Sainfoin (Onobrychis viciaefolia Scop) is a forage crop in the Leguminosae family. The leaves of this plant and other tissues have a high level of proanthocyanidins (PAs). The BAN gene encodes anthocyanidin reductase (ANR), which is a key enzyme in PA biosynthesis. This report describes cloning of a BAN homolog gene (viz., OvBAN) from local sainfoin and overexpression of this gene in alfalfa (Medicago sativa L.), a globally important forage crop. The open reading frame (ORF) of OvBAN contains 1020 bp, which is deduced to encode 339 amino acids. Alignment with amino acid sequences showed that OvBAN had 99%, 87%, and 85% identity to homologs from another Onobrychis viciaefolia variety, known as Lotus corniculatus, and Medicago sativa, respectively. The ORF of OvBAN was cloned into a binary vector under the control of the CaMV35S promoter, in which the nptII gene was used as a selectable marker. The resulting binary vector was introduced to alfalfa using Agrobacterium tumefaciens-mediated transformation. Kanamycin-resistant plants were obtained and grown in potting soil. PCR and Southern blot analyses demonstrated the integration of the OvBAN transgene in transgenic plants. Quantitative real-time PCR analysis revealed that OvBAN was highly expressed in transgenic plants. Crude enzyme was extracted from transgenic and wild-type plants. Enzyme tests showed higher ANR activity in the transgenic samples than in the wild-type. Furthermore, butanol-HCl-based cleavage analysis showed that the PA content was higher in transgenic plants than in wild-type plants. These results demonstrate that OvBAN is an appropriate gene for the enhancement of PAs in alfalfa.

皂素（Onobrychis viciaefolia Scop）是豆科植物中的草料作物。该植物和其他组织的叶片中含有高水平的原花青素（PAs）。该BAN基因编码花青素还原酶（ANR），这是在生物合成PA中的关键酶。该报告描述了从本地皂素克隆BAN同源基因（即OvBAN）并在苜蓿（苜蓿）中过表达该基因，苜蓿是全球重要的饲料作物。OvBAN的开放阅读框（ORF）包含1020 bp，据推测可编码339个氨基酸。与氨基酸序列的比对表明，OvBAN与另一种同源物具有99％，87％和85％的同一性Onobrychis viciaefolia变种，分别称为Lotus corniculatus和Medicago sativa。在CaMV35S启动子的控制下，将OvBAN的ORF克隆到二元载体中，其中npt II基因用作选择标记。使用根癌农杆菌介导的转化将所得的二元载体引入苜蓿。获得抗卡那霉素的植物并在盆栽土壤中生长。PCR和Southern印迹分析表明OvBAN转基因已整合到转基因植物中。实时定量PCR分析显示OvBAN在转基因植物中高表达。从转基因和野生型植物中提取粗酶。酶测试显示，转基因样品中的ANR活性高于野生型。此外，基于丁醇-HCl的裂解分析表明，转基因植物中的PA含量高于野生型植物。这些结果表明，OvBAN是紫花苜蓿中增强PA的合适基因。