抗坏血酸过氧化物酶基因转化苹果的研究
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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。
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.
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。
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.
引文
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魏振林. 2001 .HAV1基因转化平邑甜茶的研究.万方硕博论文数据库[硕士毕业论文].安徽:安徽农业大学
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陈祯. 2002 .高等植物转基因研究.生物学通报,38(8) :6～8
陈利锋,叶茂炳,陈永幸等,抗坏血酸与小麦抗赤霉病性的关系.植物病理学报,1997,27(2):113-118
达克东,仁德才,张松. 2001.超强表达接到豇豆胰蛋白酶抑制剂(CpT I)转化苹果的研究.园艺学报, 28 (1) :57～58
冯凤娟.2008.农杆菌介导的DHAR基因转化甜瓜和苹果的研究.西北农林科技大学[硕士学位论文] .陕西:西北农林科技大学
姜世民. 2003.体外定向进化大肠杆菌β-半乳糖苷酶和农杆菌介导DREB1A基因转化苹果的研究[硕士论文].江苏:南京农业大学
贾士荣,曹冬孙. 1992.转基因植物.植物生理学通讯,9(2):3-15
刘静,邵建柱,徐继忠.2006.农杆菌介导法将L FY基因导入苹果的研究.河北农业大学学报, 29(3):6-9
刘鹏,李勃,刘庆忠.2003.冷锻炼诱导甜椒抗冷力的生化机理研究.山东农业科学, 3
刘庆忠,赵红军,孙清荣.2001.抗真菌γ-硫堇蛋白Rs-afp1基因导入苹果获得转基因植株.农业生物技术学报,9(3):239-242
雷蓉华,马锋旺,梁东. 2007.苹果高效遗传转化受体系统的建立与优化.西北农业学报16 (6):128～132
刘淑娥. 2005.转基因技术在苹果育种中的应用.河北果树,(2):3-12
马玉华,马锋旺,马小卫,等.干旱胁迫对苹果叶片抗坏血酸含量及其代谢相关酶活性的影响.西北农林科技大学学报(自然科学版),2008,36(3):151～154
秦玲,王永熙,李明.2002.植物基因转化技术在苹果遗传改良中的应用.西北植物学报,22(4):1018-1024
庞勇,马锋旺,徐凌飞.抗坏血酸对苹果组培苗耐热性的诱导效应.中国科技论文在线,2004
邵建柱,马宝焜. 2003.转基因苹果研究进展.果树学报, 20(1): 49-53
余叔文,汤章城. 1999.植物生理与分子生物学.北京:科学出版社, 54- 76; 421- 534
余文琴,刘星辉. 2001.越冬期番石榴叶片脂质的过氧化状况.福建农业大学学报,30(3):357-361
王关林,方宏筠.植物基因工程原理与技术.北京:科学出版社, 1998, 1- 41; 194- 317
王三红,杨梦悦,顾敏.2007.农杆菌介导三价融合基因Rirol转化八棱海棠的研究.果树学报, 24(6):731～736
王泽槐,梁立峰. 1994.香蕉冷害过程叶片抗坏血酸含量及过氧化氢酶活性的变化.华南农业大学学报,15(3):71-76
魏振林. 2001 .HAV1基因转化平邑甜茶的研究.万方硕博论文数据库[硕士毕业论文].安徽:安徽农业大学
薛大煜,马艳青,黄炎武. 1996.低温胁迫对辣椒幼苗抗坏血酸含量的影响.湖南农业大学学报, 12:24
杨丽丽. 2006.根癌农杆菌介导的抗寒基因转录因子CBF转化苹果的研究.万方硕博论文数据库[硕士毕业论文].河北:河北农业大学
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