目录
摘 要 III
关键词 III
ABSTRACT IV
引言
引言
第一章 文献综述 1
1 梨非生物胁迫的研究进展 1
1.1 梨干旱胁迫的研究进展 1
1.2 杜梨在非生物胁迫研究中的优势 2
2 WRKY基因的研究进展 2
2.1 WRKY转录因子 2
2.2 转录激活 2
2.3 转录抑制 2
3 WRKY转录因子的功能论述 3
3.1 WRKY转录因子参与生物胁迫 3
3.2 WRKY转录因子参与非生物胁迫响应 3
3.3 WRKY转录因子在植物抗旱中的功能 4
4 杜梨中WRKY转录因子的研究方法和展望 5
第二章 材料与方法 6
1 植物材料和逆境处理的方法 6
2 实验方法 6
2.1 RNA提取和实时荧光定量PCR分析 6
2.2 PbrWRKY53的基因克隆 7
2.3 PbrWRKY53的亚细胞定位 7
2.4 中间表达载体的构建 8
2.5 遗传转化 8
2.6 转基因系的耐旱性评估 9
2.7干旱前后逆境基因的表达量分析 10
2.8统计分析 10
第三章 结果与分析 11
3.1 PbrWRKY53对非生物逆境的响应模式 11
3.2 PbrWRKY53的亚细胞定位 11
3.3 PbrWRKY53的超表达增强了转基因烟草的耐旱性 12
3.4 PbrWRKY53的超表达增强了转基因秋子梨的耐旱性 15
3.5 PbrWRKY53的超表达减少转基因株系ROS的积累，提高抗氧化酶活性 16
3.6 干旱处理前后野生型和转基因系响应基因的表达分析 18
第四章 讨 论 21
致 谢 24
参考文献 25
本科期间获得学术成果 30
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杜梨转录因子PbrWRKY53的功能验证
摘 要
中国是世界第一产梨大国，梨果品经济在我国国民经济中占有重要地位。然而，梨生长发育过程中易受到非生物胁迫的危害，尤其是干旱胁迫最为突出。因此，选育梨抗干旱品种显得尤其必要。随着基因工程育种技术的快速发展，定向改变植物性状成为可行的途径。抗旱基因的筛选是基因工程育种的前提和关键。根据以往的研究，WRKY是植物中转录因子的一个大家族，但大多数的WRKY家族成员目前尚不清楚。在本研究中，我们报道了从杜梨中分离得到的PbrWRKY53的功能鉴定。干旱和脱落酸对PbrWRKY53有强烈的诱导作用而盐和低温对其诱导作用不明显。亚细胞定位结果显示PbrWRKY53定位于细胞核中。遗传转化到烟草和秋子梨中发现增强了烟草和秋子梨对干旱胁迫的耐受性。与野生型相比，转基因系的失水率更低，活性氧积累量较少和拥有更高的抗氧化酶活性。此外，在烟草中超表达PbrWRKY53导致PbrNCED1的表达水平提高，同时AsA的积累量提高。综上所述，PbrWRKY53是受干旱诱导的转录因子，是植物耐旱性的正向调节因子。至少部分通过调控PbrNCED1产生AsA，在耐旱性方面发挥积极作用。
FUNCTIONAL CHARACTERIZATION OF TRANSCRIPTION FACTOR PbrWRKY53 ISOLATED FROM PYRUS BETULAEFOLIA
ABSTRACT
China is the largest pearproducing country in the world, pear fruit economy in Chinas national economy occupies an important position.However, pear is vulnerable to abiotic stress during its growth and development, especially drought stress.Therefore, it is especially necessary to breed pear drought resistant varieties.With the rapid development of genetic engineering breeding technology, directional change of plant traits has become a feasible approach.The selection of droughtresistant genes is the premise and key of genetic engineering breeding.The study show that WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report the identifification and functional characterization of PbrWRKY53 isolated from Pyrus betulaefolia. PbrWRKY53 was greatly upregulated by drought and abscisic acid, but slightly induced by salt and cold. Subcellar localization analyses showed that PbrWRKY53 was located in the nucleus. Ectopic expression of PbrWRKY53 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to drought stress. The transgenic plants exhibited better water status, less reactive oxygen species generation and higher levels of antioxidant enzyme activities and metabolites than the wild type.In addition, overexpression of PbrWRKY53 in transgenic tobacco resulted in enhanced expression level of PbrNCED1, and led to the increase in larger amount of AsA accumulation in comparison to WT. Taken together, these results demonstrated that PbrWRKY53 ,a droughtinduced transcription factor, acts as a positive regulator of drought tolerance and plays a positive role in drought tolerance, which might be, at least in part, promoting production of AsA via regulating PbrNCED1 expression.

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