【作者】 冯莎莎；

【导师】 杜国强；

【作者基本信息】 河北农业大学 ， 果树学， 2007， 硕士

【摘要】 本研究以苹果（Malus domestica Borkh．）品种富士（Malus domestica var．Fuji）和营养系砧木M₂₆试管苗为试材，采用花粉管通道法和农杆菌介导法将抗寒基因的转录因子CBFs导入苹果中。通过对影响花粉管通道法进行外源基因导入节果的适宜时期和DNA注射浓度等因素研究，初步建立了节果花粉管通道法基因转化体系；研究了潮霉素和头孢霉素对苹果不定芽再生和继代苗的影响并进行了适宜选择压的筛选，进一步完善了节果叶盘法基因转化体系；对转化株系进行了抗寒性状生理指标检测与筛选，明确了外源CBFs基因与苹果抗寒性的相关性，并获得了抗性株系，为进一步开展利用基因工程改良苹果抗寒性状工作打下了良好的基础。主要研究结果如下：1．利用花粉管通道法将携带CBF₃和GUS基因的pWBVec10a质粒DNA导入富士苹果中，探讨了DNA浓度及注射时间对转化率的影响，结果表明，当外源DNA溶液的注入时间控制在人工授粉后11～24h，浓度为1000μg／mL时有利于提高苹果花粉管通道法转化外源基因效率，GUS基因阳性表达率可达12.5％。2．抗生素种类和浓度对苹果不定芽再生和继代苗生长影响差异较大。在以潮霉素为选择标记的苹果砧木M₂₆转化体系中，宜采用300mg／L的头孢霉素和3mg／L的潮霉素作为选择培养的脱菌浓度和选择压；用300mg／L的头孢霉素和5mg／L的潮霉素作为筛选培养的脱菌浓度和选择压。3．再生株系在含5mg／L潮霉素的培养基上进行为期6个月的抗性筛选，对具有潮霉素抗性的转化株系进行GUS检测和PCR检测，结果表明：在检测的30个株系中有4个株系表现阳性反应颜色，GUS基因阳性表达率为13％；有5株扩增出和阳性对照一致的特异条带，表明CBF₃基因已整合至M₂₆的基因组DNA中。4．经低温诱导转化株系叶片中的SOD、POD活性与对照相比均有所提高，随着低温诱导时间的延长其活性呈现先上升后下降最后趋于平缓的趋势，但变化幅度不同。低温诱导7h时，转CBF₂基因M₂₆试管苗叶片的SOD活性最大；低温诱导4h时，转CBF₃基因嘎拉试管苗叶片的SOD活性最大。低温诱导3h时，转CBF₂基因M₂₆试管苗叶片的POD活性最大；低温诱导6h时，转CBF₃基因嘎拉试管苗叶片的POD活性最大。5．经低温诱导，转化株叶片的MDA含量和电解质渗透率与对照相比均有所降低，随着低温诱导时间的延长其表现出的变化幅度不同。6．经低温诱导转化株叶片中的脯氨酸含量与对照相比均有所提高，呈现上升后下降最后趋于平缓趋势。诱导时间达8h时，转CBF₂基因M₂₆试管苗叶片的脯氨酸含量最大；诱导时间达2h时，转CBF₃基因嘎拉试管苗叶片的脯氨酸含量最大。更多还原

【Abstract】 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.更多还原