Nadeau J A, Zhang X S, Li J, O'Neill S D
Division of Biological Sciences, University of California at Davis 95616, USA.
Plant Cell. 1996 Feb;8(2):213-39. doi: 10.1105/tpc.8.2.213.
A differential screening approach was used to identify seven ovule-specific cDNAs representing genes that are expressed in a stage-specific manner during ovule development. The Phalaenopsis orchid takes 80 days to complete the sequence of ovule developmental events, making it a good system to isolate stage-specific ovule genes. We constructed cDNA libraries from orchid ovule tissue during archesporial cell differentiation, megasporocyte formation, and the transition to meiosis, as well as during the final mitotic divisions of female gametophyte development. RNA gel blot hybridization analysis revealed that four clones were stage specific and expressed solely in ovule tissue, whereas one clone was specific to pollen tubes. Two other clones were not ovule specific. Sequence analysis and in situ hybridization revealed the identities and domain of expression of several of the cDNAs. O39 encodes a putative homeobox transcription factor that is expressed early in the differentiation of the ovule primordium; O40 encodes a cytochrome P450 monooxygenase (CYP78A2) that is pollen tube specific. O108 encodes a protein of unknown function that is expressed exclusively in the outer layer of the outer integument and in the female gametophyte of mature ovules. O126 encodes a glycine-rich protein that is expressed in mature ovules, and O141 encodes a cysteine proteinase that is expressed in the outer integument of ovules during seed formation. Sequences homologous to these ovule clones can now be isolated from other organisms, and this should facilitate their functional characterization.
采用差异筛选方法鉴定出7个胚珠特异性cDNA，它们代表在胚珠发育过程中以阶段特异性方式表达的基因。蝴蝶兰完成胚珠发育事件序列需要80天，这使其成为分离阶段特异性胚珠基因的良好系统。我们构建了蝴蝶兰胚珠组织在孢原细胞分化、大孢子母细胞形成、向减数分裂转变以及雌配子体发育的最终有丝分裂期间的cDNA文库。RNA凝胶印迹杂交分析表明，4个克隆具有阶段特异性，仅在胚珠组织中表达，而1个克隆对花粉管具有特异性。另外2个克隆不是胚珠特异性的。序列分析和原位杂交揭示了几个cDNA的身份和表达结构域。O39编码一种假定的同源异型框转录因子，在胚珠原基分化早期表达；O40编码一种细胞色素P450单加氧酶（CYP78A2），是花粉管特异性的。O108编码一种功能未知的蛋白质，仅在外珠被外层和成熟胚珠的雌配子体中表达。O126编码一种富含甘氨酸的蛋白质，在成熟胚珠中表达，O141编码一种半胱氨酸蛋白酶，在种子形成期间在胚珠的外珠被中表达。现在可以从其他生物体中分离出与这些胚珠克隆同源的序列，这将有助于对它们进行功能表征。
