【作者】 李颖；

【导师】 马锋旺；

【作者基本信息】 西北农林科技大学 ， 果树学， 2010， 硕士

【摘要】 抗坏血酸作为一种重要的抗氧化剂,在植物细胞的酶促和非酶促反应中都具有重要意义。植物在逆境胁迫下会产生大量具有毒害作用的活性氧,活性氧的清除系统对于植物维持其正常生理功能具有重要意义。抗坏血酸过氧化物酶(APX)是清除活性氧的关键酶,当植物体内的活性氧积累时,能快速诱导APX表达,APX催化抗坏血酸与H2O2反应而使H2O2被分解。利用生物技术培育高营养,抗逆的新品种是目前育种的主要目标之一。通过基因工程的方法提高活性氧清除酶活性,从而提高植物的抗氧化胁迫的能力,并为研究其代谢途径以及在抗坏血酸-谷胱甘肽循环中的作用奠定基础。本研究通过农杆菌介导法将抗坏血酸过氧化物酶基因导入苹果,获得转APX基因植株,为选育具有高抗逆性的新品种奠定基础。本文主要研究结果如下:1.优化了农杆菌介导的以苹果叶片为受体的遗传转化体系,获得了转APX基因苹果植株。通过研究APX基因转化中影响叶片转化效率的多种因素,确定了各主要影响因素的值。试验结果表明:2d的预培养,OD600值为0.6的工程菌液浓度,侵染时间10min,延迟筛选10d能获得较高的转化率。2.确立了潮霉素对嘎啦苹果合适的筛选压力,只需3 mg·L-1Hyg就能完全抑制其生长。3. PCR检测和Gus染色表明,APX基因已整合到苹果基因组中。更多还原

【Abstract】 Ascorbate acts as an important antioxidants in both enzymatic and non-enzymatic reactions in plant cells. Under the adverse circumstance forces, plants can produce massive ROS which has very strongly poisonous function to the plant. Ascorbate peroxidase is the critical enzyme of scavenging the reactive oxygen species. When the reactive oxygen species were accumulated, the APX expression and detoxification of H2O2 through reaction of ascorbate and H2O2 can be immediately induced. Researchers hope to enhance the ROS elimination enzyme activity through the genetic engineering method. Thus sharpens the ability of the anti-oxidation forces and it would be also helpful to get more information about the role of APX playing in AsA-GSH cycle and its metabolism pathway. In this research, gala apple with APX gene was acquired by Agrobacterium tumefacien to lay a foundation for breeding the variety which has the high resistance.The main results were as follows:1. Agrobacterium-mediated genetic transformation system was optimized, and the transgenic plantlets of apple cultivar carrying APX gene were obtained.Factors afecting the transformation frequency were studied, and some parameters were determinated. The results indicated that better transformation rate was gained when the leaves was precultivated 2 days, the OD600 of grobacterium was about 0.6, Agrobacterium infected 10 min, and 10 days of detention screening.2. The better selective efficiency was gotten when the concentration of hygromycin was 3 mg/L Hyg.3. Result s of PCR and Gus proved that the APX gene had integrated into apple genome.更多还原