贾第虫microRNA 及其靶基因的预测与验证
文献类型：学位论文
| 作者 | 张燕琼 |
| 学位类别 | 硕士 |
| 答辩日期 | 2008-08 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 文建凡 |
| 关键词 | 第虫 计算机预测 microRNAs 靶基因 |
| 其他题名 | Computational Identification of microRNAs and their Targets in Giardia lamblia |
| 中文摘要 | MicroRNAs (miRNAs)是一类长约21-25nt的非编码小分子RNAs，通过与靶基因的互补结合在转录水平及转录后水平来负调控基因表达。人们已在众多高等多细胞生物中如人、果蝇、线虫、拟南芥等鉴定出众多microRNAs分子。近来报道单细胞原生生物衣藻中也存在大量microRNAs。然而到目前为止，在被很多证据证实是最原始的单细胞真核生物贾第虫中却仍未有microRNAs的报道。那么到底贾第虫这种具有特殊进化地位的单细胞原生动物是否存在有microRNAs呢？如果存在的话，其microRNAs的特点是什么？与高等多细胞生物及单细胞衣藻的microRNA相比又有何异同点呢？贾第虫的microRNAs是否与其致病性相关呢？ 已有研究表明，贾第虫基因组中存在与RNAi相关的Argonaute（AGO）家族蛋白和Dicer酶。有意思的是，这些与siRNA引起RNAi作用关键的蛋白AGO和Dicer同样也是miRNA系统的关键成份，这就提示我们在贾第虫中很有可能也存在有miRNA并发挥功能。有研究发现在贾第虫基因组中存在大量的非编码转录物，这些大量的非编码转录物中，是否都是后来所认为的为双向启动子转录有用基因时的副产物，还是也存在一些起调控作用的RNA分子（如miRNAs等），需要进一步的研究。 本文利用生物信息学的手段，依据miRNAs的生物学特征，结合多种计算机预测的方法，在贾第虫基因组中筛选可能的microRNAs分子，结果共鉴定出50个miRNAs候选分子，这50个可能的贾第虫miRNAs不具有保守性，在已知的其他物种的miRNAs中找不到同源物。用这50个microRNAs BLASTN 贾第虫的蛋白质编码序列及其相邻5’端和3’端各200bp的序列，来寻找这些microRNAs所调控的靶基因。结果表明，寻找到的贾第虫miRNA的靶基因除很大一部分未知功能的蛋白外，还包括了很多涉及不同功能的蛋白，如VSP蛋白（various surface proteins）这样一类表面抗原蛋白，提示我们贾第虫miRNA可能与其致病性相关。接下来我们对其中14个预测的贾第虫microRNAs进行了RT-PCR检测并克隆测序，结果表明gla-mir-6, gla-mir-35在贾第虫滋养体细胞中稳定表达。我们的研究第一次用生物信息学结合实验的方法在贾第虫寻找到了microRNAs，为下一步深入研究这些microRNAs在贾第虫中的功能提供了可能。 |
| 英文摘要 | MicroRNAs (miRNAs) are a family of ~22 nucleotide small, non-coding RNAs that negatively regulate gene expression at the transcriptional and post-transcriptional level. miRNAs have been identified in many metazoan including humans, flies, nematodes and plants. Recent researches also reveal that Chlamydomonas reinhardtii, a unicellular green alga, encodes many miRNAs. Thus far, however, no miRNAs have been identified in Giardia lamblia which is considered as one of the most primitive extant eukaryotes. Does any miRNA exist in Giardia, which takes a paramount position in evolution? If yes, what are the characteristics of the miRNAs? What are the differences between the miRNA in Giardia and those in higher eukaryote and Chlamydomonas？Are the miRNA in Giardia related to the inducing of diarrhea? Several recent studies indicate that in Giardia genome, there exist RNAi-related Argonaute (AGO) protein family and Dicer. Interestingly, those key proteins which induce RNAi-AGO and Dicer-are also paramount components in miRNA system, so it implies that miRNA may also exist in Giardia and have functions. Some other researches reveal the existance of huge amount of non-coding transcripts, which are thought to be produced by bi-directional promoters ubiquitously located in Giardia genome. Whether some of the non-coding transcripts are regulational RNA molecules (such as miRNAs) requests further study. In this work, several approaches were combined to make computational prediction of potential miRNAs and their targets in Giardia lamblia. A total of 50 potential miRNAs have been identified following a range of strict filtering criteria. Using these potential miRNA sequences, we have searched their corresponding targets using BLASTN Giardia lamblia coding sequences with their 5’ and 3’ flanking 200bp sequences. Although most of the potential target genes of Giardia miRNAs encode functionally unknown proteins, other annotated target proteins are involved in various physiological functions. A case in point is VSP proteins (various surface proteins), which are a sort of surface antigen proteins. This result implies that Giardia miRNA may be pathogenicity-related. After testing the expression of 12 predicted Giardia miRNAs by RT-PCR and sequencing, we find that gla-mir-6 and gla-mir-35 are steadily expressed in Giardia trophozoite cells. Our study predicts the existance of microRNAs in Giardia by bioinformatics and also confirms the prediction by wet-lab experiments for the first time, which paves the way for studying the functions of those microRNAs in Giardia. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6320]  |
| 专题 | 昆明动物研究所_真核细胞进化基因组 |
| 推荐引用方式 GB/T 7714 | 张燕琼. 贾第虫microRNA 及其靶基因的预测与验证[D]. 北京. 中国科学院研究生院. 2008. |
入库方式： OAI收割
来源：昆明动物研究所
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