【作者】 周冬根；

【导师】 罗玉萍；

【作者基本信息】 南昌大学 ， 微生物学， 2007， 硕士

【摘要】 微小RNA(microRNA)是近年来在动植物以及病毒中被广泛研究的一类长度为18-25nt的非编码小分子RNA。许多研究表明，这些microRNA(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，几乎涉及到了所有的细胞水平方面的功能。由于目前在各个物种中所鉴定的microRNA的数量远远不及理论上的饱和值，所以对microRNA的分离鉴定依然是microRNA研究的一个热点。科学家在2001年便完成了对狗基因组的测序，而且通过全基因组比较分析，发现人与犬类基因组具有相似的核苷酸差异水平，为研究人类相关疾病提供了一个很好的平台，同时犬类物种间具有很大的表型多样性。最为重要的是，目前在miRNA数据库(miRBase)中只报道了6个miRNA基因。以上这些都为在犬类基因组中鉴定miRNA提供了理论平台和现实意义。由于miRNA具有时空表达特异性，以及mRNA的降解产物和其它非编码RNA的干扰，所以采用实验RNA组学的方法具有一定的局限性。因而我们这里采用计算机RNA组学的方法利用成熟miRNA的序列保守性以及前体结构具有发夹二级结构的性质在犬类基因组中鉴定出357个miRNA分子，现将研究结果小结如下：1．所鉴定的357个miRNA序列分布在犬类的33条染色体，其中在基因组中属于单拷贝的序列是281个，属于多拷贝的序列是35个；分布在基因间隔区的序列是222个；分布在内含子中的序列是125个，其中与基因链互补的序列有75个，与基因链反向的有50个；分布在外显子上的序列是6个；分布在非翻译区(UTR)上的序列是3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分析所有预测的序列的所在基因组位点是否能够形成符合miRNA标准的二级结构。折叠结果表明：绝大多数序列能够形成典型的发夹结构。狗基因组中与人同源的miRNA由于在人类中已经通过实验证实可以稳定表达。同时通过EST数据库搜索发现8个新的miRNA可以稳定表达。而且我们对其中5个新的miRNA分子进行RT-PCR分析，两个microRNA分子得到预期条带，说明可以稳定表达。3．通过分析发现狗基因组中这300个与人miRNA具有同源的序列形成149个家族，其中miR-154、miR-17和let-7家族分别含有15、13和10个成员。大多数家族中只含有一个成员。这与植物miRNA形成家族的形式完全相反。动物家族间的成员在序列上呈现出三种形式：成员间呈现多拷贝形式，或者是成员间序列上是相似的，又或者是他们具有同源性。4．利用启动子分析软件对所有的miRNA进行了分析，最后鉴定出了142个miRNA形成了53个簇，由上游的启动子控制表达，其中28个簇由多个同源的miRNA基因组成，18个miRNA簇形成8个不同的同源簇。进一步的分析表明有些簇是通过重复的形式产生多拷贝。5．研究结果表明8号染色体上存在一个与人具有高度同源的印迹区域，在这个区域发现一个最大的由41个miRNA组成的miRNA基因簇，另外两个簇则位于该簇的上游，分别是由5个miRNA组成的基因簇以及C／D snoRNA基因簇。进一步分析表明这最大的microRNA基因簇由三个基因家族组成，这三个家族分别是由不同的microRNA通过串联重复而产生的。6．研究发现，15个miRNA位于基因组的重复区域。其中7个位于LINE元件中，其它大多数位于SINE中。它们有些完全位于重复元件内，有些则与元件部分重叠。而且还有2个miRNA位于rRNA基因的内含子中。7．通过分析发现大多数miRNA在动物的进化过程中都是保守的。研究发现，大多数miRNA是通过局部串联重复以及非局部重复形式而进化的。串联重复在邻近区产生多个拷贝，而非局部重复发生在不同染色体之间，从而产生基因或者基因簇的迁移。8．对所鉴定的miRNA靶序列的预测分析表明，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。更为重要的是，我们鉴定出了狗特异性的靶序列，它们可能参与了狗生长过程中的一些特殊过程。最后，我们发现miR-186能够调节其宿主蛋白的表达，从而影响其表达量。这是一种自我调节的反馈机制，相互制约，相互影响。更多还原

【Abstract】 McroRNAs, as a family of non-coding small RNA 18-25nt in length, have beenprofoundly studied in recent years in plants, animals and virus. As an importantregulator in eukaryote organism, many study work had done to prove that microRNAhave abroad roles in the development of organism involving every aspect of cellphysical activity. The characterization of miRNA is still a hot academic domain forthe cause of the identified microRNA far from saturation. The scientists hadcompleted sequencing of dog genome, and through human/dog genomic pairalignment, it is found that dogs and humans have similar levels of overall nucleotidediversity, meanwhile, the species display extreme phenotypic diversity, therefore, allprovide a good flat for research disease related with human. More importantly, sixmiRNA were characterized in miRBase thus far. Above all, these provide a greatacademic and realistic meaning for idemifying miRNAs in dog genome.As for time- and tissue- speciality of miRNA expression and mis-leading ofdegradation of mRNA and other non-coding RNAs, there is limitation by usingexperimental RNomics. Therefore, we apply computational RNomics to predictmiRNA in dog based on sequence conservation of mature miRNAs and theirprecursor displaying hairpin structure, leading to identification of 357 novel miRNAs.The following is the summarization of our result:1. The loci of all 357 microRNA sequences ha genome lie in all 33 chromosomes ofgenome. Among these miRNAs, 281 sequences belong to single-copy in doggenome and the other 35 sequences belong to multi-copy. The number ofsequences in intergenic region is 222, in intron is 125, anti-sense to gene is 50,sense to gene is 75, and in exon is 6, in UTR is 3. All precursors ranged from 65to 120 nt in length, with average length of 80 nt.2. We have analyzed whether all miRNA could form stem-loop when joined withflanking sequence in genome through m-fold programs and the results showedthat a majority of miRNA have this property. Homologue of human miRNAs hadbeen validated by experiment, indicating that they can express stably. Meanwhile,8 novel miRNAs were found by searching EST database. Furthermore, we carryout RT-PCR assay of 5 novel miRNA, two of which can obtain the expected result,suggesting that they can express. 3. Trough analysis, we found that the 300 homologue of human miRNA belong to149 families, and miR-154, miR-17 and let-7 family have 15, 13 and 10,respectively. Most of families only have one members, the case is different fromplant. Members of animal family display three mode, identical, closely related orhomology.4. For conforming miRNAs are transcripted under common promoter, we analysis.these miRNAs using promoter analysis program, leading to identification of 142miRNAs grouping into 53 clusters controlled by promoter in upstream, amongwhich 28 clusters is made up of multi homologus miRNAs and 18 miRNA clusterform 8 distinct paralogous clusters. Further analysis indicate that some clusterhave multi-copies through duplication.5. Interestingly, we found a large miRNA cluster made up of 41 miRNAs, whichreside in imprinted region of chromosome 8 high conserved between human anddog, and another two clusters lie in upstream of this cluster, a miRNA clustercomposed of 5 miRNA and C/D snoRNA cluster.6. We found that 15 miRNA occur in repetitive region in genome, among which 7 liein LINE element and the others reside in SINE elements. Some of themcompletely reside in repetitive region, and some overlap with element. TwomiRNAs reside in intron of rRNA gene.7. A majority of miRNAs are conserved among animal through analysis. Most ofmiRNAs evolved by local tandem duplication and non-local duplication. Localtandem duplications result in paralogous sequences that are located on the sametranscript. Non-local duplications result in paralogous genes or gene clusters ondifferent chromosomes.8. We predict target of identified miRNAs using 3-UTR. Further analysis indicatethat a majority of target involved in almost all cellular processes, for example,development, metabolism, regulatory factor and signal transduction. The mostriched GO term is transcriptional factor. Meanwhile, some miRNAs might areinvolved in processes related with cancer. Most importantly, we identified targetgene unique to dog, which regulate special processes during dog development.Finally, we found that miR-186 can regulate expression of its host protein, resultin turnout of its protein. This is a self-regulatory feedback mechanism.更多还原