Plant Alternative Splicing and Abiotic Stress (PASAS)

遗传变异是生物多样性和表型变异的重要基础。植物如何应对外界刺激，如病原体/害虫的攻击或逆境条件，取决于植物物种的基因含量和基因表达的调控。基因在许多不同的水平上受到调控。一个重要的水平是基因的开启或关闭，或上升或下降，称为转录控制。第二个水平发生在基因转录或复制成RNA之后，称为转录后控制。有许多不同的转录后控制机制，选择性剪接（AS）是最重要的机制之一。选择性剪接是指基因转录物的不同部分以不同的组合连接在一起，从而从一个基因中产生多个信使RNA （mRNA）。由此产生的mrna可以被翻译成具有不同功能的蛋白质，或者可以作为降解的目标。因此，AS增加了生物体蛋白质组的复杂性，并可以调节mRNA的水平。选择性剪接影响植物发育、生存能力和对包括非生物胁迫在内的外界条件的适应性。然而，尽管植物中的AS对转录物水平和差异蛋白功能的产生有影响，但它作为全球基因表达控制的主要贡献者在很大程度上被忽视了。目前的估计表明，相当大比例的植物基因（35%）经历了选择性剪接。我们已经表明，这显然被低估了，因此实际上有更多的基因经历AS，许多基因有更多的AS事件，而不是迄今为止所描述的。此外，许多基因的不同剪接模式受不同的应激条件和参与剪接的蛋白质水平的改变所调节。因此，有必要共同努力发现和识别AS事件，并开发出在全球和基因特异性水平上准确测量AS变化的方法。我们将通过应用基因组学和生物信息学工具来解决这个问题：454测序、全基因组平铺阵列、用于量化AS转录物水平的RT-PCR系统和用于比较est的算法，以及经历不同非生物胁迫和剪接因子表达改变的植物。我们将识别新的，未注释的AS事件，评估其提供AS定量分析的潜力的技术，并使用这些工具来研究非生物胁迫下AS的变化。我们还将使用这些技术来扩展我们对特定的交换作用的替代剪接因子在调节AS中的作用的研究。该提案与ERA-PG会议的主题直接相关，并汇集了已经参与植物选择性剪接研究和非生物胁迫的四个研究小组。可测量的结果将是新颖的，以前描述的AS事件存储在适当的数据库中；研究AS变化的技术；AS事件及其频率和后果的数据库；以及许多拟南芥基因转录水平与AS之间的关系。这项研究的一个总体结果将是显著提高对AS重要性的认识，并需要考虑AS及其在植物基因表达转录后控制中的影响。了解基因调控各方面的复杂性和微妙性对于理解植物如何生长和生存以及预测对环境变化的反应非常重要，这种认识是未来植物育种政策的基石。

Genetic variation is an important basis for biodiversity and phenotypic variation. How plants respond to external stimuli such as attack by pathogens/pests or stress conditions depends on the gene content of the plant species and the regulation of expression of the genes. Genes are regulated at many different levels. One important level is where genes are turned on or off or up or down - called transcriptional control. A second level occurs after the gene is transcribed or copied into RNA - called post-transcriptional control. There are many different mechanisms of post-transcriptional control and alternative splicing (AS) is one of the most important. Alternative splicing is where different portions of a gene transcript are joined in different combinations to generate more than one messenger RNA (mRNA) from a gene. The resultant mRNAs can be translated into proteins with different functions or can be targeted for degradation. Thus, AS increases the proteome complexity of an organism and can regulate mRNA levels. Alternative splicing affects all aspects of plant development, viability and adaptability to external conditions including abiotic stress. Nevertheless, AS in plants has been largely over-looked as a major contributor to global gene expression control despite its influence on transcript levels and on generation of differential protein functions. Current estimates suggest that a significant proportion of plant genes (35%) undergo alternative splicing. We have shown that this is clearly underestimated such that substantially more genes undergo AS and many genes have more AS events than have been described to date. In addition, alternative splicing patterns of many genes are regulated by different stress conditions and by altered levels of proteins which are involved in splicing. It is, therefore, essential to have a concerted effort to discover and identify AS events and to develop methods to accurately measure changes in AS at global and gene-specific levels. We will approach this by applying genomics and bioinformatics tools: 454 sequencing, whole genome tiling arrays, an RT-PCR system to quantify AS transcript levels and algorithms for comparing ESTs, to plants experiencing different abiotic stresses and with altered expression of splicing factors. We will identify new, un-annotated AS events, assess the technologies for their potential to provide quantitative analysis of AS and use these tools to study changes in AS under abiotic stress. We will also use these technologies to extend our examinations of the roles of specific trans-acting alternative splicing factors in regulating AS. The proposal is directly relevant to the themes of the ERA-PG call and brings together four research groups already involved in plant alternative splicing research and abiotic stress. The measurable outcomes will be novel, previously undescribed AS events deposited in appropriate databases; techniques for studying changes in AS; a database of AS events with their frequency and consequences; and information on the relationship between transcript levels and AS for many Arabidopsis genes. An overall outcome of this research will be a significantly increased awareness of the importance of AS and the need to consider AS and its consequences in post-transcriptional control of plant gene expression. Knowledge of the complexity and subtlety of all aspects of gene regulation is important in understanding how plants grow and survive and in the prediction of responses to changing environments and such awareness is the cornerstone of future plant breeding policies.