Lan Lefu, Chen Wei, Lai Ying, Suo Jinfeng, Kong Zhaosheng, Li Can, Lu Ying, Zhang Yujun, Zhao Xiangyu, Zhang Xiansheng, Zhang Yansheng, Han Bin, Cheng Jing, Xue Yongbiao
Institute of Genetics and Development Biology, Chinese Academy of Science and National Center for Plant Gene Research, Beijing 100080, China.
Plant Mol Biol. 2004 Mar;54(4):471-87. doi: 10.1023/B:PLAN.0000038254.58491.c7.
To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.
为了在全基因组范围内监测水稻授粉和受精过程中的基因表达谱，我们从水稻的绿色/黄化茎和雌蕊（授粉后0-5小时，5hP）中生成了73424个高质量的表达序列标签（EST），随后用于构建包含约10000个独特水稻基因的cDNA微阵列。该微阵列用于分析未授粉雌蕊（UP）、授粉后5小时（5hP）和授粉后5天（5DAP）、花药、茎、根、10日龄胚（10EM）和10日龄胚乳（10EN）中的基因表达。聚类分析表明，花药的基因表达谱与根更相似，而不是与雌蕊相似，并且大多数雌蕊优先表达的基因对授粉和/或受精有反应。有253个EST在授粉和受精过程中表现出差异表达（e +/- 2倍变化），其中约70%可以被赋予推定功能。我们还回收了20个与先前鉴定的授粉相关和/或育性相关基因相似的基因以及先前未涉及的基因。微阵列和实时PCR分析表明，该阵列的灵敏度估计为每个细胞1-5个mRNA拷贝，两种方法之间差异表达的基因显示出高度相关性。我们的结果表明，这里构建的cDNA微阵列是可靠的，可用于监测水稻中的基因表达谱。此外，授粉过程中差异表达的基因代表了解析水稻这一重要生物学过程分子机制的候选基因。
