(yǎng)至對(duì)數(shù)期,與He La細(xì)胞共孵育,分析比較兩種細(xì)菌對(duì)上皮細(xì)胞侵襲能力間的差異。8.rpo H m RNA的穩(wěn)定性分析:將AS-RpoH高表達(dá)菌株和對(duì)照株培養(yǎng)至對(duì)數(shù)期,q RT-PCR分析rpo H m RNA的水平。結(jié)果:1.RT-PCR和Northern Blot檢測(cè)發(fā)現(xiàn),AS-RpoH在S.Typhi中存在表達(dá),長(zhǎng)約3000 nt;5′RACE結(jié)果顯示轉(zhuǎn)錄起始點(diǎn)位于yhh K編碼區(qū)上游411 nt處;RT-PCR結(jié)果表明AS-RpoH的轉(zhuǎn)錄終止位點(diǎn)在距離5′端3461~3508 nt;yhh K編碼區(qū)探針做Northern Blot分析發(fā)現(xiàn),其雜交條帶與AS-RpoH表達(dá)高峰區(qū)探針雜交條帶長(zhǎng)度基本一致。2.Northern Blot和q RT-PCR結(jié)果表明,AS-RpoH在不同生長(zhǎng)時(shí)期下的表達(dá)水平無(wú)明顯差異,在酸應(yīng)激下的表達(dá)水平顯著下降,在熱應(yīng)激、氧應(yīng)激和高滲應(yīng)激下的表達(dá)無(wú)明顯變化。3.q RT-PCR結(jié)果表明,與WT相比,Δrpo S中AS-RpoH表達(dá)下調(diào),Δrpo E中AS-RpoH表達(dá)無(wú)變化。在氧應(yīng)激和高滲應(yīng)激下,WT、Δrpo S、Δrpo E中AS-RpoH表達(dá)無(wú)明顯變化;在酸應(yīng)激下,WT和Δrpo E中AS-RpoH表達(dá)下調(diào),Δrpo S中AS-RpoH的表達(dá)無(wú)變化。4.成功構(gòu)建AS-RpoH高表達(dá)菌株(WT-p BAD-AS-RpoH)和空質(zhì)粒對(duì)照株(WT-p BAD)。5.動(dòng)力實(shí)驗(yàn)顯示,AS-RpoH高表達(dá)菌株和對(duì)照株動(dòng)力無(wú)明顯變化。6.生長(zhǎng)曲線測(cè)定結(jié)果顯示,不同應(yīng)激下AS-RpoH高表達(dá)菌株和對(duì)照株的生長(zhǎng)狀況無(wú)明顯差異。7.與對(duì)照株相比,AS-RpoH高表達(dá)菌株對(duì)He La細(xì)胞的侵襲能力減弱。8.q RT-PCR結(jié)果表明,高表達(dá)AS-RpoH可以提高rpo H m RNA的穩(wěn)定性。結(jié)論:在S.Typhi中新鑒定了一個(gè)長(zhǎng)鏈反義RNA AS-RpoH,其分子全長(zhǎng)在3461~3508 nt之間,且為yhh K的3′非翻譯區(qū);rpo S有可能參與調(diào)控酸應(yīng)激下AS-RpoH的表達(dá)調(diào)節(jié);AS-RpoH能使細(xì)菌對(duì)He La細(xì)胞的侵襲能力下降,同時(shí)其可提高rpo H m RNA的穩(wěn)定性。
[Abstract]:Objective: Non-coding RNA (ncRNA) plays an important role in gene expression regulation network and has been widely found and studied in many bacteria. Through the analysis of transcriptional RNA-seq of Salmonella typhimurium (S. Typhi) under different stress conditions, we found reverse-coding RNA of RPO H gene, named AS-RpoH. The purpose of this study was to identify AS-RpoH and investigate its molecular expression and function in S.Typhi. METHODS: 1. AS-RpoH molecular characterization analysis: AS-RpoH specific primers and hybridization probes were designed, and the expression of AS-RpoH in S.Typhi was confirmed by RT-PCR and Northern Blot, and the transcription of AS-RpoH was investigated by 5'RACE and RT-PCR. The expression characteristics of AS-RpoH were analyzed by Northern Blot and real-time fluorescence quantitative PCR (q RT-PCR), and the expression levels of AS-RpoH in logarithmic wild strains (OD600 0.4) were analyzed by Q RT-PCR. Analysis of the effect of sigma factor on AS-RpoH expression: Select sigma factor deletion mutants (rpo E and RPO S) which have been prepared in the early stage of the laboratory, and analyze the expression characteristics of AS-RpoH in different defective strains under different conditions by Q RT-PCR. 4. Prepare AS-RpoH high-expression strain: Cover the coding region of RPO H in ncRNA AS-RpoH A pair of primers were designed and primers were added with Noc I and Xho I digestion sites. The AS-RpoH gene fragments were amplified by PCR and inserted into the P BAD/Myc-His A vector. After digestion, PCR and sequencing, the recombinant vector (p BAD-AS-RpoH) and empty vector (p BAD/Myc-His A) were electrotransformed into S.Typhi wild strain to construct a highly expressed strain (WT-p-p-p). BAD-AS-RpoH and blank plasmid control strain (WT-p BAD). 5. Dynamics test: 0.3% isotonic semi-solid dynamic medium was prepared. The strains with high expression of AS-RpoH and the control strains were inoculated on the power plate respectively and cultured at 37 C for a certain period of time. 6. Growth curve was compared according to the diameter of the bacterial circle. The growth of AS-RpoH high-expression strains and control strains under different conditions was observed by using OD600 as ordinate and time as abscissa. 7. Invasion test of He La epithelial cells: AS-RpoH high-expression strains and control strains were cultured to logarithmic phase, incubated with He La cells, and the invasiveness of the two bacteria to epithelial cells was analyzed and compared. Differentiation. 8. Stability analysis of RPO H m RNA: AS-RpoH high-expression strains and control strains were cultured to logarithmic phase, and the RPO H m RNA level was analyzed by Q RT-PCR. Results: 1. RT-PCR and Northern Blot detection showed that AS-RpoH was expressed in S. CR results showed that the transcriptional termination sites of AS-RpoH ranged from 3461 to 3508 NT at the 5'-terminal; Northern Blot analysis showed that the length of hybridization bands of yhh K coding region probe was basically the same as that of AS-RpoH peak region probe. 2. Northern Blot and Q RT-PCR results showed that the expression level of AS-RpoH had no significant difference at different growth stages. The expression of AS-RpoH in RPO S was down-regulated and that in RPO E was unchanged compared with that in WT. The expression of AS-RpoH in WT, RPO S and RPO E did not change significantly under oxygen stress and hyperosmotic stress. Under WT and RPO E, AS-RpoH expression was down-regulated, and the expression of AS-RpoH in RPO S was unchanged. 4. High expression strain of AS-RpoH (WT-p BAD-AS-RpoH) and blank plasmid control strain (WT-p BAD) were successfully constructed. 5. Dynamic test showed that the high expression strain of AS-RpoH and the control strain had no significant change in dynamics. 6. The growth curve showed that AS-RpoH high expression strain and control strain had no significant change under different stress. Compared with the control strain, the invasion ability of AS-RpoH-overexpressing strain to He La cells was weakened. 8. Q RT-PCR results showed that the high expression of AS-RpoH could improve the stability of rpo-H m RNA. Conclusion: A long-chain antisense RNA AS-RpoH was identified in S. Typhi, and its molecular length was 3461-3508 nt. RpoS may be involved in regulating the expression of AS-RpoH under acid stress; AS-RpoH can decrease the invasiveness of bacteria to He-La cells and improve the stability of rpo-H m RNA.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378

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9 陳建才;傷寒沙門菌SNPs分子分型分析和鼠傷寒沙門菌分子流行病學(xué)特點(diǎn)研究[D];蘇州大學(xué);2012年

10 廖莉;傷寒沙門菌質(zhì)粒對(duì)細(xì)菌生物膜形成的影響及分子機(jī)制研究[D];蘇州大學(xué);2012年

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