原花青素是黄烷-3-醇的寡聚或多聚物，包括A型和B型两种结构类型。至今，关于原花青素聚合作用机理的问题仍然没有解决，其主要原因之一是没有合适的模式系统。本课题前期工作中利用PAP1转基因烟草建立了一个优良的模式系统--转基因红色细胞系：该细胞虽不能合成原花青素，但具有与原花青素聚合作用相关的关键酶，能将儿茶素或表儿茶素转化为A型原花青素化合物。据此，本课题拟从转基因红色细胞中分离A型原花青素缩合酶（APCE），测定其多肽氨基酸序列；克隆编码该酶的全长基因，通过体外重组表达及其酶活性分析，APCE转基因植物高表达和基因沉默分析，GFP-APCE融合蛋白的亚细胞定位等来阐明该酶的生物学功能。本课题将首次揭示一种新的催化A型原花青素聚合的缩合酶，对该酶功能的表征能直接鉴定一些参与原花青素聚合的延伸单元前体分子。课题的开展将解析A型原花青素聚合作用的酶促反应机理，增进对原花青素生物合成机制的理解。

Proanthocyanidins (PAs) are oligomers or polymers of flavan-3-ols and include two types of structures, A-type and B-type. A-type PAs are featured with two types of linkages between units in oligomers or polymers, whereas B-type PAs are characterized with only one type of linkage between units. To date, many questions pertaining to the polymerization of the two types of PAs still remain unanswered. Two of the most fundamental unanswered questions are "What molecule is the precursor of the PA extension units?" and "What kind of enzymes polymerizes the formation of PAs?" The answers to these two questions are the keys to understand the polymerization of PAs. Until recently, the main barrier to obtain answers has been the lack of an appropriate research system. To overcome this barrier, we obtained transgenic red cells from PAP1 transgenic red plants, which were previously demonstrated to express a protein complex involved in the polymerization of PAs. The suspension-cultured red cells biosynthesized high levels of anthocyanins, leucoanthocyanidins, and anthocyanidins. More importantly, these red cells expressed enzymes polymerizing the formation of PAs although they did not produce PAs due to lacking endogenous epicatechin and catechin. From suspension-cultured transgenic red cells, we had isolated a protein that converted epicatechin or catechin to A-type procyanidin compounds. We termed this protein A-type procyanidin condensing enzyme (APCE). Tryptic peptide sequencing of this protein obtained a partial amino acid sequence and a result of Blast search showed that this enzyme was a peroxidase-like protein. Underlying this application, we will clone the gene encoding this enzyme from suspension-cultured red cells and produce a large amount of recombinant protein to determine the enzymatic properties. We will use transgenic approaches, RNA interference and subcellular localization of recombinant fusion protein to characterize the gene. This study will enhance to solve a long-standing problem in plant biochemistry: the mechanism of polymerization of PAs. This study will be for the first time to characterize an enzyme that polymerizes the formation of A-type procyanidins. The cloning and functional characterization of the gene encoding A-type procyanidin condensing enzyme will enhance the understanding of PA biosynthesis and metabolic engineering.