Maize Full-Length cDNA Project

全长互补DNA序列对玉米基因组中基因的准确注释和未来的功能鉴定至关重要。该项目将产生大约30,000个关注生殖发育和应激诱导转录本的FlcDNA序列。使用13种器官类型(从花序原基到未成熟种子)的RNA构建的Gateway兼容骨干中的商业文库，通过帽和尾的选择高度丰富了全长cDNA的表达，将被标准化，以增加稀有转录本的表达。我们将使用相同的方法制作一个新的文库，这些文库来自其他花朵发育阶段、胚胎、盾片和经过胁迫诱导处理的7日龄幼苗。将根据(1)360,000个5‘和3’表达序列标签(EST)组件，通过与公开可获得的EST、FlcDNA和水稻和玉米的基因组序列比对，以及(2)将特定生殖阶段的RNA与360,000个过滤排列的克隆杂交，以丰富稀有转录本。该项目的成果包括：(1)从18万个假定的FlcDNA中获得360,000个5‘和3’EST；(2)30,000个FlcDNA克隆和序列；(3)FlcDNA注释，包括可用的阶段特异性表达和可能的作图信息，以及一个基于网络的基因组浏览器。玉米Flc DNA资源将具有广泛的潜在用途，从功能研究到理解谷物多样化过程中的基因进化，以及探索参与植物繁殖和环境胁迫的基因调控网络。项目成果的可获得性将在http://www.genome.arizona.edu上创建一个基于网络的展示，以提供对项目成果和项目进度的访问。包括克隆、扩增文库和高密度过滤器在内的所有生物资源也将通过亚利桑那州基因组研究所的BAC/EST资源中心(www.genome.arizona.edu/order)公开提供。

Full-length complementary DNA (FLcDNA) sequences are crucial for accurate annotation and future functional characterization of the genes in the maize genome. This project will generate approximately 30,000 FLcDNA sequences focusing on reproductive development and stress-induced transcripts. A commercial library in a Gateway-compatible backbone built using RNA from 13 organs types (from inflorescence primordia to immature seeds), highly enriched for full-length cDNA representation by cap and tail selection, will be normalized to increase representation of rare transcripts. A new library from additional floral developmental stages, embryo, scutellum, and 7-day seedlings with stress inducer treatments will be made using the same methods. Candidate clones for finishing will be picked based on (1) 360,000 5' and 3' Expressed Sequence Tag (EST) assemblies generated in this project by alignment to publicly available ESTs, FLcDNAs and genomic sequences of rice and maize, and (2) hybridization of RNA from specific reproductive stages to 360,000 filter-arrayed clones in order to enrich rare transcripts. Iterative primer walking will be employed on each candidate FLcDNA, and a transposon method will also be utilized to complete finish larger insert and difficult clones with phred 40 quality.The outcomes from the project to be delivered to the community include: (1) 360,000 5' and 3' ESTs from 180,000 presumptive FLcDNAs, (2) 30,000 FLcDNA clones and sequences, and (3) FLcDNA annotation, including available stage-specificity of expression and possible mapping information, and a web-based genome browser. The maize FLcDNA resources will have a wide variety of potential uses from functional studies towards understanding gene evolution during cereal diversification, and exploration of gene regulatory networks involved in plant reproduction and environmental stresses. Availability of project outcomesA web-based display will be created at http://www.genome.arizona.edu to provide access to the project results and project progress. All biological resources including clones, amplified libraries, and high-density filters will also make publicly available through BAC/EST resource center at Arizona Genomics Institute (www.genome.arizona.edu/order).