Abstract: Hydrated lettuce seeds were the model materials for studying freezing tolerance and cryopreservation of seeds with high moisture content. Previous studies had shown that fatty acid biosynthesis pathway was involved in freezing tolerance of hydrated lettuce seeds. In this study, the moisture content of the lettuce seeds was tested after imbibition for different time. Then the difference in germination rate of seeds with different water contents after programmed cooling treatment was examined and the expression of genes involved in fatty acid synthesis (FAD2, FAD3, PPT, ELOVL), and cold response gene ICE1 were studied. The results showed that the moisture content of seeds increased with imbibition time, and the water absorbing rate showed a ‘fast-slow-fast’ trend. Cooled to the same freezing temperature (-20℃, -22℃) by programmed cooling, the seeds that imbibed less than 6 h exhibited relatively high germination rate. However, the germination rate of seeds that imbibed for more than 8 h was significantly reduced after the cooling treatment. The moisture content of imbibed seeds was saturated at 8 h, and the seeds were more sensitive in this state, which showed the effect of moisture content on freezing tolerance. After cooling treatment, gene expression was studied which indicated that the fatty acid desaturase genes (FAD2, FAD3), palmitoyl-protein thioesterase genes (PPT), and long-chain fatty acid elongase genes (ELOVL) were gradually up-regulated under freezing treatment with the extension of imbibition time for the seeds. The expression of above genes achieved the highest level when seeds imbibed for 10 h, because the hydrated seeds suffered the most serious freezing injury due to the high moisture content. Meanwhile, the expression of the antifreeze genes (ICE1) of seeds also showed an increasing tendency in imbibed cooled seeds, which achieved the highest level at 10 h. This study showed that when the moisture content of seeds was higher, the seeds would be subjected to higher freezing damage. In the same time, the seeds had a certain freezing resistance. Therefore, synthesis of more unsaturated fatty acids and antifreeze proteins by over-expression of related genes to protect the cell membrane would improve the freezing tolerance of imbibed seeds.

Key words: Lactuca sativa seed, water content, unsaturated fatty acids, cold resistance gene

摘要： 以生菜(Lactuca sativa)种子为研究对象，通过不同时间的吸水处理分析其含水量变化，再通过程序降温处理，分析不同含水量种子发芽率的差异，以及脂肪酸合成有关基因(FAD2、FAD3、PPT、ELOVL)和冷调节基因ICE1的表达。结果表明，种子含水量随吸水时间增加而升高。程序降温至同样的低温冷冻条件下(-20℃、-22℃)，吸水时间小于6 h的种子发芽率较高，而吸水8 h以上的种子发芽率显著降低。种子吸水8 h含水量处于饱和状态，在此状态下种子对低温较为敏感,说明含水量对种子耐冻性有影响。冷冻处理后生菜种子基因表达检测结果表明，脂肪酸去饱和酶基因(FAD2、FAD3)、蛋白质棕榈酰基硫脂酶相关基因(PPT）、长链脂肪酸延伸酶相关基因(ELOVL)的表达水平均随着种子含水量增加呈上升趋势，吸胀10 h的种子表达量最高，此时种子由于高含水量所受冷冻伤害最大。基因ICE1在冷冻处理种子中的表达也随着吸水时间增加而升高，在吸水10 h时种子中表达量到最高水平。综上，种子含水量越高，所受冷冻伤害越大。但种子在低温条件下具有一定的抗冷反应，可通过相关基因的过表达调控合成更多不饱和脂肪酸、抗冻蛋白等提高含水种子耐冻性。

关键词: 生菜种子, 含水量, 不饱和脂肪酸, 抗冷基因