靶向水稻脂氧合酶OsRCI-1的活性小分子化合物筛选体系的建立
Establishment of a screening system for active chemicals based on activity detection of rice lipoxygenase OsRCI-1

为明确虫害诱导水稻脂氧合酶OsRCI-1的生化特性，通过构建水稻脂氧合酶OsRCI-1重组蛋白原核表达体系，对重组蛋白催化条件进行优化，并研究小分子化合物对OsRCI-1活性的影响。结果表明，缓冲液pH和反应温度都能影响水稻OsRCI-1蛋白的催化活性，当缓冲液pH为7.0时催化活性最佳，为169.9 mol·mg-1·min-1；在检测的20~50℃反应温度范围内，在25℃和45℃时出现2个催化活性高峰，分别为68.2 mol·mg-1·min-1和70.4 mol·mg-1·min-1。利用建立的OsRCI-1活性检测体系筛选出4种小分子化合物，即水杨苷异羟肟酸、水杨酸、叔丁基对二苯酚和茉莉酸，均对水稻OsRCI-1活性具有调控作用，抑制中浓度依次为0.12、418.67、232.84、2 747.19 μmol/L。表明本试验所建立的靶向水稻脂氧合酶活性化合物鉴定体系可用于活性小分子化合物的快速、高通量筛选。
In order to determine the biochemical characteristics of rice lipoxygenase OsRCI-1, a prokaryotic expression system of recombinant OsRCI-1 protein was established and the catalytic conditions of OsRCI-1 were optimized, and the effects of chemicals on the activity of OsRCI-1 were analyzed. The results showed that the pH of catalytic buffer and temperature could greatly affect the activity of OsRCI-1, with the highest catalytic activity (169.9 mol·mg-1·min-1) observed at pH 7.0; in the range of 20-50℃, the activity of OsRCI-1 showed two peaks (68.2 mol·mg-1·min-1 and 70.4 mol·mg-1·min-1) at 25℃ and 45℃, respectively. By using the OsRCI-1 activity detection system, four active chemicals including salicylhydroxamic acid, salicylic acid, tertiary butylhydroquinone, and jasmonic acid were identified, which could regulate OsRCI-1 activity, with a half inhibitory concentration (IC50) of 0.12, 418.67, 232.84, and 2 747.19 μmol/L, respectively. To sum up, the screening system for OsRCI-1 activity was a quick and high-throughput system and could be used for identification of active chemicals against rice lipoxygenase.