伤寒沙门菌长链反义RNA AS-RpoH分子特性及功能初步研究

发布时间：2018-08-28 20:10

【摘要】：目的:非编码RNA(non-coding RNA,ncRNA)在基因表达调节网络中发挥重要的作用,在很多细菌中已被广泛发现和研究。本研究组通过对伤寒沙门菌(S.Typhi)不同应激条件下的转录组RNA-seq的分析,发现rpo H基因对侧反向编码的RNA,命名AS-RpoH,本论文旨在对AS-RpoH进一步鉴定,并对其分子表达特性及在S.Typhi中的功能展开初步研究。方法:1.AS-RpoH分子特性分析:设计AS-RpoH特异性引物和杂交探针,用RT-PCR和Northern Blot证实AS-RpoH在S.Typhi中的表达存在;用5′RACE实验和RT-PCR探究AS-RpoH的转录起始和可能的终止位点,分析其分子全长和基因定位。2.AS-RpoH表达特性分析:用Northern Blot和实时荧光定量PCR(q RT-PCR)技术分析S.Typhi在不同生长时相的AS-RpoH的表达水平;用q RT-PCR分析对数期野生株(OD600 0.4)在酸、氧、高渗及热应激环境刺激下AS-RpoH的表达情况。3.分析sigma因子对AS-RpoH表达影响:选用实验室前期已经制备的sigma因子的缺失变异株(Δrpo E和Δrpo S),用q RT-PCR分析不同条件下不同缺陷株中AS-RpoH的表达特点。4.制备AS-RpoH高表达菌株:在ncRNA AS-RpoH中覆盖rpo H编码区序列处设计一对引物,引物加上NocⅠ和Xho I酶切位点。经PCR扩增AS-RpoH基因片段,通过酶切和连接,插入至p BAD/Myc-His A载体,经过酶切、PCR及测序鉴定后,将重组载体(p BAD-AS-RpoH)和空载体(p BAD/Myc-His A)电转入S.Typhi野生株中,构建高表达菌株(WT-p BAD-AS-RpoH)和空质粒对照株(WT-p BAD)。5.动力试验:制备0.3%的等渗半固体动力培养基,吸取1μL培养至对数生长期的AS-RpoH高表达菌株和对照株菌液,分别接种于动力板,37℃培养一定时间后根据细菌圈直径来比较细菌的动力差异。6.生长曲线测定:以时间为横坐标,OD600为纵坐标描制曲线,观测AS-RpoH高表达菌株和对照株在不同条件下的生长情况。7.He La上皮细胞侵袭实验:将AS-RpoH高表达菌株和对照株培养至对数期,与He La细胞共孵育,分析比较两种细菌对上皮细胞侵袭能力间的差异。8.rpo H m RNA的稳定性分析:将AS-RpoH高表达菌株和对照株培养至对数期,q RT-PCR分析rpo H m RNA的水平。结果:1.RT-PCR和Northern Blot检测发现,AS-RpoH在S.Typhi中存在表达,长约3000 nt;5′RACE结果显示转录起始点位于yhh K编码区上游411 nt处;RT-PCR结果表明AS-RpoH的转录终止位点在距离5′端3461~3508 nt;yhh K编码区探针做Northern Blot分析发现,其杂交条带与AS-RpoH表达高峰区探针杂交条带长度基本一致。2.Northern Blot和q RT-PCR结果表明,AS-RpoH在不同生长时期下的表达水平无明显差异,在酸应激下的表达水平显著下降,在热应激、氧应激和高渗应激下的表达无明显变化。3.q RT-PCR结果表明,与WT相比,Δrpo S中AS-RpoH表达下调,Δrpo E中AS-RpoH表达无变化。在氧应激和高渗应激下,WT、Δrpo S、Δrpo E中AS-RpoH表达无明显变化;在酸应激下,WT和Δrpo E中AS-RpoH表达下调,Δrpo S中AS-RpoH的表达无变化。4.成功构建AS-RpoH高表达菌株(WT-p BAD-AS-RpoH)和空质粒对照株(WT-p BAD)。5.动力实验显示,AS-RpoH高表达菌株和对照株动力无明显变化。6.生长曲线测定结果显示,不同应激下AS-RpoH高表达菌株和对照株的生长状况无明显差异。7.与对照株相比,AS-RpoH高表达菌株对He La细胞的侵袭能力减弱。8.q RT-PCR结果表明,高表达AS-RpoH可以提高rpo H m RNA的稳定性。结论:在S.Typhi中新鉴定了一个长链反义RNA AS-RpoH,其分子全长在3461~3508 nt之间,且为yhh K的3′非翻译区;rpo S有可能参与调控酸应激下AS-RpoH的表达调节;AS-RpoH能使细菌对He La细胞的侵袭能力下降,同时其可提高rpo H m RNA的稳定性。
[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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

【相似文献】