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.
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