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以苹果叶片总RNA为模板,通过克隆获得抗苹果斑点落叶病基因Mal d 1。Mal d 1开放阅读框长度为480 bp,编码159个氨基酸残基,包含2个外显子和1个内含子。蛋白结构分析显示,Mal d 1蛋白包含bet v 1-like结构域。荧光定量PCR分析结果表明,Mal d 1在苹果叶、花、果皮、果肉中均有表达,但各器官中表达水平存在差异;在不同发育时期的果皮中都有表达,表达水平随时间呈现先上升后下降的趋势;在抗性品种叶片中表达水平较高;在叶片人工接种病菌条件下表达量随时间的延长明显高于对照组。利用PRI101-AN载体和农杆菌介导的遗传转化方法转化苹果愈伤组织,转基因愈伤组织的抗病鉴定结果显示,Mal d 1过量表达增强愈伤组织对苹果斑点落叶病的抗性。
The total RNA of apple leaves was used as a template to obtain Mald 1, an anti-apple leaf spot disease resistance gene. The Mal d 1 open reading frame (ORF) was 480 bp in length and encoded 159 amino acid residues, including 2 exons and 1 intron. Protein structure analysis showed that the Mal d 1 protein contains the bet v 1-like domain. The results of real-time quantitative PCR showed that Mal d 1 was expressed in apple leaves, flowers, pericarp and pulp, but the expression levels of different organs were different. The expression of Mal 1 was found in the pericarp at different developmental stages, And then decreased. The expression level in leaves of resistant varieties was higher than that of control. The expression level of leaves in artificial inoculated leaves was significantly longer than that in control. The transformation of apple callus with PRI101-AN vector and agrobacterium-mediated genetic transformation method showed that Mal d 1 overexpression enhanced the resistance of callus to apple leaf spot disease.