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

该项目旨在开发一种大规模表征植物基因的系统。参与代谢途径的基因在农业分子育种中的应用得到了广泛的表征。为了识别特定的基因，人们可以通过重复的层析步骤来纯化目标蛋白，并制造出与特定区域相对应的肽。另一种方法是寻找在几个生物体中相关蛋白质中保守的特定结构域，并合成简并引物。然而，由于丢失了表达基因的信息，这种方法不能用于众多基因的特征分析。在本项目中，我们随机分离了水稻和拟南芥的cDNA，并通过寻找与其他蛋白质的相似性来进行特征分析。从悬浮培养或不同组织中提取mRNA，将其直接克隆到pUC载体中。随机分离cDNA片段，利用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，我们还开发了一个氨基酸相似性识别软件系统。根据已分类蛋白家族中典型结构域的信息，设计简并引物，通过常规筛选水稻和拟南芥基因文库，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，这些候选基因可能有助于培育耐逆、抗病或高产的转基因植物。因此，这些基因肯定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.