Institute of Crop Science, Chinese Academy of Agricultural Sciences, No. 80 South Xueyuan Road, Haidian, Beijing 100081, China.
Gembloux Agro-Bio Tech, University of Liège, 5030 Gembloux, Belgium.
Int J Mol Sci. 2020 Dec 16;21(24):9594. doi: 10.3390/ijms21249594.
Lunasin is a soybean-derived peptide that exhibits anticancer bioactivity in different cancer cells and has been identified in different plants. However, recent studies revealed through molecular and chemical analyses that lunasin was absent in wheat and other cereals. In this study, the soybean-derived lunasin was cloned into pCAMBIA3300 and we transferred the expression vector into wheat via an -mediated transformation. The identification of transgenic wheat was detected by polymerase chain reaction, Western blot analysis, and ultra-performance liquid chromatography with tandem mass spectrometry. An enzyme-linked immunosorbent assay showed that lunasin content in transgenic wheat L32-3, L32-6, and L33-1 was 308.63, 436.78, and 349.07 µg/g, respectively, while lunasin was not detected in wild-type wheat. Lunasin enrichment from transgenic wheat displayed an increased anti-proliferative activity compared with peptide enrichment from wild-type wheat in HT-29 cells. Moreover, the results of a real-time quantitative polymerase chain reaction showed a significant elevation in p21, Bax, and caspase-3 expression, while Bcl-2 was significantly downregulated. In conclusion, soybean-derived lunasin was successfully expressed in wheat via -mediated transformation and may exert anti-proliferative activity by regulating the apoptosis pathway in HT-29 cells, which provides an effective approach to compensate for the absence of lunasin in wheat.
亮氨酸脑啡肽是一种来源于大豆的肽，在不同的癌细胞中表现出抗癌的生物活性，并在不同的植物中被鉴定出来。然而，最近的研究通过分子和化学分析表明，亮氨酸脑啡肽不存在于小麦和其他谷物中。在这项研究中，来源于大豆的亮氨酸脑啡肽被克隆到 pCAMBIA3300 中，我们通过农杆菌介导的转化将表达载体转入小麦中。通过聚合酶链反应、Western blot 分析和超高效液相色谱-串联质谱对转基因小麦进行了鉴定。酶联免疫吸附试验显示，转基因小麦 L32-3、L32-6 和 L33-1 中的亮氨酸脑啡肽含量分别为 308.63、436.78 和 349.07 µg/g，而野生型小麦中未检测到亮氨酸脑啡肽。与野生型小麦肽富集物相比，从转基因小麦中富集的亮氨酸脑啡肽在 HT-29 细胞中表现出更高的抗增殖活性。此外，实时定量聚合酶链反应的结果显示，p21、Bax 和 caspase-3 的表达显著升高，而 Bcl-2 的表达显著下调。总之，通过农杆菌介导的转化，成功地在小麦中表达了来源于大豆的亮氨酸脑啡肽，它可能通过调节 HT-29 细胞中的凋亡途径发挥抗增殖活性，为弥补小麦中亮氨酸脑啡肽的缺乏提供了一种有效的方法。
