Research In Genetic Transformation Of Tocopherol Cyclase Gene(VTE1) From Arabidopsis And Stress Tolerance In Transgenic Tobacco

Tocopherol is also called vitamin E (VE), it is one kind of the important lipophilic antioxidant on cell membrane for plants under the stressful environments. Tocopherol cyclase (TC) catalyzes the conversion of various phytyl quinol pathway intermediates to their corresponding tocopherols through the formation of the chromanol ring. In this study, tobacco W38 was transformed with a construct containing a cDNA insert encoding tocopherol cyclase (VTE1) from Arabidopsis under the control of the cauliflower mosaic virus (CaMV) 35S promoter and we examine if the content of VE in transgenic tobacco plants increase with the over-expression of VTE1, and then study the improve effect of VTE1 from Arabidopsis on the content of VE in tobacco. Based on the fact that the level of tocopherols vary with the stress level outside, we studied the endurance of transgenic tobacco plants to high light and salinity, in order to provide the exact evidence for the relationship between the tocopherols of high content and the stress condition, form a solid foundation for the study of the mechanism and utility of endurance in plants, The main results were as follows:The ten transgenic tobacco plants were confirmed by Southern blot and RT-PCR analyses, which lead the same PCR product of 495 bp as the positive control. Compared to the wild type controls, the content of vitamin E in transgenic lines was two times higher than that in the wild type, the most was 11.32 times, which showed that TC is the important limiting enzyme in the biosynthesi s of tocopherols. The over-expression of VTE1 could increase the content of tocopherols effectively.The endurance of transgenic plants was higher than that of the wild type to salinity stress on their phenotype, which showed on the withered areas of leaves. In different salinities(150,250 mmol/L), the content of tocopherols in transgenic lines was 1.3-1.8 higher than that in wild type, which indicated that the over-expression of VTE1 had increased the salt stress tolerance in tobacco plants.Meanwhile, the content of proline in wild type increased with the increase of salinity, but it increased first and then decreased in transgenic tobacco. The content of proline in wild type and transgenic lines in 150 mmol/L are 2.43 and 9.94 times higher than those in 0 mmol/L, respectively. They are 4.99 and 6.44 times higher in 250 mmol/L than in 0 mmol/L.When we compared the content of VE in wild type with that in transgenic plants, both them are treated with high light, the results showed that the content of VE in transgenic lines is consistently higher than that in wild type during different illuminating time, some is 6.2 times higher.In the different treat time of the high light, the activity of superoxide dismutase (SOD) in transgenic plants and wild type were increased first and then decreased, then increased at last. The activity in wild type was consistently higher than that in transgenic tobacco.The activity of peroxidase(POD) increased in both them in different illumination of high light. After 30h treatment of high light, the increasement of POD activity in wild type was higher than that in transgenic plants.The content of monodehydroascorbate(MDA) also increased both of them, and it reached the high level after 30h. It indicateded that the plants were injured more seriously, but the injurement of transgenic plants were light than wild type. Data abovementioned showed that there was a crosstalk in tocopherols and other antioxidants in protecting plants from high light.In conclusion, the over-expression of VTE1 could elevate the tolerance of tobacco plants to salinity and high light stress. To control the level of ROS and to protect cells under stress conditions, plant tissues contain enzymatic ROS scavenging mechanisms in plant include SOD, POD and CAT and a network of low molecular mass antioxidants such as tocopherols, and complicated mechanisms were in the two protective system. There was some relationship between antioxidant levels and the activity of antioxidant regenerating enzymes. When the activity of one kind of enzymatic/nonenzymatic scavengers decreased, the other one would enhance the level for the oxidants accumulation. Because of different mechanisms, there would be difference of respondent duration and intensity under kinds of stress for every antioxidant. Based on our experiments, short illumination of high light could induce high expression of tocopherols, and then the equilibrium between production and removal of ROS may be kept by VE and enzymatic scavengers.