A型原花青素聚合相关缩合酶基因的克隆与功能分析

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.

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。

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。本课题前期工作中建立了PAP1转基因烟草红色细胞系（6R）和白色细胞系（6W），6R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。项目首先开展了转基因6R细胞和6W白色细胞的比较转录组学研究，通过生物信息学手段从6R细胞获得与A型原花青素聚合相关的关键基因PAL1、CHS、DFR和ANR以及PAP1、TT8、GL3、TTG1、EGL3和MYBL2等转录因子，并探讨了这些基因在两种表型不同的细胞中的表达模式。基于此，利用RT-PCR和RACE-PCR技术分别从华南美丽葡萄和陆地棉中克隆了DFR和ANR全长基因，在大肠杆菌中进行了可溶性表达，重组酶经纯化后进行了体外酶活性分析。然后分别将这些基因转入烟草或拟南芥突变体中过表达，发现遗传互补原花青素生物合成的途径；采用病毒介导的基因沉默技术原位干扰相应基因的表达确证其生物学功能。同时将这些基因与GFP融合在洋葱表皮细胞中瞬时表达确定了其在细胞质中发挥作用。另外，详细研究了两种植物不同器官和不同发育时期的原花青素分布与组成，发现其聚合反应中延伸单元的前体分子结构类型多样。项目的实施全面解析了原花青素聚合相关合成酶DFR、ANR的功能，首次揭示A型原花青素聚合反应延伸因子前体分子的多样性。研究结果为详细阐明原花青素生物合成机制积累了基础数据，为通过代谢工程进行原花青素相关性状的植物品种改良和分子育种提供了理论依据。