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 Overall design: 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授粉后1天（1 dpa）的棉花纤维中提取总RNA的方法。基于该方法提取的RNA及其他棉花mRNA所合成的表达序列标签（Expressed Sequence Tags, EST）经测序后被组装为重叠群（contigs）。由目标文库特异性EST构成的重叠群以及特异性单序列（singletons）被用于制备基因芯片（microarray）。将基因芯片与源自完整1 dpa胚珠和1 dpa纤维的标记核酸进行杂交，以筛选纤维起始阶段差异表达调控的基因。纳入已知在棉花纤维中特异性表达的基因，以验证基因芯片实验的可靠性与准确性。对差异表达基因进行基因本体（Gene Ontology, GO）富集分析，验证了此前报道的纤维起始相关生物学特征，例如蛋白质生物合成过程的显著增强。GO富集分析显示细胞膜相关生物学过程显著上调，该结果得到纤维起始细胞内质网（Endoplasmic Reticulum, ER）染色强度升高的实验佐证。对纤维起始细胞中差异调控的单个基因进行表达分析，结果提示钙离子可能参与棉花纤维发育过程，该推测也得到纤维起始细胞内钙离子水平显著升高的实验支持。
关键词：比较杂交
实验设计概述：该基因芯片覆盖11143个基因模型。其中，1个基因模型由5种不同的寡核苷酸探针表征，8604个基因模型由4种不同的寡核苷酸探针表征，1706个基因模型由3种不同的寡核苷酸探针表征，831个基因模型由3条相同的寡核苷酸探针表征，另有1个基因模型由2种不同的寡核苷酸探针表征。每张基因芯片均与源自大田种植植株不同日期采收的1 dpa纤维或1 dpa胚珠的RNA提取得到的标记核酸进行杂交，其中包含一次荧光染料互换（dye swap）对照实验。