【作者】 张强；

【导师】 葸玉琴；

【作者基本信息】 西北师范大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 microRNAs(miRNAs)是近年来发现的一种非编码的小RNA，长度仅18-24个核苷酸左右，普遍介导基因转录后的调节，参与调控机体各种生理进程。miRNA与疾病如肿瘤的发生发展密切相关。研究发现血液循环中也存在miRNA，循环miRNA可以作为肿瘤无创诊断的生物学标志物。目前对未知miRNA序列的发现主要以生物信息学预测为主，而直接克隆法较少见。运用RNA反转录5’末端的转换模板技术（Switching Mechanism At5’endof the RNA Transcript, SMART），对鸡肝脏中的microRNA进行了筛选分离，获取了新的microRNA序列。实验中，首先给microRNA加上poly(A)尾巴，结合SMART技术反转录得到microRNA的cDNA文库，再将长度适宜的片段连接到质粒，最后转化大肠杆菌克隆测序，获得了3条未知序列。经查证，这3条序列在miRBase中没有任何记录，其前体的二级结构预测都能形成茎环结构，符合microRNA前体的结构特征，为新发现的microRNA。接着，针对获得的3条microRNA新序列，运用了3个常用的靶基因预测软件miRanda、TargetScan和MirTarget2分别进行靶基因预测。将3个软件均能够预测到的靶基因作为候选靶基因分析microRNA的生物学功能。功能分析结果显示，这些靶基因显著富集在102个GO分类和13条信号通路中。靶基因广泛参与信号转导、新陈代谢、细胞粘附和跨膜转运等生命活动过程，而且与胚胎干细胞的生长分化密切相关。实验为microRNA的直接克隆以及microRNA的靶基因功能分析提供了方法学参考。更多还原

【Abstract】 MicroRNAs(miRNAs)are non-encoded small RNAs which discovered in recentyears,about18-24nucleotides in length.They mediate the post-transcriptional genesmodulation generally and regulate various physiological processes in the body.miRNAs areclosely related to the occurrence and development of human diseases such as tumour.Studiesfound that there are miRNAs in the blood circulation,and circulated miRNAs can act as anew non-invasive turnout diagnosis biomarkers.Currently the discovery of unknown miRNAsequences mainly base on bioinformatics prediction, direct cloning method was rarely.Switching mechanism at5’end of the RNA transcript(SMART) method has been used toobtain new microRNAs sequences from chicken’s liver. In the experiment, RNA fractionswere extracted and polyadenylated by poly(A) polymerase at first; then, cDNA library whichwas suitable for PCR has been constructed by SMART reverse transcript technology; afterthat, the target cDNA fragments were recovered and cloned into vector for sequencing, andthree microRNAs were discovered. Sequence analysis showed that all of them were notexisted in miRbase,but their precursors can form stem-loop structures,therefore thesequences can be classified into new microRNAs.Next,three commonly used target gene prediction software which are miRanda、TargetScan and MirTarget have been used to predict the target gene of the three newmicroRNAs.And then,using target gene sequences which three software can alsopredicted to analysis biology function of three microRNAs.The result showed that targetgene significantly enrichment in102GO classification and13signal path.Most targetgenes involved in many life activities which include signal transduction, metabolism, celladhesion and across the plasma membrane,and also closely related to the growth ordifferentiation of embryonic stem cell. The result provide methodology reference for thedirect cloning of microRNA and microRNA target gene function analysis.更多还原