Genome-wide analysis of short RNAs as modulators in dehydration stress tolerance using tolerant and genetic model systems

干旱胁迫是世界范围内严重影响作物生产力的常见环境条件。预测是，干旱压力，以降雨不可预测的变化或与不断增长的城市人口争夺淡水的形式，将继续是可能影响全球作物产量的主要单一非生物因素。干旱胁迫几乎影响植物生长和代谢的各个方面。植物对水分亏缺的反应取决于胁迫的持续时间和程度、生长阶段和胁迫时间等因素。大多数环境压力导致缺水压力。冻土可以减少水分吸收，从而产生水分胁迫；以同样的方式，土壤中的盐积累降低了水势，使土壤水的可用性降低。为了在缺水条件下生存，植物必须保持水分状态以维持离子稳态。对不同胁迫的共同响应表明，在水分亏缺胁迫条件下，基因产物具有相似的功能。脱水过程中基因表达的研究表明，存在促进植物胁迫反应的相互作用的信号感知和转导途径。内源性脱落酸（ABA）水平的增加是水分缺乏的结果，被认为与信号转导有关。到目前为止，已经分析了有关转基因方法提高抗旱性的蛋白质编码区，但很明显，缺少重要的调控决定因素。sRNAs最近被认为是基因表达的重要调控成分。有两类sRNAs: microRNAs （miRNAs）和短干扰rna （sirna）。mirna是内源性调节性srna，源于内源性前体转录物的茎环区域。mirna对蛋白质编码基因的信息进行退火，从而导致mrna的切割。对sRNAs的全基因组分析将使我们能够识别在水分充足和干燥的组织中不同积累的新型sRNAs。因此，我们将利用高通量测序技术从两种不同的耐脱水模式物种（金车前草和M. truncatula）的水分充足和脱水的组织中克隆和测序sRNAs。对sRNAs的全基因组分析将使我们能够识别在水分充足和干燥的组织中不同积累的新型sRNAs。已知和新型sRNAs的表达谱将通过微阵列杂交确定，并通过Northern blots验证。靶基因将被预测或通过实验确定在水分充足和脱水的组织中不同积累的sRNAs。将通过过表达sRNA或sRNA不敏感靶基因来分析sRNA调控已验证靶基因的生物学相关性。几种基因组学工具集成在这种方法中，如高通量测序和微阵列杂交。如果没有这些工具，这个项目是不可行的，因为sRNA调控的规模需要全基因组分析。所选方法的优势在于，它将揭示新的sRNAs以及已知和新的sRNAs在抗旱性中的功能。虽然使用没有完整基因组序列的植物物种开展该项目更加困难，但其优势在于它允许(i)鉴定出在拟南芥和水稻中不存在的新sRNAs； （ii）对两种耐旱模式物种的sRNAs的分析将揭示调节脱水耐受性的不同网络。

Drought stress is a common adverse environmental condition that seriously affects crop productivity worldwide. The prediction is that the drought stress, in the form of unpredictable changes in rainfall or competition for fresh water with growing urban populations, will continue to be the major single abiotic factor likely to affect crop yields globally. Drought stress affects practically every aspect of plant growth and metabolism. Plant responses to water deficit depend upon factors such as duration and degree of stress, growth stage and time of stress exposure. Most environmental stresses result in water-deficit stress. Frozen soil can reduce water uptake and thus produce water stress; in the same way, salt accumulation in the soil decreases the water potential that makes soil water less available. In order to survive under water deficit conditions, plants have to maintain their water status to maintain ion homeostasis. The common responses to different stresses indicate similar functions of the gene products for plants under stress conditions involving water deficit. The existence of interacting signal perception and transduction pathways, which promote the plant stress response, is suggested by studies on gene expression during dehydration. Endogenous abscisic acid (ABA) levels increase as result of water deficit and it is thought to be involved in signal transduction. Up to now the protein coding regions have been analysed with respect to transgenic approaches to improve drought tolerance, but it has become clear that important regulatory determinants are missing. sRNAs have been recently recognised as important regulatory components of gene expression. There are two classes of sRNAs: microRNAs (miRNAs) and short interfering RNAs (siRNAs). miRNAs are endogenous regulatory sRNAs that derive from stem-loop regions of endogenous precursor transcripts. miRNAs anneal to the messages of protein-coding genes which result in the cleavage of the mRNAs. The genome wide analysis of sRNAs will allow us to identify novel sRNAs that are differentially accumulated in well watered and dried tissues. Therefore, sRNAs will be cloned and sequenced from well watered and dehydrated tissues of two different dehydration tolerant model species (C. plantagineum and M. truncatula) using high-throughput sequencing technology. The genome wide analysis of sRNAs will allow us to identify novel sRNAs that are differentially accumulated in well watered and dried tissues. The expression profile of known and novel sRNAs will be determined by microarray hybridisation and validated by Northern blots. Target genes will be predicted or experimentally determined for sRNAs that are differentially accumulated in well watered and dehydrated tissues. The biological relevance of the sRNA regulation of validated target genes will be analysed by over-expressing sRNAs or sRNA insensitive target genes. Several genomics tools are integrated in this approach, such as high-throughput sequencing and microarray hybridisation. This project is not feasible without these tools because the scale of sRNA regulation requires genome wide analysis. The advantage of the chosen methodology is that it will reveal novel sRNAs and the function of known and novel sRNAs in drought tolerance. Although it is more difficult to carry out this project using plant species without complete genome sequences, the advantages are that it allows (i) the identification of novel sRNAs that do not exist in Arabidopsis and rice and (ii) the analysis of sRNAs in two drought tolerant model species will reveal different networks regulating dehydration tolerance.