Oryza sativa Japonica Group Transcriptome or Gene expression. Oryza sativa Japonica Group

High-throughput sequencing of small RNAs from rice was used to identify distinct miRNAs that are responsive to elicitors from the fungal pathogen Magnaporthe oryzae. [Expression profiling by array] We used microarrays to determine the expression behaviour of target genes for elicitor-regulated miRNAs. [High throughput sequencing] High-throughput sequencing of rice small RNAs was performed in two different tissues, leaves and roots, and two different time point of elicitor treatment, 30' and 2h Amplicons were prepared by 5´and 3´adaptor ligation in which the 5'-adaptor contained a 'barcode' consisting of a 4-nucleotide identifier sequence for each sample. The libraries containing unique barcodes were combined and subjected to pyrosequencing (454 Life SciencesTM, Roche) Overall design: [Expression profiling by array] Leaves from rice plants were harvested at two time points after the onset of treatment (30' and 2h) with elicitors of Magnaporthe oryzae 18.1 and used for RNA extraction and hybridization on Affymetrix microarrays. Mock inoculations were performed with sterile water for control experiments. Three biological replicates were analyzed. Each sample represented a pool of approximately 150 rice plants. [High throughput sequencing] 8 samples examined: leaves and roots, treated or not with elicitors at two different time points, 30' and 2h (2x2x2)

本研究通过对水稻小RNA进行高通量测序，筛选鉴定响应稻瘟病菌（Magnaporthe oryzae）激发子的特异性microRNA (miRNA)。【芯片表达谱分析】我们借助微阵列技术，分析受激发子调控的miRNA的靶基因表达模式。【高通量测序】本实验选取水稻叶片与根部两种组织，分别在激发子处理后的30分钟与2小时两个时间点开展水稻小RNA高通量测序。测序文库通过5'端与3'端接头连接制备，其中5'接头带有用于区分各样本的4核苷酸标签（barcode）。携带唯一标签的文库混合后，采用焦磷酸测序法（pyrosequencing，454 Life Sciences™，罗氏Roche）进行测序。总体实验设计：【芯片表达谱分析】水稻植株经稻瘟病菌激发子18.1处理后，于处理启动后的30分钟与2小时两个时间点采集叶片样本，提取RNA后进行Affymetrix芯片杂交检测。对照组采用无菌水进行模拟接种，共设置3次生物学重复，每个样本由约150株水稻植株混合制备。【高通量测序】本次实验共检测8个样本：涵盖叶片与根部两种组织，各设置激发子处理组与未处理对照组，并覆盖30分钟、2小时两个时间点，实验设计为2×2×2方案。