【作者】 章文蔚；

【导师】 罗玉萍；

【作者基本信息】 南昌大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 MicroRNAs(miRNAs)是广泛存在于真核生物细胞的一类内源性小分子单链非编码RNA(non-coding RNA，ncRNA)，大小为21 nt左右。研究表明，这些miRNA作为一种真核生物体内的重要的调节因子，在生物体内发挥着重要的调节作用，并影响着生物体生命活动的各个方面。虽然目前通过实验和计算机的方法在植物和动物中发现了众多miRNA分子，但是在各个物种中所鉴定的miRNA的数量远不及理论上的饱和值，所以对miRNA的分离鉴定依然是miRNA研究的一个热点。马铃薯(S．tuberosum L．)属茄属多年生草本植物，它作为世界上广为栽培且种植面积位居第四的重要粮食作物，在农业生产和人民生活中均占有极其重要的地位，然而马铃薯极易受植物病毒的危害而造成种质退化和减产。近期的研究表明miRNA与植物每个阶段的生长发育息息相关，且参与植物的防御和应答外界环境压力胁迫，所以对马铃薯miRNA的研究有着非常重要的现实和理论意义。而对马铃薯miRNA研究的第一步就是找出马铃薯中尽可能多的miRNA，以及它们作用的靶标。本研究利用计算机RNA组学方法在马铃薯EST数据库、GSS数据库和nr数据库中搜寻保守的马铃薯miRNA分子，然后采用RT-PCR手段对预测结果进行验证，并观察它们在不同组织中的表达情况。利用在线分析软件分析马铃薯miRNA的靶序列，并对靶序列的功能进行预测分析，结果小结如下：1．在数据库中共搜寻出48条保守的马铃薯miRNA候选分子，其中30条miRNA来源于EST数据库，11条miRNA来自于GSS数据库，7条miRNA鉴定于nr数据库。这些miRNA候选分子与已知拟南芥或水稻的miRNA有着高度同源性。2．在研究马铃薯miRNA分子中，发现一些仅存在于双子叶植物中的miRNA分子，如miR403、miR869和miR870。通过比对分析发现，仅成熟的植物miRNA分子表现较为保守，而其前体变化较大。3．使用在线预测植物miRNA的软件——microHARVESTER，在44种不同植物物种中预测出136条保守的miRNA候选分子。通过计算分析发现，大部分候选分子的MFEI值大于0.7，可信度较高。4．通过分析这48条马铃薯miRNA，最终筛选出186个潜在靶分子，其中～20％的靶分子为一些在生长发育过程中起重要调控作用的转录因子；～30％的靶分子参与调控马铃薯体内的代谢过程、信号转导、对外界环境胁迫的应答以及调控自身合成；～24％的靶分子我们未预测出其功能。5．针对不同的植物RNA提取具有不同要求的情况，以富含淀粉、多糖物质的马铃薯根为材料，在现有植物RNA提取方法的基础上进行改良。RNA电泳分析，纯度鉴定及RT-PCR结果显示，该方法提取的RNA质量好，纯度高，能应用于后续实验。6．通过对12条miRNA候选分子进行RT-PCR分析，发现miRNA在不同组织中存在着特异性表达情况，如miR157a、miR390、miR414a和miR395，分析结果表明这些miRNA分子在马铃薯生长发育过程中的存在着不同水平的表达。更多还原

【Abstract】 MicroRNAs (miRNAs) are an extensive class of endogenous non-coding RNAs (ncRNAs) found in eukaryotic organisms, which play important roles in many biological processes as posttranscriptional regulators of gene expression. Although several hundred miRNAs have been predicted and identified by computational and experimentally methods both in animals and plants, search for new miRNAs is still a hot academic domain because miRNAs identified so far are far from saturated based on theoretical prediction. Potao (Solarium tuberosum L.) is the fourth biggest crop that can be used as grain and vegetables in the world. However, viruses and diseases are considered worldwide as the main cause of potato severe agricultural loss and seed degeneration. One recent survey has indicated that miRNAs are associated with every stage of plant’s growth and development, particularly in response to pathogen invasion and environmental stress. Therefore, predictation and identification of potato miRNAs and their targets have been the subject of considerable interest.We have applied computational RNomics to predict potato miRNAs from expressed sequence tags (EST), genomic survey sequence (GSS), and non-redundant (nr) databases. To validate the prediction of miRNAs in potato, we performed RT-PCR assay to verify expression of mature miRNAs. Finally, we used miRU to predict the potential target genes for these miRNA candidates. The main results are described as below:1. We identified 48 potential miRNAs in potato. Among them, 30 miRNAs were identified in EST database, 11 miRNAs in GSS database and 7 miRNAs in nr database. Our results indicate that the majority of potential miRNAs have homologues in previously known A. thaliana and O. sativa.2. Our results indicate that several miRNAs are conserved among families of dicots, such as miR403, miR869, and miR870. And we found that only these mature miRNAs are highly conserved in plants instead of miRNA precursors.3. Additionally, we have predicted 136 conserved miRNAs in 44 plant species by microHARVESTER which was utilized on line. Most of these potential miRNAs have higher MFEI values(>0.7).4. We found 186 potential targets in potato. Of these predicted targets,～20% are associated with the members of the transcription factor gene families with known or predicted functions, mostly in development, and～30% are related to metabolic and cellular processes, signal transduction, defense/stress functions and their own biogenesis. We also identified a significant number(～24%) of the predicted targets with unknown functions, suggesting possible new roles for conserved miRNAs in potato.5. The efficient and economical method for isolation of plant RNAs was developed based on previous methods. The result of agarose gel electrophoresis of extracted RNA from potato root showed that the improved method was most efficient compared with guanidine isothiocyanate solution and the FastRNA Green method. Successful RT-PCR showed that the RNAs isolated with this method were in good integrity and purity, and can satisfy the needs of most molecular biological experiments.6. Performing RT-PCR assay of 12 miRNAs, we found that several miRNAs have different expression level among different organs, such as miR157a, miR390, miR414a, and miR395. These results suggest that the examined miRNAs have diverse expression patterns during development.更多还原