Transformation Of Transcription Factor CBF Gene And Analysis Of Coldhardness-related Indexes In Trangenic Lines Of Apple

Using the pollen tube pathway transformation and agrobacteria-mediatedtransformation, the transcription factor CBFs gene, which was considered to relate to coldhardness induction, was transferred into Fuji and apple stock M₂₆. In order to study theoptimum introducing period and the concentration of DNA, the technique of pollen tubepath way was used to introduce gene into apple, all proved that it is feasible to study fruittrees. The effects of Hyg and Cef on adventitious buds and plantlet and the impact ofselection pressure screening, further improve the apple leaf disc gene conversion system;right lines into the cold hardiness traits physiological indexes detection and screening,clear pair of exogenous gene and cold resistance of apple relevance, and was resistantstrains, To further develop the use of genetic engineering to improve the cold tolerance ofapple lay a good foundation.The main results were as follows:1. Using pollen tube pathway transformation, plasmid pWBVec10a containing geneCBF₃ and report gene GUS was introduced into Fuji. The effects of inversion rate by theoptimum introducing period and the concentration of DNA were studied. The resultsshowed that the best time to inoculate was at 11 hours to 24 hours after pollination, and thebest concentration of DNA was 1000μg/ml. the rate of expression of GUS was gained with12.5%.2. The effects of different antibiotic and concentration on adventitious buds andplantlet were studied to optimize the selection pressure. In the transformation system ofapple stock M₂₆. 300mg/L of Cef and 3mg/L of Hyg were added to as suitable selectionpressure in de-bacteria culture, 300mg/L of Cef and 5mg/L of Hyg were added to assuitable screen pressure in de-bacteria culture.3. Regenerative buds had been growing in culture added 5mg/L of Hyg for 6 months,the transgenic lines with the hygromycin-resistant ability were further tested by GUSanalysis and PCR analysis. The result shows: there were 30 hygromycin-resistant lines,four plants showed GUS⁺, the rate of expression of GUS was gained with 13%; five plantsshowed specific positive band as same as the plasmid control, the result indicated that theCBF gene has been integrated into the apple genome.4. The leaves of transgenic plants could maintain higher SOD, POD activity thancontrol though cold acclimation, the activity increased early, then decreased and at last itkept stabilization, but in different extent. The level of SOD activity of M₂₆ carring CBF₂gene increased and reached to the maximum at the 7h; but the Gala carting CBF₃ gene wasat the 4h. The level of POD activity of M₂₆, carring CBF₂ gene increased and reached to the maximum at the 3h; but the Gala carring CBF₃ gene was at the 6h.5. Though cold acclimationm, the transgenic plants could maintain lower MDAcontent and electrolytic leakage than control, but in different extent.6. The leaves of transgenic plants could maintain higher proline content than controlthough cold acclimation, the content increased early, then decreased and at last it keptstabilization. The level of proline content of M₂₆ carring CBF₂ gene increased and reachedto the maximum at the 8h; but the Gala carring CBF₃ gene was at the 2h.