Genes differentially expressed in fiber 1 day post anthesis (dpa)

A method was developed to isolate RNA from 1 dpa fiber. ESTs derived from this and other cotton mRNAs were sequenced and assembled into contigs. Contigs composed of EST unique to libraries of interest and unique singletons were represented on a microarray. Microarrays were hybridized with labed nucleic acids derived from whole 1 dpa ovules and 1 dpa fiber to identify genes differentially regulated during fiber initiation. Genes with known expression in fiber were included to validate the microarray. Analysis of the gene ontology (GO) of differentially expressed genes suported previously reported aspects of fiber initiation such as an increase in protein biosynthesis. An increase in the "membrane" was indicated by the GO analysis ans supported by increased staining of fiber initial ER. Analysis of individule genes differentially regulated in fiber initials indicated calcium may play a role in fiber deveopment which was also supported by an increase in calcium in fiber initials. Keywords: comparative hybridization The microarray represents 11143 gene models. One gene model is represented by 5 different oligonucleotides, 8604 gene models are represented by 4 different oligonucleotides, 1706 gene models are represented by 3 different oligonucleotides, 831 gene models are represented by 3 of the same oligonucleotides and one gene model is represented by 2 different oligonucleotides. Each micro array is hybridized with a labelled nucleic acid derived form RNA isolated from 1 dpa fiber or 1 dpa ovules harvested on different days from field grown plants. One is a dye swap.

本研究开发了一种从开花后1天（1 day post-anthesis, 1 dpa）纤维中分离RNA的方法。将由此获得的表达序列标签（Expressed Sequence Tags, ESTs）与其他棉花mRNA进行测序，并组装为重叠群（contigs）。将仅在目标文库中存在的EST组成的重叠群以及独特的单序列（singletons）点制于基因芯片（microarray）上。分别使用源自完整1 dpa胚珠与1 dpa纤维的标记核酸对基因芯片进行杂交，以鉴定纤维起始发育过程中差异调控的基因。选取已被证实于纤维中表达的基因作为芯片验证的阳性对照。对差异表达基因进行基因本体（Gene Ontology, GO）分析，其结果验证了此前已报道的纤维起始发育特征，例如蛋白质生物合成水平的提升。GO分析还提示“膜”相关组分的丰度上升，这一结果得到了纤维起始细胞内质网（Endoplasmic Reticulum, ER）染色增强的佐证。对纤维起始细胞中差异调控的单个基因进行分析后发现，钙离子可能参与纤维发育过程，而纤维起始细胞内钙离子水平的升高也支持这一推测。关键词：比较杂交（comparative hybridization）。该基因芯片共涵盖11143个基因模型，其中5种不同寡核苷酸对应1个基因模型，4种不同寡核苷酸对应8604个基因模型，3种不同寡核苷酸对应1706个基因模型，3种相同寡核苷酸对应831个基因模型，2种不同寡核苷酸对应1个基因模型。每张基因芯片均使用源自大田种植植株不同天数收获的1 dpa纤维或1 dpa胚珠的RNA所制备的标记核酸进行杂交，其中包含一组染料互换（dye swap）实验。