蛋白酶抑制剂对含水生菜种子耐冻性的影响

选取生菜(Lactuca sativa)种子作为试材，外源添加蛋白酶抑制剂2-硝基苯甲酸5,5'-Dithiobis-(2-nitrobenzoic acid)，DTNB]对种子吸胀处理，通过程序降温，分析2-硝基苯甲酸对低温下种子发芽率及生理活性的影响。结果表明，低温下含水生菜种子的致死温度为–20 ℃；外源添加2-硝基苯甲酸2 mmol·L–1时种子发芽率最高，即对种子活性的保护效果显著；在此浓度下种子内SOD活性比对照提高1.38倍，羟基自由基清除能力提高1.17倍；与对照组相比产生两种新的蛋白11 S种子贮藏球蛋白Jug r4和11 S种子贮藏球蛋白2，均属于球蛋白家族，可提高含水种子的耐冻性；低温下种子内积累更小分子量的球蛋白多肽，对种子具有低温保护效果。综上，低温条件下生菜种子产生一定的抗冷反应，外源添加2 mmol·L–1 2-硝基苯甲酸可提高含水种子发芽率及生理活性，产生抗冻蛋白，积累更小分子量的球蛋白多肽进而提高种子抗冻性。

Effect of Protease Inhibitor on Freezing Tolerance of Hydrated Lettuce Seeds

Lactuca sativa seeds were selected as test materials, and exogenous protease inhibitor 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was added to soak the seeds. The effects of DTNB on seed germination rate and physiological activity at low temperature were analyzed by programmed cooling. The results showed that the lethal temperature of lettuce seeds was –20 ℃ at low temperature. Exogenous DTNB 2 mmol·L–1 had the highest germination rate, that is, it had a significant protective effect on seed activity. At this concentration, the Superoxide dismutase (SOD) activity in seeds was 1.38 times higher than that in the control, and the hydroxyl radical scavenging ability was 1.17 times higher than that in the control. Compared with the control, two proteins, 11 S globulin seed storage protein Jug r4 (XP_023733339.1) and 11 S globulin seed storage protein 2 (XP_023769779.1), belonging to globulin family, were expressed after 2 mmol·L–1 DTNB imbibing, which could improve the freezing resistance of the seeds. SDS-PAGE experiment showed that seeds accumulated small molecular globulin polypeptides at low temperature, which had a chilling effect on the seeds. In conclusion, seeds had a certain freezing tolerance under low temperature. Exogenous addition of 2 mmol·L–1 DTNB could improve the germination rate, physiological activity, antifreeze protein generation, and accumulation of globulin polypeptides with smaller molecular weight of hydrated lettuce seeds, so as to improve the freezing resistance of seeds.