Large-scale isolation of plant genes for the use in molecular breeding

本项目旨在建立一个大规模的植物基因表征系统。为了在农业分子育种中应用，对参与代谢途径的基因进行了深入的表征。为了确定一个特定的基因，可以通过重复的层析步骤纯化目标蛋白，并得到与特定区域相对应的肽。另一种方法是在几种生物中寻找相关蛋白中保守的特定结构域，合成简并引物。然而，为了对许多基因进行表征，由于表达基因的信息丢失，这种方法无法应用。在本项目中，我们随机分离水稻和拟南芥的cdna，并通过寻找与其他蛋白质的相似性来对其进行鉴定。从悬浮培养或不同组织中分离mRNA，将cdna直接克隆到pUC载体中。随机分离cDNA片段，使用GenBank数据库进行序列分析。为了有效地识别相关基因或蛋白质，我们还开发了一个软件系统来识别氨基酸相似性。根据预测的功能对鉴定的cdna进行分类，并进一步进行保守结构域的搜索。根据分类蛋白家族中发现的典型结构域信息，合成简并引物，并通过常规筛选水稻和拟南芥cDNA文库，筛选出包含这些结构域的其他相关基因。这些基因的特性为我们提供了一系列候选基因，这些候选基因可能对制造耐胁迫、抗病或高产的转基因植物有用。因此，这些基因无疑是农业领域分子育种的有力工具。

This project aimed to develop a system to characterize plant genes in a large-scale. Genes involved in metabolic pathways were intensively characterized for the use in molecular breeding in agriculture. To identify a specific gene, one can purify the target protein by repeated chromatography steps and make a peptide corresponding to a particular region. Another way is to search for a specific domain which is conserved in related proteins among several organisms and synthesize degenerate primers. However, to characterize numerous genes, such methods cannot be applied because of the loss of information of expressed genes.In this project, cDNAs of rice and Arabidopsis were randomly isolated and characterized by searching for similarity to other proteins. mRNA was isolated from suspension culture or different tissues, and cDNAs were directly cloned into the pUC vector. cDNA fragments were randomly isolated and subjected to sequence analysis using the GenBank database. To efficiently identify a related gene or protein, we also developed a software system for identification of amino acid similarities. Characterized cDNAs were categorized by predicted functions and further subjected to search for conserved domains.Based on the information of typical domains found in categorized protein family, degenerate primers were synthesized, and other related genes including such domains were fished out by conventional screening of rice and Arabidopsis cDNA libraries. Characterization of these genes provided us a series of candidates which may be useful for making transgenic plants, that are stress-tolerant, disease-resistant, or producing a high yield. Therefore, these genes are certainly powerful tools for molecular breeding in the field of agriculture.