Characterisation of tomato short RNAs involved in fruit development

细胞中的大多数RNA分子参与蛋白质的产生（核糖体、转移或信使RNA），但也有具有其他功能的RNA分子。最近发现的一类非编码rna被称为微rna (miRNAs)，因为它们非常短（19-24个核苷酸）。这些mirna可以识别特定的mrna，因为它们具有部分互补的序列。由于mirna和mrna之间的相互作用，靶mrna的表达显著降低。大多数植物mirna调节植物正常发育所需的转录因子的产生。在拟南芥和水稻中已经发现了许多mirna，并且在一个物种中发现的一些mirna在另一个物种中不存在，这表明存在仅存在于某一类植物中的mirna。我们假设mirna可以调节拟南芥和水稻缺失的不同植物特性的发育。如果这是真的，那么可以在其他物种中发现调节有价值植物性状的新型mirna。由于肉质果实在农艺上的重要性，我们在肉质果实发育上检验了这一假设。番茄是肉质果实发育和成熟的模式植物；因此，我们对来自番茄果实和叶片的50多万个短rna进行了测序。我们已经证明，保守的mirna存在于水果中，其中一个靶向水果成熟所需的已知转录因子。我们还鉴定了四个新的mirna，这些mirna在拟南芥和其他植物基因组中缺失，并表明其中一个靶向CTR家族（参与果实成熟的基因）成员。此外，我们还发现了另一类短RNA的新候选物，它们通过与mirna不同的生物发生途径产生。这些被称为ta- sirna，是由前体非编码rna分阶段产生的。我们的初步工作表明，肉质果实的发育过程是由miRNA调控的，我们希望通过转基因番茄植株产生更多的miRNA或抵抗miRNA靶向的靶基因来确定我们所展示的两个miRNA在靶向成熟基因中的生物学作用。我们还建议验证和表征ta-siRNA候选者。进一步的深度测序工作将建立果实发育和成熟过程中短rna的表达谱，并确定受已知转录因子调控的短rna参与果实发育。最后，我们开发了一种新的技术来生成被短rna切割的mrna文库，我们将把这种方法应用于番茄果实组织。这个工具将有助于目标预测，并将对作物研究产生重大影响。大多数作物物种的基因组是未知的，该工具将使研究人员能够研究这些物种中的短RNA靶点。了解果实发育和成熟过程中的这一调控层可能有助于改善肉质果实的关键特性。

Most of the RNA molecules in cells are involved in protein production (ribosomal, transfer or messenger RNAs), however there are RNA molecules with other functions. A very recently discovered class of non-coding RNAs have been called microRNAs (miRNAs) because they are very short (19-24 nucleotides). These miRNAs can recognise specific mRNAs because they have partially complementary sequences to them. As a result of this interaction between miRNAs and mRNAs, the expression of the targeted mRNAs is significantly reduced. Most plant miRNAs regulate the production of transcription factors required for normal development of plants. Many miRNAs have been identified in arabidopsis and rice and several miRNAs found in one species were absent from the other suggesting that there are miRNAs which are present only in a certain group of plants. We hypothesized that miRNAs could regulate the development of different plant characteristics that are missing from arabidopsis and rice. If this was true, novel miRNAs regulating valuable plant traits could be discovered in other species. We tested this hypothesis on fleshy fruit development because of its agronomic importance. Tomato is the model plant for fleshy fruit development and ripening; therefore we sequenced more than half a million short RNAs from tomato fruit and leaf. We have demonstrated that conserved miRNAs are present in fruit and that one of them targeted a known transcription factor required for fruit ripening. We also identified four novel miRNAs, which are absent from arabidopsis and other plant genomes and showed that one of them targeted a CTR family (genes that are involved in fruit ripening) member. In addition we identified novel candidates of another class of short RNA that are produced through a different biogenesis pathway than miRNAs. These are called ta-siRNAs and are produced in a phased manner from precursor non-coding RNAs. Our preliminary work demonstrated that the process of fleshy fruit development is regulated by miRNAs and we would like to establish the biological role of the two miRNAs we showed to target ripening genes through transgenic tomato plants that produce more miRNAs or target genes that are resistant to miRNA targeting. We also propose to validate and characterise the ta-siRNA candidates. Further deep sequencing work will establish the expression profile of short RNAs during fruit development and ripening and also identify short RNAs that are regulated by known transcription factors involved in fruit development. Finally, we developed a novel technique to generate a library of mRNAs that are cleaved by short RNAs and we will apply this protocol for tomato fruit tissue. This tool will help target prediction and will have a big impact for crop research. Most crop species' genome is not known and this tool will enable researchers to investigate short RNA targets in these species. Understanding this layer of regulation in fruit development and ripening may lead to the improvement of key characteristics of fleshy fruits.