Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia V0H 1Z0, Canada.
BMC Plant Biol. 2014 Mar 11;14:62. doi: 10.1186/1471-2229-14-62.
Polyphenol oxidase (PPO), often encoded by a multi-gene family, causes oxidative browning, a significant problem in many food products. Low-browning potatoes were produced previously through suppression of PPO gene expression, but the contribution of individual PPO gene isoform to the oxidative browning process was unknown. Here we investigated the contributions of different PPO genes to total PPO protein activity, and the correlations between PPO protein level, PPO activity and tuber tissue browning potential by suppression of all previously characterized potato PPO genes, both individually and in combination using artificial microRNAs (amiRNAs) technology.
Survey of the potato genome database revealed 9 PPO-like gene models, named StuPPO1 to StuPPO9 in this report. StuPPO1, StuPPO2, StuPPO3 and StuPPO4 are allelic to the characterized POTP1/P2, POT32, POT33 and POT72, respectively. Fewer ESTs were found to support the transcriptions of StuPPO5 to StuPPO8. StuPPO9 related ESTs were expressed at significant higher levels in pathogen-infected potato tissues. A series of browning phenotypes were obtained by suppressing StuPPO1 to StuPPO4 genes alone and in combination. Down-regulation of one or several of the PPO genes did not usually cause up-regulation of the other PPO genes in the transgenic potato tubers, but resulted in reduced PPO protein levels. The different PPO genes did not contribute equally to the total PPO protein content in the tuber tissues, with StuPPO2 accounting for ~ 55% as the major contributor, followed by StuPPO1, ~ 25-30% and StuPPO3 and StuPPO4 together with less than 15%. Strongly positive correlations between PPO protein level, PPO activity and browning potential were demonstrated in our analysis. Low PPO activity and low-browning potatoes were produced by simultaneous down-regulation of StuPPO2 to StuPPO4, but the greatest reduction occurred when StuPPO1 to StuPPO4 were all suppressed.
StuPPO1 to StuPPO4 genes contributed to browning reactions in tuber tissues but their effect was not equal. Different PPO genes may be regulated independently reflecting their diversified functions. Our results show that amiRNAs can be used to suppress closely related members of highly conserved multi-gene family. This approach also suggests a new strategy for breeding low-browning crops using small DNA inserts.
多酚氧化酶（PPO）通常由多基因家族编码，会导致许多食品发生氧化褐变，这是一个重大问题。先前通过抑制 PPO 基因表达生产出低褐变马铃薯，但单个 PPO 基因同工型对氧化褐变过程的贡献尚不清楚。在此，我们通过使用人工 microRNA（amiRNA）技术，单独和组合抑制所有先前鉴定的马铃薯 PPO 基因，研究了不同 PPO 基因对总 PPO 蛋白活性的贡献，以及 PPO 蛋白水平、PPO 活性和块茎组织褐变潜力之间的相关性。
对马铃薯基因组数据库的调查显示，有 9 个 PPO 样基因模型，在本报告中分别命名为 StuPPO1 至 StuPPO9。StuPPO1、StuPPO2、StuPPO3 和 StuPPO4 分别与已鉴定的 POTP1/P2、POT32、POT33 和 POT72 等位。发现较少的 EST 支持 StuPPO5 至 StuPPO8 的转录。StuPPO9 相关的 EST 在受病原体感染的马铃薯组织中表达水平显著较高。单独和组合抑制 StuPPO1 至 StuPPO4 基因获得了一系列褐变表型。在转基因马铃薯块茎中，一个或几个 PPO 基因的下调通常不会导致其他 PPO 基因的上调，但会导致 PPO 蛋白水平降低。不同的 PPO 基因对块茎组织中总 PPO 蛋白含量的贡献并不均等，StuPPO2 占约 55%，是主要贡献者，其次是 StuPPO1，约 25-30%，StuPPO3 和 StuPPO4 一起占不到 15%。我们的分析表明，PPO 蛋白水平、PPO 活性和褐变潜力之间存在很强的正相关性。通过同时下调 StuPPO2 至 StuPPO4，可产生低 PPO 活性和低褐变马铃薯，但当同时抑制 StuPPO1 至 StuPPO4 时，褐变减少最为明显。
StuPPO1 至 StuPPO4 基因对块茎组织中的褐变反应有贡献，但作用并不均等。不同的 PPO 基因可能独立调控，反映了它们多样化的功能。我们的结果表明，amiRNA 可用于抑制高度保守的多基因家族中的密切相关成员。该方法还为利用小 DNA 插入物培育低褐变作物提供了一种新策略。
