Skinner Debra J, Gasser Charles S
Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.
BMC Plant Biol. 2009 Mar 16;9:29. doi: 10.1186/1471-2229-9-29.
Arabidopsis ovules comprise four morphologically distinct parts: the nucellus, which contains the embryo sac, two integuments that become the seed coat, and the funiculus that anchors the ovule within the carpel. Analysis of developmental mutants has shown that ovule morphogenesis relies on tightly regulated genetic interactions that can serve as a model for developmental regulation. Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes. Expression-based gene discovery can be used access such obscured genes.
Affymetrix microarrays were used for expression-based gene discovery to identify sets of genes expressed in either or both integuments. The genes were identified by comparison of pistil mRNA from wild type with mRNA from two mutants; inner no outer (ino, which lacks the outer integument), and aintegumenta (ant, which lacks both integuments). Pools of pistils representing early and late stages of ovule development were evaluated and data from the three genotypes were used to designate genes that were predominantly expressed in the integuments using pair-wise and cluster analyses. Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization.
The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino. Robust microarray averaging (RMA) analysis of array data provided the most reliable comparisons, especially for weakly expressed genes. The studies yielded an over-abundance of transcriptional regulators in the identified genes, and these form a set of candidate genes for evaluation of roles in ovule development using reverse genetics.
拟南芥胚珠由四个形态上不同的部分组成：包含胚囊的珠心、形成种皮的两层珠被以及将胚珠固定在心皮内的珠柄。对发育突变体的分析表明，胚珠形态发生依赖于严格调控的基因相互作用，这些相互作用可作为发育调控的模型。冗余性、多效性效应和微妙的表型可能会妨碍在筛选表型变化时鉴定影响某些过程的突变体。基于表达的基因发现可用于获取此类隐蔽基因。
使用Affymetrix微阵列进行基于表达的基因发现，以鉴定在一层或两层珠被中表达的基因集。通过比较野生型雌蕊mRNA与两个突变体（内无外（ino，缺乏外珠被）和无珠被（ant，缺乏两层珠被））的mRNA来鉴定这些基因。评估了代表胚珠发育早期和晚期阶段的雌蕊池，并使用来自三种基因型的数据，通过成对和聚类分析来指定主要在珠被中表达的基因。发现约两百个基因很可能在这些结构中优先表达，并且通过逆转录酶聚合酶链反应和原位杂交证实了表达类别的预测性质。
结果表明，当与已知基因表达模式的预测相结合，或与更特异的突变体ino结合使用时，有可能利用对植物表型有广泛影响的突变体ant来鉴定在胚珠中特异性表达的基因。对阵列数据进行稳健的微阵列平均（RMA）分析提供了最可靠的比较，特别是对于弱表达基因。这些研究在所鉴定的基因中产生了过多的转录调节因子，这些转录调节因子形成了一组候选基因，可用于通过反向遗传学评估其在胚珠发育中的作用。
