应用酵母单杂交系统分离水稻蜡质基因5'调控区结合蛋白的基因

在水稻蜡质基因５’上游区中一段３１ｂｐ 核苷酸序列能与水稻未成熟种子核蛋白特异结合。为了克隆这一核蛋白基因，以此３１ ｂｐ 序列构建成“鱼饵”质粒，从水稻ｃＤＮＡ文库中筛选到１３ 个阳性克隆。根据这些阳性克隆中插入ｃＤＮＡ 片段的相互杂交结果，对这些克隆进行了分组。

Isolation of the Genes Encording DNA Binding Protein Interacting with 5' Upstream Region of the Rice Waxy Gene by Using Yeast One hybrid System

We have previously reported that a 31 bp DNA fragment within the 5' upstream region of rice waxy gene could interact specifically with nuclear proteins extracted from immature rice seeds. This 31 bp DNA fragment was used as a bait in a yeast one hybrid system to isolate the genes encoding trans acting factors regulating rice waxy gene expression. This yeast one hybrid system consists of two plasmids: the p178 plasmid consisted of two copies of 31 bp oligonucleotids adjacent to the mini promoter Pcyc1 directing lacZgene expression, in the second pPC86 plasmid, a rice cDNA library was ligated to a gene encoding GAL4 transactivating domain as a translational fusion to form a rice cDNA GAL4 library (Fig.1). A yeast strain harboured the p178 plasmid was then transformed with the pPC86 plasmid containing cDNA GAL4 library, and grown on medium containing X gal to select blue colonies (Figs.2 and 3). After several round selections, thirteen positive clones were obtained.These thirteen cDNA clones can be divided into four groups according to their homology by Southern blot analysis.