| Cucumber (Cucumis sativus L.) is an important vegetable and planted all over the world. Cucumber belongs to thermophilic crops and is easily suffered chilling damage in early spring in solar greenhouse as well as in open field. Many papers reported that cold acclimation can enhance cold tolerances of cucumber whereas the proper temperature was in debate. All life activities of plant are controlled by genetic materials. At present, the cold tolerance genes have been cloned in many plants and found that the activator of transcription CBF can activate expression of cold tolerance genes and the cold tolerance of plants can be increased by CBF over-expression. In this research, we chose three different low temperatures to train cucumber seedlings and try to find the optimum low temperature and time. At the same time, we cloned CBF1 gene from cucumber seedlings during cold acclimation, and CBF1 gene sense expression vector was constructed and transferred into cucumber inbred line, the cold tolerance of the transferred plants were increased significantly.The details were as follows:1.S11 (cold tolerance and self-bred line) and S22 (cold sensitive and self-bred line) seedlings were treated under three low day/night temperature combinations (10/4℃, 10/6℃and 10/8℃) at three true leaves stage. The results showed that the activities of SOD, POD, CAT and root activity,Vc content decreased significantly, the chilling injury index and electrolyticleakage increased significantly under the treatment of 10/4℃,these indicated that cold damage has arisen in cucumber seedling leaves under this treatment. The activities of SOD, POD, CAT and the content of Vc, soluble protein, soluble sugar increased significantly under the treatments of 10/6℃and 10/8℃, thus reduced the active oxygen damages. At the same time, the MDA contents and chilling injury index decreased significantly, but root activity increased, all these indicated that the cold tolerance of cucumber seedlings can be enhanced by use of cold acclimation under the treatments of 10/6℃and 10/8℃. In general, the best cold acclimation condition for S11 was 10/6℃and lasting for 3d, as for S22 was 10/6℃and lasting for 2d.2. Using S11 as test material, experiments were conducted to study the effect of cold acclimation on mesophyll cell ultrastructure of cucumber seedlings. The results showed that in normal growth conditions, the grana lamella in cucumber seedlings leaves were arranged regularly with more grana, chloroplast structure and the whole membrane system was complete. The chloroplast structure were changed, the grana lamella become loose and disorder under cold stress, and the chloroplast membrane was injured seriously under cold stress. Furthermore, the damage to membrane system was increased instead of repaired. Cold acclimation enhanced cold tolerances of cucumber seedlings. Compared to cucumber seedling without cold acclimation, the grana lamella in cucumber seedlings with cold acclimation were arranged relative closely, the whole membrane system was more complete, the damage to chloroplast was slighter and the damage can be repaired in a short time under cold stress. All these indicated that cold acclimation with optimum temperature can maintained the integrity of membrane system (especially chloroplast) under cold stress, thus alleviated the cold damage to cucumber seedlings.3. Using the high cold tolerance cucumber plant as test material. PCR primers were designed based on the conservation sequence of CBF1 gene in arabidopsis thaliana. By means of RT-PCR methods, we had cloned CBF1 gene from cucumber seedlings leaves with cold acclimation, and the gene contained 671bp and 213 amino acids determined by this gene. The gene had 100 percent homology to that of arabidopsis thaliana in coding region, and had minor difference in start codon 5′-upstream region. The gene also has high homology compared to that of chorispora bungeana, olimarabidopsis pumila and Chinese small iris.The pCBF1 was digested with SacI and SacII, and then CBF1 gene was obtained. After the double digestion to PROk2 with SacI/BamHI, fragments were ligated. Then the sense expression vector of activating transcription factor of cucumber was constructed and ordered pROK2-CBF1. With this expression vector, we can transfer the gene to other plants to increase their resistance to low temperature, drought and salt.4. The target gene was transferred into cucumber self-bred line (disease resistance and cold sensitive) by pollen-tube pathway via ovary-injecting. Two transgenic plants were found by use of preliminary screening of Kan, PCR and Southern hybridization detection, and the conversion is 0.05%. The optimum time for ovary-injecting was one day before flowering, and can get transgenic plants at this time. The separation law of transformation generation was not as agreement with the Mendelian genetic law, the reason may be gene silencing. The transformants probability of appearance increased with adding of generation.5. Under cold stress, the activities of SOD, POD and the contents of proline, soluble protein of T1 generation increased significantly compared with those of the wild type. The generation of T2 grew longer time under nature low temperature and generation of T3 had strong growth potential under low temperature in winter compared with the wild type, and can live through the winter, while the wild type all died. The cold tolerance was enhanced and the disease resistance to downy mildew, powdery mildew was kept in transgenic plants. At present, the generation of T3 was obtained.
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