【作者】 高佳莉；

【导师】 罗玉萍；

【作者基本信息】 南昌大学 ， 微生物学， 2007， 硕士

【摘要】 microRNA(miRNA)是一种长约22nt的非编码RNA，通过与靶基因的3′UTR区结合来调控靶基因的表达。目前已证实miRNA在生物体生长、发育和疾病发生等过程中发挥着重要的作用。由于现在在各个物种中已鉴定的miRNA的数量离实际的饱合值还很远，所以对miRNA的分离鉴定仍然是miRNA研究的一个重点和热点。动物miRNA基因在进化上具有高度的保守性，许多序列都可以在亲缘关系相近的物种中找到，甚至在亲缘关系较远的物种中都有发现。牛全基因组的测序在2005年便已完成，这为计算机搜寻miRNA提供了前提条件。因而，我们可以利用已鉴定的种子序列，通过生物信息学同源性搜索的方法来搜寻牛的miRNA基因，并且应用软件搜寻候选基因的靶序列，系统分析miRNA的基因组织形式。鉴定和分析牛miRNA基因及其靶基因，有助于我们进一步了解miRNA对牛生长发育的影响与作用，有利于通过基因工程手段改良牛的品种与质量，并增加经济效益。此外，通过全基因组比较分析，发现人与牛基因组具有相似的核苷酸差异水平，这为研究人类相关疾病提供了一个较好的平台。本文采用计算机RNA组学和实验RNA组学相结合的方法，利用成熟miRNA的序列保守性以及前体结构具有发夹二级结构的特性在牛基因组中鉴定出289个miRNA分子，现将研究结果小结如下：1．所鉴定的289个牛miRNA基因分布在牛基因组30条染色体上，其中以21、19和X染色体上数量最多，分别为48、20和19个。基因组中，190个miRNA分布于于基因间隔区；分布在内含子区的序列有92个，其中与基因链互补的有49个，与基因链反向的有43个；位于外显子的有2个；分布在非翻译区(UTR)的为2个；基因组织形式暂未知序列2个。所有前体序列长度从65到120个核苷酸长度，平均长度在80个核苷酸左右。2．通过mfold分析所有预测的miRNA序列是否能够形成符合miRNA标准的发夹环状二级结构。折叠结果表明，绝大多数序列能够形成典型的发夹结构。通过RT-PCR实验验证7个候选miRNA前体的表达情况。在牛心、肝、脾和肺四个组织中，miRNA前体大都能稳定表达，但是也存在一些在不同组织中特异性表达的情况，如miR-130a，miR-134，miR-368和miR-410。3．序列分析发现牛基因组中289个序列形成145个基因家族。let-7和miR-17家族分别有11和7个成员，而大多数miRNA家族，例如miR-124，miR-7，miR-218家族只有一个成员。这与植物miRNA形成家族的形式完全相反。动物家族成员之间通常是多拷贝或者是序列同源的。4．利用启动子分析软件对所有的289个miRNA进行分析，发现其中130个基因形成40个基因簇。其中最大的基因簇位于21号染色体上，由32个基因组成的。进一步分析表明，基因簇很可能是由于进化过程中的单个基因的重复、变异和缺失形成的。同时，基因簇本身的重复形成多个同源簇。5．通过分析发现大多数miRNA在动物的进化过程中都是保守的。大多是通过串联重复和局部重复的方式复制进化的。串联重复是在同一染色体上邻近区域形成多个拷贝；而局部重复则是发生在不同染色体之间，从而引起基因或者基因簇的迁移。6．通过miranda程序批处理序列数据，鉴定出843个miRNA靶作用位点。大部分靶基因与miRNA不完全互补，暗示它们可能被翻译抑制；少数情况是miRNA与靶序列完全配对，这些miRNA则可能行使类似siRNA的靶序列切割功能。大多数miRNA所调节的靶序列参与了几乎生物体所有的细胞过程，比如生长发育、代谢、调控因子的调节以及信号转导作用等。其中参与的最多的功能可能是调节转录因子的表达。同时有些miRNA可能参与了一些癌症相关的过程。更多还原

【Abstract】 MicroRNAs(miRNAs), a class of noncoding RNA of approximately 22 nucleotides in length, can regulate gene expression by binding to the region of 3’UTR of the target mRNAs. Increasing evidence indicates that microRNA have been implicated in a variety of critical biological processes, including development, cell differentiation, and oncogenesis. The characterization of miRNA is still a hot academic domain because miRNAs identified so far are far from saturated. MiRNA in mammalian is highly conversative during evolution, not only in highly relative species, but also in widely divergent species. The genome of cow has been completely sequenced and annotated, which provides advantage to search new cow microRNAs and their target genes by computational analysis. Alignment show that cows and humans have similar levels of overall nucleotide diversity, so cow may be served as a model organism for the study of human disease.We apply comutational RNomies and experiment Rnomics to predict and validate miRNA in cow based on sequence conservation of mature miRNAs and their precursor displaying hairpin structure, leading to identification of 289 homology miRNAs.The following is the summarization of our result:1. The loci of all 289 microRNA sequences in genome lie in all 30 chromosomes of genome. Among these miRNAs, 48 in chromosome 21, 20 in chromosome 19 and 19 in chromosomeⅩ. The genomic organization of these candidate miRNA genes is divergent:190 are located in the intergenic regions; 92 in intron regions (49 in sense chain and 43 in anti-sense chain); 2 in exon region; 2 in UTR region and 2 in unknown region. All precursors ranged from 65 to 120 nt in length, with average length of 80 nt.2. Computational analysis showed that a majority of miRNA identified in this work can be folded to the typical stem-loop secondary structure of the miRNA family. Performing RT-PCR assay of 7 miRNAs, we found that several miRNAs have different expression level among different organs, such as miR-130a, miR-134, miR-335, and miR-655. 3. Analysis showed that the 289 homologue miRNA belong to 145 families, and let-7 and miR-17 family have 11 an7 members, respectively. Most of families only have one member, which is different from plant. Members of animal family display multi-copy, closely related or homology.4. Using promoter analysis program, we found that of the 289 miRNA identified in this work, 130 are grouped into 40 clusters controlled by promoter in upstream. Further analysis indicate that duplication, mutation and depletion have happen during evolution.And then, duplication of clusters themselves form distinct paralogous clusters.5. A majority of miRNAs are conserved among animal. Most of miRNAs evolved by local tandem duplication and non-local duplication. Local tandem duplications result in paralogous sequences that are located on the same transcript. Non-local duplications result in paralogous genes or gene clusters on different chromosomes.6. We use miranda for sequence batch processing, 843 potential target in cow has been pretested.A majority of targets are partly matched,which suggest that they may have been translation depression;a few miRNA matched with target completly ,they may cause mRNA degradation. A majority of target involved in almost all cellular processes, for example, development, metabolism, regulatory factor and signal transduction. The most riched GO term is transcriptional factor. Meanwhile, some miRNAs might are involved in processes related with cancer.更多还原