【作者】 杨梦悦；

【导师】 章镇； 房经贵；

【作者基本信息】 南京农业大学 ， 果树学， 2006， 硕士

【摘要】 本研究以三价融合基因Rirol为目的基因，通过农杆菌介导方法转化八棱海棠，以期获得矮化、耐缺铁、抗冷、旱和盐碱的优良转基因苹果砧木。通过研究影响农杆菌介导的八棱海棠遗传转化的因素，优化了八棱海棠遗传转化体系，建立了高效遗传转化体系，获得了携带Rirol基因的苹果砧木新种质。实验内容和研究结果如下： 1．建立了八棱海棠高效的遗传转化体系： 研究了农杆菌菌株类型、vir基因诱导因子、Km加入时间、白化处理及悬浮液pH值对八棱海棠遗传转化的影响，建立了以LBA4404为侵染菌，以MS₀+KH₂PO₄0.1mmol／L+1.0％蔗糖+0.5％葡萄糖+1mmol／L脯氨酸+75μmol／L OH-AS pH5.6为悬浮液活化菌株，侵染后叶片放到再生培养基上（MS+BA4.0mg／L+NAA0.2mg／L）共培养两天，然后转入延后培养基（MS+BA4.0mg／L+NAA0.2mg／L+cef 250mg／L），延后到再生苗高2cm时，将苗转入选择培养基（MS+BA4.0mg／L+NAA0.2mg／L+Km50mg／L）进行选择培养的遗传转化体系。实验结果表明，抗性苗出现频率达8.4％。 2．获得了携带Rirol基因的苹果砧木新种质： 在上述体系优化过程中获得了84个卡那霉素抗性转化株系，其中GUS检测阳性的株系有65株。对65株GUS阳性株系中的24个株进行PCR扩增检测，19个株系出现了与阳性质粒相同的条带。随机取5株PCR阳性株系，进行Southern杂交检测，有4个株系出现了杂交条带。PCR检测和Southern杂交检测结果表明，Rirol基因已整合到八棱海棠转化植株的基因组中。对Souther杂交检测呈阳性的4个株系进行RT-PCR检测，4个株系均能扩增出目的条带，表明导入基因已在转录水平表达。对这4个株系进行了耐盐性检测，发现4个转基因株系的耐盐性与对照相比均有不同程度的提高。更多还原

【Abstract】 In order to obtain some transgenic apple rootstocks of dwarfing, iron-deficiency tolerance, cold, drought and saline resistance, the trinal fusion gene （Rirol） was used as the purpose gene, transformation of Malus robusta by Agrobacterium-mediated, optimized the transformation system of Malus Robusta, established a high-efficient transformation system, obtained the transgenic plants with Rirol gene of Malus robusta. The experiment contents and the research results as follows:1. A high-efficient transformation system of Malus robusta was developed:Effect of the type of Agrobacterium strain, vir induce factor, time of adding kanmycin, plant whitening process, and the pH value of suspention liquid of Malus robusta transformation were studied. Established a transformation system of taking LBA4404 as infection strain, with MS₀+0.1 mmols/ L of KH₂PO₄+1.0 %cane sugars+0.5% glucose+ the 1 mmol/ L proline+75μmol/ L OH- AS （pH5.6） for the suspention liquid to activate the strain, leaves were dipped into agrobacterium suspension, then were coculture on regeneration culture medium （MS + BA4.0mg/L + NAA0.2mg/L） for two days, selection culture on selective medium （MS+BA4.0mg/L+NAA0.2mg/L+Km50mg/L） after seedling height was 2 centimeters. The result indicted that the appearing frequency of resistance seedling was 8.4%.2. Obtained the transgenic plants with Rirol gene of Malus robusta:84 putative transgenic Malus robusta lines with the Roril gene via selecting of kanamycin were obtained during optimizing process described above. Among them a total 65 positive lines via selecting of GUS. 24 of which were verified by PCR amplification, Among them, 19 of which were confirmed to carry the same fragment as positive plasmid. Random take 5 PCR positive lines, 4 of them were confirmed to carry the transgene by Southern blot analysis. According the result of PCR and Southern blot analysis, the Rirol gene had been integrated into the plant genome of transgene Malus robusta. All these 4 Southern positive lines could be detected the aim fragment by RT-PCR, the result indicated更多还原