【作者】 程汝滨；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 药物化学， 2009， 博士

【摘要】 非编码小RNA分子(small non-coding RNA)是近年来在生物体内发现的一类新型RNA分子,其大小一般在400bp以下,本身不编码蛋白质,而是直接参与生命活动的调控。目前非编码小RNA已成为生命科学研究的前沿和热点。在真核生物已发现了大量的非编码小RNA分子,如microRNA、siRNA和piRNA。近年来在原核生物中,尤其是细菌中,也发现了一类与真核生物中非编码小RNA类似的小RNA分子,称为反式作用小RNA(trans-encoded sRNA);它们通过与靶基因配对结合影响靶基因的翻译效率和稳定性。在真核生物中,RNA干扰(RNAi)技术作为一种简便的基因功能研究方法已经得到了广泛的应用,更为重要的是,它为疾病的治疗和预防提供了一种新的技术和方法。然而,目前在原核生物中进行基因功能研究的工具依然是传统的同源重组和转座技术。因此,在原核生物中建立一种类似于真核生物RNAi的基因功能研究技术具有重要的理论意义和明确的应用前景。在本论文中,我们利用人工设计的反式作用小RNA(artificial trans-encoded sRNA,atsRNA)进行了细菌基因沉默的研究。首先,通过对已知细菌内源反式作用小RNA的生物信息学分析,得到了它们在一级和二级结构上的共同特征,并以此建立了atsRNA的设计原则。atsRNA由靶基因配对结合区域、Hfq结合位点和不依赖于Rho因子的转录终止末端三个模块构成;根据每个模块的一级结构特征设计atsRNA的三个组成部分,然后按模块的顺序随机组合构建atsRNA多样性文库;对库中atsRNA进行二级结构预测,根据二级结构的设计标准从库中筛选并合成候选atsRNA。根据上述atsRNA设计原则,我们针对外源基因EGFP和内源基因uidA分别设计合成了一系列atsRNA,插入小RNA表达载体后,转入宿主菌,并检测它们对细菌靶基因的干扰效率。实验结果表明,大部分atsRNA有效地抑制了靶基因的表达。在此基础上,我们对atsRNA的作用机制进行了系统的研究。(一)atsRNA突变体实验结果证明,atsRNA的二级结构(如靶基因配对结合区域的茎环结构、配对区域内loop环内碱基个数等)和稳定性对atsRNA的干扰活性至关重要(;二)在构成atsRNA的三个模块结构中,靶基因配对结合区域和Hfq结合位点是atsRNA发挥基因沉默作用所必须的,且Hfq结合位点对于atsRNA在体内的稳定性非常重要。(三)利用hfq缺失菌株,我们发现atsRNA介导的基因沉默是Hfq依赖型的,且Hfq能够与atsRNA直接结合而增加其稳定性。(四)atsRNA能够直接抑制靶基因的翻译和降解靶mRNA,atsRNA介导的靶mRNA的降解是RNase E依赖型的;并且在atsRNA介导的基因沉默中,atsRNA对靶基因翻译的阻断作用起主导作用,而靶mRNA的降解不是atsRNA引起靶基因沉默所必须的。(五)为了进一步证明atsRNA能够作为一种细菌基因功能研究的工具,我们针对细菌体内的必须基因murA, trmA和ygjD设计合成了一系列的atsRNA,定量PCR的结果表明atsRNA明显的降低了靶基因的mRNA表达水平,且atsRNA的表达能够有效地抑制宿主细胞的生长速度,说明atsRNA的表达有效的降低了靶基因的表达。综上所述,本论文首次利用人工设计的反式作用小RNA成功地对细菌的靶基因进行了高效和特异性的沉默,从而建立了一种高效、简便的细菌功能基因的研究技术。随着细菌内源性小RNA的研究进展,人们发现小RNA在细菌的致病力和环境适应力方面发挥了重要的调节作用。针对细菌的致病基因或致病基因的调控基因设计相应的atsRNA,将有可能阻断或抑制细菌的致病力,达到抗感染的目的。因此,atsRNA基因沉默技术的建立不但具有重要的理论意义,而且具有广泛的应用前景,将为细菌感染的治疗提供了一种崭新的途径,同时为解决日益加剧的细菌耐药难题创造条件。更多还原

【Abstract】 Small noncoding RNAs (sRNAs) are widespread in both eukaryotes and prokaryotes. These small noncoding RNAs, which function as regulators of gene expression, constitute a structurally diverse class of molecules that are typically shorter than 400 nucleotides (nt) in length and do not contain expressed open reading frames (ORFs). The eukaryotic small noncoding RNAs, such as microRNA (miRNA), short interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) have making a splash during the past few years. Recently, with the development of the experimental and computational approaches, hundreds of sRNAs have been identified in prokaryotes, especially in bacterial. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become an extraordinarily powerful RNA silencing tool for elucidating and manipulating gene functions in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes in which homologous recombination and transposon mutagenesis remain to be the major tools of deciphering the function of genes.In this study, we described firstly the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of the natural bacterial sRNAs, we have developed the principle and process for atsRNA design. atsRNA was designed to be a modular structure composed of three elements: mRNA basepairing region, Hfq binding site and typical Rho-independent terminator. The three component parts were selected randomly and then assembled into a series of atsRNA candidates whose secondary structures were then predicted by MFOLD program. atsRNAs should be selected from the atsRNA candidates according to predicted secondary structure and certain other selecting criteria. The complementary DNA oligonucleotides corresponding to the designed atsRNAs and cloned into a plasmid vector under the control of tac promoter for expression of atsRNAs. To evaluate the feasibility of this method, an exogenous EGFP gene on a multi-copy plasmid and an endogenous uidA gene encoding beta-glucuronidase were used as targets for the atsRNAs. Most, if not all, atsRNAs inhibited the expression of the target genes to effectively when they were expressed by IPTG addition in E. coli.Further studies demonstrated that the secondary structure (e.g. stem-loop structure of mRNA basepairing region, the number of unpaired nucleotides in a loop structure) and stability were crucial for the activity of atsRNA. Additionally, mutations in either mRNA basepairing regions or Hfq binding sites abolished the ability to repress the expression of target genes. This result also indicated that atsRNA acted by basepairing with target mRNA. The arsRNA-mediated gene silencing was Hfq dependent and Hfq could stabilize the atsRNA by binding to the atsRNA directing. atsRNA led to translational repression and RNase E dependent degradation of target mRNA, and the translation inhibition was the primary event for gene silencing. As for certain natural regulatory RNAs, degradation of the mRNA does not contribute to the efficiency of repression.Finally, in order to substantiate our findings, we generated atsRNAs for silencing of three essential genes murA, trmA and ygjD. We succeeded to cause the growth inhibition of E. coli cells by expressing atsRNAs complementary to several essential genes, suggesting that these atsRNAs inhibit efficiently the expression of these essential genes. All together, our findings demonstrated that atsRNA was an effective RNA tool for specific gene silencing in bacteria.Recent studies have demonstrated that numerous endogenous trans-encoded sRNAs have crucial roles in bacterial stress responses and virulence regulation. Therefore, atsRNAs targeting against virulence genes would function effectively in bacterial pathogens and it could potentially serve as antibiotics. Given the emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics, atsRNAs will provide an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium.更多还原