【作者】 杨国栋；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 MicroRNAs（miRNA）是一类长约21~24nt的小分子RNA。miRNA具有重要的调控基因表达功能，可降解靶基因的mRNA或抑制蛋白质的翻译。但目前对于miRNA基因本身的转录调控知之甚少。由此，我们通过系统分析植物miRNA在基因组上的分布结构、pri-miRNA、miRNA启动子、调控miRNA的转录因子等各方面的信息，建立了植物miRNA基因组学数据库pmiRGD （plant miRNAGenomics Database），以期为深入理解MIRNA基因转录过程的调控机制提供全方位信息支持。Intronic miRNA位于其它转录单元（宿主基因）的内含子内，与靶基因、宿主基因之间存在复杂的调控关系。而植物intronic miRNA的研究却非常少。我们以模式植物拟南芥、水稻为例，系统分析了植物intronic miRNA的特征、表达特性和功能。这些信息将推进植物intronic miRNA的研究。本研究的主要结果如下：（一） miRNA基因组学数据库pmiRGD的构建（1） miRNA前体序列和基因组序列的获取：18种植物的9299个miRNA前体序列来自miRBase、PMRD数据库，以及近期的文献；相应植物的基因组注释信息来自TAIR10、RGAP6.1、及phytozome6.0等植物基因组数据库。（2） miRNA与其它转录单元的位置关系分析：利用BLASTN将pre-miRNA完全匹配到基因组，及相关注释基因或内含子内上，寻找miRNA的宿主基因。共有7255个pre-miRNA完全匹配到基因组上7940个互不重叠的位置及相应的宿主基因，其中约10%的miRNA定位于其它转录基因的内含子内。（3）miRNA簇：位于同一宿主基因有义链上的miRNA为同一基因簇，67个基因内miRNA位于32个miRNA簇；其它位于基因组的同一条链上，分别以1kb、2kb、3kb为最大间隔距离（MID）确定为同一基因簇，724个基因间miRNA位于318个miRNA簇（MID=3kb）。（4）pri-miRNA：通过广泛的文献检索，共获得拟南芥、玉米中，127个MIRNA基因的328个RACE验证的pri-miRNA。此外，BLASTN结果中，969个miRNA分布于941个宿主基因的有意义链上，这些宿主基因的mRNA也作为相应miRNA的原初转录物（pri-miRNA）。（5）转录起始位点确定及启动子序列获得：根据pri-miRNA确定miRNA基因的转录起始位点（TSS）。获取TSS上游最长1kb范围内的基因间序列作为启动子。对其它未确定TSS的miRNA，获取pre-miRNA上游最长2kb范围内的基因间序列作为启动子。（6）转录因子结合位点（TFBS）鉴定：利用P-Match和TF-scan两个程序分析miRNA启动子区域内可能的TFBS。P-Match利用TRANSFAC6.0植物启动子元件数据库为基础进行预测；TF-scan利用Megraw等人构建的99个转录因子结合位点序列的矩阵进行预测。在miRNA的启动子上发现了大量的转录因子结合位点。（7）miRNA的表达模式分析：利用植物小RNA高通量测序的数据，分析miRNA在不同发育阶段、不同组织的表达模式。目前pmiRGD仅提供水稻和拟南芥miRNA在根、地上部、花中的表达模式。PmiRGD数据库网页使用Microsoft Visual Studio2008编写，Access2003数据库存储数据，系统运行在Windows2003服务器。网页设计简洁易用，免费对用户开放，网址：www.plantmirgo.org.（二）拟南芥、水稻intronic miRNA特征分析（1）从已有的数据库（miRBase v18、PMRD v1.0）和文献中，共获得1495（拟南芥）和2760（水稻）个pre-miRNA序列。利用BLASTN发现了37个拟南芥和181个水稻intonic miRNA位于蛋白编码基因的内含子。分别选取16个（拟南芥）和10个（水稻）intronic miRNA进行RT-PCR实验，证明了这些miRNA来源于真正的内含子。（2）基因结构分析发现，拟南芥2个intronic miRNA位于同一个基因簇；水稻13个intronic miRNA分别位于6个miRNA簇。这些位于同一基因簇的miRNA大多来自不同的基因家族，暗示它们可以作用于不同的靶基因。同时，染色体定位分析表明，拟南芥73%（27/37），和水稻55%（99/181）的intronic miRNA定位于基因组片段复制区域，这暗示了基因组或染色体的片段复制事件可能对于intronic miRNA的起源和进化起到了非常重要的作用。（3）含有miRNA的内含子长度分析结果显示，拟南芥79.2%的的内含子小于1kb（最大3298bp）。91.7%的pre-miRNA上游序列小于1kb。水稻83%的内含子小于3kb（最长12626bp），71.4%的上游区域小于1kb。这些数据说明植物的内含子比较小。取内含子范围内miRNA的上游序列进行启动子预测，拟南芥1个（1/37）、水稻14个（14/181）内含子预测到有启动子存在。由此可见，植物中较小的内含子和少数可能的启动子，暗示大多数intronic miRNA在宿主基因内部不存在独立的转录单位。（4）经MPSS数据库分析发现，拟南芥的19个、水稻的48个intronic miRNA检测到了表达，这说明intronic miRNA是真实表达的。利用Genevestigator数据库，分析宿主基因在植物不同发育阶段的表达模式。结果显示，大多数基因在几乎所有的发育时期都表达，且有较高的表达水平。另外，发现了拟南芥1个基因、水稻26个基因只在植物发育的某一个或两个阶段特异表达。宿主基因的这些表达特性，暗示了与其共表达的intronic miRNA在植物生长发育过程中重要作用。（5）利用降解组测序数据，鉴定了拟南芥13个成熟体intronic miRNA的41个靶基因及水稻148个成熟体intronic miRNA的494个靶基因。并且有一些靶基因是功能非常重要的转录因子。这也显示了intronic miRNA参与的基因表达调控途径的重要性。更多还原

【Abstract】 microRNAs (miRNAs) are small noncoding RNAs with a length of approximately21-24nucleotides which can regulate the expression of certain target genes, either by messengerRNA (mRNA) degradation or by translation repression. The expression of miRNAs in plantsinvolves transcription from MIRNA loci by RNA polymerase (pol II), multi-step processing ofthe primary transcripts by the DCL1complex. However, the knowledge about transcriptionalregulation of plant miRNAs is limited. In this project, we developed a plant miRNA genomicsdatabase (pmiRGD). The pmiRGD is a comprehensive resource which provides informationabout miRNA genomic organization, experimentally verified primary transcripts, putativetranscription factor binding sites (TFBSs), and deep sequencing data for miRNA. Theinterplay of these various information sources concerning genomic features associated withMIRNA genes and their expression profiles could provide extremely important clew for usersto discover the transcriptional regulation and function of miRNAs in planta.The majority of miRNAs are localized within intronic regions of protein-coding genes(host genes) and have diverse functions in regulating important cellular processes in animals.To date, few plant intronic miRNAs have been studied functionally. In the present study, wecarried out a genome-wide analysis with a particular focus on the characterization of intronicmiRNAs in rice and Arabidopsis.The main results were as follows:I Construction of the pmiRGD database(1) miRNA data collection: The pmiRGD collects available plant miRNA data depositedin public database and gleaned from the recent literatures.9,299pre-miRNA sequences wereretrieved from miRBase (release17), PMRD and six literatures published in recent years.Moreover, the up-to-data genome assembly and corresponding annotation files of plant species were carefully chosen, and retrieved from TAIR10, RGAP6.1and Phytozome6.0.(2) miRNA genomic organization: For each miRNA, we identified the genomic locationand putative overlapping gene by querying the genome assembly and correspondingannotated sequences using the appropriate Perl object and running a BLASTN analysis.Overall,7,255miRNAs hairpin sequences match to7,940unique genomic locations, andabout10%miRNAs reside within the introns of other genes.(3) miRNA clusters: We classified the pri-miRNAs into two groups: miRNA clusters fromintergenic regions and intragenic regions.67intragenic miRNAs reside within32clusters,and754intergenic miRNAs reside within318clusters (MID=3kb).(4) miRNA primary transcripts:328experimentally verified primary transcript sequencesfor127MIRNA genes were identified from three published literatures in Arabidopsis thalianaand Zea mays. In addition, the mRNA of941unique host gene were retrieved as pri-miRNAsfor969intragenic miRNAs.(5) Transcription start site (TSS) and promoter sequences: We identified the genomiclocation of the TSS associated with aforementioned miRNA primary transcripts. We choose tofocus on the1kb upstream region for miRNA promoter sequences. Similarly, if no primarytranscripts were identified, sequences are extracted in range (-2000,0) with respect to eachpre-miRNA, but shortened if necessary so as not to overlap with any upstream gene3′-UTR.(6) Identification of Transcription Factor Binding Sites (TFBSs): In order to identifyputative TFBS near the TSS of miRNA primary transcripts, we employed two freely availableprograms, P-Match and TF-scan. All the position weight matrices (PWMs) of plant promoterelements from TRANSFAC6.0and99PWMs constructed by Megraw et al., were matched tomiRNA promoters by P-Match and TF-scan, respectively.(7) miRNA expression profiling: In our pmiRGD database, we have already addeddeep-sequencing data concerning miRNA expression profiling in different developmentalstages in Arabidopsis and rice. We extracted the read sequences and counts from the GEOdatabase and map the reads to the set of miRNA precursor sequences using Bowtie allowingat most two mismatches between the read and the hairpin sequence.The pmiRGD website was constructed using Hypertext Markup Language (HTML) in theMicrosoft Visual Studio2008environment, and graphical user interface (GUI) interact with Access2003database engine. pmiRGD can be freely accessed at http://www.plantmirgo.org.II Genome-wide analysis of intronic microRNAs in Arabidopsis and rice(1) To identify intronic miRNA genome-widely, we collect1495and2760miRNAprecursors from miRBase (release18), PMRD and recent literatures for Arabidopsis and rice,respectively. BLAST result revealed that37and181intronic miRNAs were found within thesense strands of the intronic regions of protein-coding genes in Arabidopsis and rice,respectively. RT-PCR results suggest that14and10intronic miRNAs were reliable inArabidopsis and rice, respectively.(2) Gene structure revealed that one cluster was found in Arabidopsis, and13out of181intronic miRNAs resided within six clusters in rice. The results also indicate that most ofclusters contain polycistronic transcription units derived from different miRNA families,which imply these intronic miRNAs can target different mRNA, and may be involved inextremely complex regulation of genetic networks and pathways. Chromosomal distributionof intronic miRNAs suggests that55%of the rice intronic miRNAs,73%of the Arabidopsisintronic miRNAs might have evolved from putative genome segmental duplication events.(3) In Arabidopsis,79.2%of introns carrying miRNAs were shorter than1kb, and most ofupstream sequences of intronic miRNA hairpin within intron in Arabidopsis were shorter than1kb (91.7%). In rice,83%of them were shorter than3kb, and71.4%upstream sequences ofmiRNAs were shorter than1kb. Furthermore, we also predicted promoters within theupstream sequences of intronic miRNA. Only one promoter in Arabidopsis, and14promotersin rice were predicted. Together, short introns and few predicted promoter sequences indicatethat most of intronic miRNAs have no independent transcription units within the intronicregions in plants.(4) The expression of19and48intronic miRNAs were retrieved in MPSS database forArabidopsis and rice, respectively. The results revealed that the intronic miRNAs aretranscribed in high level. Moreover, the host protein-coding genes were examined in differentstages of development using Genevestigator. Expression profiles of21out of36host genes inArabidopsis, and152out of175in rice were retrieved. The results revealed that the majorityof host genes are expressed in all (or most of) stages of development with higher level. Interestingly,1and26host genes present with very specific expression patterns inArabidopsis and rice, respectively. Expression pattern analysis of host genes suggests that theintronic miRNAs might play an important role in plant development.(5) Using degradome sequencing data, the putative target genes of intronic miRNAs wereidentified. The results showed that some target genes encode important transcription factors,which suggests that the intronic miRNAs might be involved in the regulation of geneticnetworks and pathways.更多还原