枇杷EjAO基因的克隆与表达分析

Molecular Cloning and Expression Analysis of an EjAO Gene in Loqua(Eriobotrya japonica)

果实的成熟和衰老是个复杂的生物学过程,一般认为活性氧(ROS)在其中起着重要作用。本研究采用cDNA末端快速扩增技术(RACE)和RT-PCR相结合的方法,从白肉枇杷成熟期果实中成功克隆到一个编码抗坏血酸氧化酶(AO)的基因,命名为EjAO。该基因全长2 174 bp,包含了长度为1 605 bp的开放阅读框。该阅读框编码的氨基酸数目为534个,所形成的蛋白质预测分子量为59.39 kD。实时荧光定量PCR结果显示EjAO在根和果实中高丰度表达,并且在白肉枇杷成熟期果实中的表达量远远高于红肉枇杷。核苷酸多态性分析显示EjAO基因在红肉枇杷和白肉枇杷共有五处多态性位点,其中T719C和A730C均导致氨基酸发生改变,推测可能对EjAO的酶学活性产生影响。EjAO的克隆和多态性位点的鉴定为阐明枇杷果实成熟与衰老机制及枇杷遗传改良育种提供一定的理论依据。

Fruit ripening an d senescence is a complex biological process. It is generally believed that reactive oxygen species（ROS） play an important role in the process. In the present study, a full-length cDNA encoding ascorbate oxidase（AO）, named EjAO, was obtained from the mature fruit of white-flesh loquat by using rapid amplification of cDNA ends（RACE） and RT-PCR methods. EjAO was 2 174 bp in length, containing a 1 605 bp open reading frame. The number of amino acids encoding by the open reading frame was 534, consisting of a protein with a 59.39 kD predicted molecular mass. The results of quantitative real-time PCR showed that the amount of EjAO was notably abundantly in root and fruit. In mature fruit of white-flesh loquat, the relative expression level of EjAO was significantly higher than that in red-flesh loquat. Nucleotide polymorphisms analysis of EjAO detected five polymorphic sites between red-and white-flesh loquat. Of which, mutation of T719 C and A730 C both caused the change of a mino acid, hinting that its might influence the enzymatic activity of EjAO between two loquat varieties. The molecular cloning and nucleotide polymorphisms detection of EjAO provided a theoretical basis for elucidation of the mechanism of loquat fruit ripening and molecular breeding in loquat.