抗苹果斑点落叶病基因Mal d 1的克隆及功能鉴定

Cloning and Function Identification of Apple Alternaria Blotch Resistant Gene Mal d 1

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 Mal d 1 gene,which might be involved in the Alternaria blotch resistance in apple,was amplified and sequenced using the total RNA of apple leaves as template. Sequence analyses showed that it was 480 bp in length,contained two exons,and was characterized by bet v 1-like domain. The expressions of Mal d 1 were detected,but varied in apple leaves,flowers,fruit peels and pulps based on q RT-PCR analyses. Along with the development of fruits,it expression in fruit peels was increased first and then decreased. In the inoculated leaves with Alternaria alternata,Mal d 1 was significantly upregulated compared to the control counterpart,indicating is potential role in the resistance against Alternaria blotch. Furthermore,overexpression of Mal d 1 in transgenic callus,which was obtained using PRI101-AN as the expression vector by Agrobacterium-mediated transformation method,could greatly enhance the resistance to Alternaria blotch.