Characterisation of tomato short RNAs involved in fruit development

细胞中的大多数RNA分子参与蛋白质的生产(核糖体、转运体或信使RNA)，但也有其他功能的RNA分子。最近发现的一类非编码RNA被称为microRNAs(MiRNAs)，因为它们非常短(19-24个核苷酸)。这些miRNAs可以识别特定的mRNAs，因为它们具有部分互补的序列。由于miRNAs和mRNAs之间的这种相互作用，靶向mRNAs的表达显著减少。大多数植物miRNAs调控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miRNAs，而在一个物种中发现的几个miRNAs在另一个物种中缺失，这表明存在只存在于特定植物群体中的miRNAs。我们假设miRNAs可以调节不同植物特性的发育，而这些特性在拟南芥和水稻中是缺失的。如果这是真的，那么可以在其他物种中发现调节有价值的植物特征的新miRNAs。由于这一假说在农学上的重要性，我们对这一假说进行了检验。番茄是肉质果实发育和成熟的模式植物，因此我们从番茄果实和叶片中测序了50多万个短RNA。我们已经证明了保守的miRNAs存在于水果中，其中一个针对的是水果成熟所需的已知转录因子。我们还鉴定了四个新的miRNAs，它们是拟南芥和其他植物基因组中不存在的，其中一个以CTR家族(参与果实成熟的基因)为靶标。此外，我们还鉴定了另一类短RNA的新候选者，这些RNA是通过不同于miRNAs的生物发生途径产生的。这些被称为ta-siRNA，由前体非编码RNA分阶段产生。我们的初步工作表明，肉质果实的发育过程受miRNAs的调控，我们希望通过转基因番茄植株产生更多的miRNAs或抗miRNA靶向的靶基因，来确定我们展示的两个miRNAs的生物学作用。我们还建议验证和表征ta-siRNA候选者。进一步的测序工作将建立短RNA在果实发育和成熟过程中的表达谱，并识别受参与果实发育的已知转录因子调控的短RNA。最后，我们开发了一种新的技术来生成被短RNA切割的mRNA库，并将该协议应用于番茄果实组织。这一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都是未知的，这一工具将使研究人员能够研究这些物种的短RNA靶标。了解果实发育和成熟过程中的这一层调控，可能有助于改善肉质水果的关键特性。

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.