Transcription profiling of fertilization and early seed development events in a solanaceous species using a 7.7K cDNA microarray from Solanum chacoense ovules

To provide a broad analysis of gene expression changes in developing embryos from a solanaceous species, we produced amplicon-derived microarrays with 7741 ESTs isolated from Solanum chacoense ovules bearing embryos from all developmental stages. Our aims were to: 1) identify genes expressed in a tissue-specific and temporal-specific manner; 2) define clusters of genes showing similar patterns of spatial and temporal expression; and 3) identify stage-specific or transition-specific candidate genes for further functional genomic analyses.We analyzed gene expression during S. chacoense embryogenesis in a series of experiments with probes derived from ovules isolated before and after fertilization (from 0 to 22 days after pollination), and from leaves, anthers, and styles. From the 6374 unigenes present in our array, 1024 genes were differentially expressed (≥ ±2 fold change, p value ≤ 0.01) in fertilized ovules and only limited expression overlap was observed between these genes and the genes expressed in the other tissues tested, with more than three quarters of the fertilization-regulated genes specifically or predominantly expressed in ovules. During embryogenesis three major expression profiles corresponding to early, middle and late stages of embryo development were identified. From the early and middle stages, a large number of genes corresponding to cell cycle, DNA processing, signal transduction, and transcriptional regulation were found. Defense and stress response-related genes were found in all stages of embryo development. Protein biosynthesis genes, genes coding for ribosomal proteins and other components of the translation machinery were highly expressed in embryos during the early stage. Genes for protein degradation were overrepresented later in the middle and late stages of embryo development. As expected, storage protein transcripts accumulated predominantly in the late stage of embryo development. Our analysis provides the first study in a solanaceous species of the transcriptional program that takes place during the early phases of plant reproductive development, including all embryogenesis steps during a comprehensive time-course. Our comparative expression profiling strategy between fertilized and unfertilized ovules identified a subset of genes specifically or predominantly expressed in ovules while a closer analysis between each consecutive time points allowed the identification of a subset of stage-specific and transition-specific genes. We produced amplicon-derived microarrays with 7741 ESTs isolated from Solanum chacoense ovules bearing embryos from all developmental stages. To monitor the expression pattern from genes involved in fertilization and embryogenesis processes, flowers were hand-pollinated and ovules were isolated every two days during a 22 days period after pollination. Four independent biological replicates were produced from each time points. In addition, to isolate genes specifically or predominantly expressed in ovules, four biological replicates of leaf, anther, and style tissue mRNA preparations were individually hybridized against unfertilized ovule mRNAs and compared with the data obtained from unfertilized and fertilized ovules at various time points after pollination. To estimate reproducibility and to produce control data for statistical analysis, a large number of unfertilized ovules were isolated and separated between seven independent control groups. RNA from randomly selected pairs of control was hybridized on six microarrays. 6374 unigenes present in our array in duplicate.

为全面分析某茄科（solanaceous）物种发育中胚胎的基因表达变化，我们利用从携带全发育阶段胚胎的查科茄（Solanum chacoense）胚珠中分离得到的7741条表达序列标签（Expressed Sequence Tag, EST），制备了扩增子来源的微阵列（microarray）。本研究的目标如下：1）鉴定以组织特异性和时间特异性模式表达的基因；2）定义呈现相似时空表达模式的基因簇；3）筛选阶段特异性或过渡特异性候选基因，以供后续功能基因组学研究。

我们通过一系列实验分析了查科茄胚胎发生过程中的基因表达，所用探针分别取自授粉后0至22天受精前后分离的胚珠，以及叶片、花药和花柱。在本微阵列包含的6374个单基因簇（unigene）中，有1024个基因在受精胚珠中呈现差异表达（倍数变化≥±2倍，P值≤0.01）；且这些基因与其他检测组织中表达的基因仅存在有限的表达重叠，超过四分之三的受精调控基因特异性或主要在胚珠中表达。

在胚胎发生过程中，我们鉴定出对应胚胎发育早期、中期和晚期的三大主要表达谱。早期与中期阶段，发现大量涉及细胞周期、DNA加工、信号转导及转录调控的基因。胚胎发育各阶段均存在与防御和应激反应相关的基因。早期胚胎中，蛋白质生物合成相关基因（包括编码核糖体蛋白及翻译机器其他组分的基因）呈高表达状态。蛋白质降解相关基因则在胚胎发育中期和后期更为富集。如预期一致，贮藏蛋白转录本主要在胚胎发育晚期积累。

本研究是首个在茄科物种中开展的、覆盖植物生殖发育早期阶段（包含完整时间序列的全部胚胎发生步骤）的转录调控程序研究。我们通过受精与未受精胚珠的比较表达谱分析，筛选出一批特异性或主要在胚珠中表达的基因；同时通过对各连续时间点的细致分析，鉴定出一批阶段特异性及过渡特异性基因。

如前文所述，我们利用携带全发育阶段胚胎的查科茄胚珠中分离的7741条表达序列标签制备了扩增子来源的微阵列。为监测参与受精及胚胎发生过程的基因表达模式，我们对授粉后22天内每2天采集的手工授粉花朵分离胚珠。每个时间点设置4次独立生物学重复（biological replicate）。此外，为筛选特异性或主要在胚珠中表达的基因，我们将叶片、花药和花柱组织的mRNA制备物各设置4次生物学重复，分别与未受精胚珠的mRNA进行杂交，并将所得数据与授粉后各时间点未受精及受精胚珠的数据进行比较。为评估实验重复性并生成统计分析（statistical analysis）所需的对照数据，我们分离了大量未受精胚珠，并将其分为7个独立的对照组。从随机选取的对照组样本对中提取的RNA，在6张微阵列上进行了杂交。本微阵列包含的6374个单基因簇均设置了双重复。