| Lanzhou lily （Lilium davidii var. unicolor）, which is a variety of Lilium davidii, belongs to genus Lilium of family Liliaceae. It is a peculiar variety in China and can be used as a kind of food, medicine and ornamental plant. What is more, it is the crown of the edible lily. Lanzhou lily is the most widely cultivated lily used for vegetable. However, there is no report about the genetic transformation of Lanzhou lily worldwide.This study took the edible Lanzhou lily as material. ASA biosynthesis and metabolism related enzyme genes （Ascorbate peroxidase APX and L-gactono-1, 4-lactone dehydrogenase GLDH） were transferred into Lanzhou lily. Transgenic plants of APX and GLDH were gained by screening and detection. Meanwhile, plant physiological mechanism of the transgenic and non-transgenic lily was studied under salt stress. Through genetic modification, the study aiming of increasing the salt tolerance of Lanzhou lily, and improve the resistance and the nutritional value.The main content and result of experiment are as follows:1. Two plant expression vector apple APX plant expression vector pCAMBIA-APX and GLDH expression vector pCAMBIA-GLDH were constructed. The target gene promoter was the CaMV35s gene, the terminator for the nopaline synthase. Hyg was used as a resistant selection marker together with a GUS marker gene2. By studying the effects of transgenic APX conversion efficiency of scale leaves, a number of factors, including Hyg concentration, the concentration of inhibitory factors, were established, and it is concluded that 10 mg L-1 Hyg, 500mg L-1Cef, 2d pre-culture, 3d of the co-culture, OD600 value 0.4 and 20 min of Lily infection were the best genetic transformation system, and gained the highest number of resistant plants after selection.3. Ten resistant lines, which were positive on the GUS staining, were identified by PCR amplification, the results showed that 10 strains amplified the specific bands of about 900 bp, while the control had no specific bands. The results proved preliminarily that the APX gene had been successfully transformed to Lanzhou lily. Respectively, 10 PCR-positive lines were further identified with Southern blot ting analysis; there are two strains with one hybrid zone, and one strain with two lines. This confirmed that APX gene was transferred into the genome of Lanzhou lily with different copies.4. Physiological responses under 200 mM salt stresses were determined in transgenic and control plant. The results showed that the APX activity of transgenic plant was significantly higher than the control plants; the speed of cell membrane permeability and MDA contents increasement was much lower than the control plants. This indicates that the salt tolerance of transgenic plants was higher than control plants. The SOD activity had no significant difference between the transgenic and control plants.5. The impact of GLDH gene conversion in the conversion efficiency of scale leaves, including the bombardment pressure and bombardment distance, was studied, it was established that 10 mg L-1 Hyg, 500 mg L-1 Cef, bombardment distance of 6 cm, bombardment pressure of 1100 psi and an amount of 300μg per gun of Lanzhou lily for the gene gun transformation system.6. Thirty-five positive strains with GUS staining were examined with PCR amplification, and all of them amplified the specific bands of around 850bp, while the control plants had no specific bands. This initially proved that the foreign gene was transferred into lily successfully. Respectively, 35 PCR-positive lines of Lily were detected by Southern blotting; one hybrid zone was detected in 7 strains, and two hybrid zones in 1 strain. This confirmed that GLDH gene has been transferred to the genome of Lanzhou lily.7. GLDH activity and ASA contents of the control and transgenic lily were determined, the results showed that GLDH activity increased 5-6 times, ASA contents increased by 2-7 times.8. The trends of antioxidant activity of antioxidant enzymes under drought stress in Lanzhou lily. The activity of SOD and CAT rose and then dropped and were highest at the fourth day. The activity of POD was the trends and highest at the fifth day. APX, DHAR, MDHAR and GR showed rose and then decreased, which DHAR and MDHAR maximum in the fifth day, APX and GR in the fourth day maximum. ASA and GSH antioxidant contents were the highest in the fourth day. |
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