Mechanism of B Activation of the Maize Anthocyanin Pathway

我们的目标是了解调控蛋白B协调诱导玉米花青素生物合成酶表达的机制，B基因产物是编码花青素生物合成酶的几个基因（结构基因）mRNA稳态积累所必需的。我们最近证明了B基因在刺激结构基因的转录中起着重要作用。利用携带结构基因启动子序列与荧光素酶融合的质粒和产生B蛋白的质粒进行的瞬时共转化研究表明，B蛋白介导了大约100倍的荧光素酶活性诱导。推导出的B蛋白氨基酸序列（从cDNA序列中获得）揭示了在几种DNA结合蛋白中发现的myc同源区域和转录激活因子特征的负电荷区域。体外和体内试验将用于：检查B蛋白的体内修饰及其亚细胞位置和组织分布；解剖结构基因转录控制所需的B蛋白区域；确定这些区域的功能（转录激活、DNA结合、蛋白/蛋白相互作用等）；评估B蛋白是否直接与结构基因中的调控区域相互作用；鉴定与B相互作用的其他蛋白质；并研究除了结构基因的转录外，B是否还会影响花青素的生物合成。玉米花青素生物合成途径是高等植物中少数已知调控蛋白及其靶点的途径之一。因此，研究B利用的机制将揭示高等植物调控基因表达的重要信息。

Our goal is to understand the mechanisms by which the expression of the maize anthocyanin biosynthetic enzymes is coordinately induced by the regulatory protein B. The B gene product is required for the steady state accumulation of mRNA from several genes encoding anthocyanin biosynthetic enzymes (structural genes), and we have recently demonstrated that the B gene plays a major role in stimulating the transcription of the structural genes. Transient cotransformation studies using plasmids carrying structural gene promoter sequences fused to luciferase, and plasmids which produce B protein, demonstrate B mediates an approximately 100 fold induction in luciferase activity. The deduced amino acid sequence of the B protein (obtained from the cDNA sequence) revealed a myc homologous region found in several DNA binding proteins and a negatively charged region characteristic of transcriptional activators. Both in vitro and in vivo assays will be used to: examine the in vivo modification of the B protein and its subcellular location and tissue distribution; dissect the regions of the B protein necessary for transcriptional control of the structural genes; determine the functions of these regions (transcriptional activation, DNA binding, protein/protein interactions, etc.); assess whether the B protein interacts directly with regulatory regions found in the structural genes; identify other proteins interacting with B; and investigate if B influences anthocyanin biosynthesis at levels in addition to transcription of the structural genes. The maize anthocyanin biosynthetic pathway represents one of a small number of pathways in higher plants for which regulatory proteins and their targets are known. Thus, studying the mechanisms utilized by B should reveal important information regarding regulated gene expression in higher plants.