Expression profile in response to salt-treatment in wheat using 22k microarray

Analysis of transcripts in response to salt treatment. In order to design the 22k wheat oligo-DNA microarray, a total of 148,676 expressed sequence tags of common wheat were collected from the database of the Wheat Genomics Consortium of Japan. These were grouped into 34,064 contigs, which were then used to design an oligonucleotide DNA microarray. Following a multi-step selection of the sense strand, 21,939 60-mer oligo DNA probes were selected for attachment on the microarray slide. This 22k oligo DNA microarray was used to examine the transcriptional response of wheat to salt stress. More than 95% of the probes gave reproducible hybridization signals when targeted with RNAs extracted from salt-treated wheat shoots and roots. With the microarray, we identified 1,811 genes whose expressions changed more than two-fold in response to salt. These included genes known to mediate the response to salt as well as unknown genes, and they were classified into 12 major groups by hierarchical clustering. These gene expression patterns were also confirmed by real-time reverse transcription (RT)-PCR. Many of the genes with unknown function were clustered together with genes known to be involved in the response to salt stress. Thus, analysis of gene expression patterns combined with gene ontology should help identify the function of the unknown genes. Also, functional analysis of these wheat genes should provide new insight into the response to salt stress. Finally, these results indicate that the 22k oligo DNA microarray is a reliable method for monitoring global gene expression patterns in wheat. Keywords: time cource, stress response Microarray hybridization was performed by a competitive two-color method including color-swap experiments. Chinese Spring wheat was grown for two weeks and treated with 150mM NaCl for 0, 1, 6 and 24 hours. RNA samples were extracted from roots and shoots.Each experiment was design for comparison in control and treated sample.盐胁迫响应转录本分析。为构建22k小麦寡聚DNA微阵列(22k wheat oligo-DNA microarray)，研究人员从日本小麦基因组联盟(Wheat Genomics Consortium of Japan)数据库中，共收集到148676条普通小麦表达序列标签(expressed sequence tags, EST)。这些序列被聚类为34064个重叠群(contigs)，随后用于设计寡核苷酸DNA微阵列。经过正义链多步筛选后，最终选取21939条60聚体寡聚DNA探针，用于固定于微阵列玻片。 本22k寡聚DNA微阵列被用于解析小麦对盐胁迫的转录响应。以盐处理小麦幼苗地上部与根部提取的RNA为靶标时，超过95%的探针可获得可重复的杂交信号。借助该微阵列，研究人员共鉴定出1811个盐胁迫下表达量变化超过2倍的基因，其中既包含已知的盐胁迫响应介导基因，也包含未知功能基因。通过层级聚类(hierarchical clustering)，将这些基因划分为12个主要类群。实时逆转录(RT)-PCR验证了上述基因表达模式的可靠性。 大量未知功能基因与已知参与盐胁迫响应的基因聚为一类，因此结合基因本体论(gene ontology, GO)的基因表达模式分析，将有助于解析未知功能基因的生物学功能。此外，对这些小麦基因的功能分析，可为盐胁迫响应机制研究提供新的视角。综上，本研究结果表明，这款22k寡聚DNA微阵列是监测小麦全基因组基因表达模式的可靠工具。 关键词：时间进程(time course)、胁迫响应(stress response) 微阵列杂交采用竞争性双色杂交法开展，包含荧光反转对照(color-swap)实验。 中国春小麦(Chinese Spring wheat)幼苗生长两周后，用150mM NaCl分别处理0、1、6和24小时。 分别提取根部与地上部的RNA样本。 每组实验均设置对照与处理样本用于比较分析。