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脂氧合酶(lipoxygenase,LOX)是植物十八碳酸代谢途径的关键酶,与种子老化密切相关。洋葱(Allium cepa)种子为短寿命种子。研究LOX基因与洋葱种子发育及老化的关系具有重要的意义。本研究以洋葱授粉后的子房和种子为材料,利用RT-PCR结合快速克隆c DNA末端(rapid-amplification of c DNA ends,RACE)技术,获得Ac LOX1(Gen Bank登录号:KU363822)基因全长。用生物信息学方法对其基因序列特征进行分析;利用RT-PCR和q RT-PCR分析Ac LOX1在洋葱不同部位、种子发育过程以及种子储存过程中的表达模式。结果表明,该基因全长为2 847 bp,包含一个2 619 bp的开放阅读框,编码873个氨基酸。氨基酸序列分析表明,Ac LOX1具有PLAT_LH2(polycystin-1,lipoxygenase,alpha-toxin_lipoxygenase homology)和LOX两种结构域;与桉树(Eucalyptus grandis)、杏(Prunus dulcis)、葡萄(Vitis vinifera)等物种的LOX基因氨基酸序列同源性高达66%~73%。系统进化树分析表明,Ac LOX1与其他物种的9-LOX聚在一起。时空表达模式表明,Ac LOX1基因在洋葱各组织中均有表达,在授粉后5~25 d随着时间增长其表达量增加,但在授粉后25 d到成熟及储存一年的种子中少量持续表达。本实验初步证实了Ac LOX1对洋葱种子发育具有促进作用且与种子老化相关联,为阐明洋葱种子短寿命的作用机制提供了理论依据。
Lipoxygenase (LOX) is a key enzyme in the plant octadecanoic acid metabolic pathway, which is closely related to seed aging. Allium cepa seeds are short-lived seeds. It is of great importance to study the relationship between LOX gene and onion seed development and aging. In this study, the ovules and seeds after pollination of onion were used as materials, and the gene of Ac LOX1 (Gen Bank accession number: KU363822) was obtained by RT-PCR combined with rapid-amplification of c DNA ends (RACE) long. Bioinformatics methods were used to analyze the gene sequences of Ac LOX1. The expression patterns of Ac LOX1 in different parts of onion, seed development and seed storage were analyzed by RT-PCR and q RT-PCR. The results showed that the full length of this gene was 2 847 bp, containing a 2 619 bp open reading frame encoding 873 amino acids. Amino acid sequence analysis showed that Ac LOX1 has two domains of PLAT_LH2 (polyoxystin-1, lipoxygenase, alpha-toxin_lipoxygenase homology) and LOX. LOXs of Ac LOX1 and Euxalyptus grandis, Prunus dulcis, Vitis vinifera, Gene amino acid sequence homology as high as 66% to 73%. Phylogenetic tree analysis showed that Ac LOX1 clustered with 9-LOX in other species. The expression patterns of Ac LOX1 in onion tissues showed that the expression of Ac LOX1 gene increased with the passage of time from 5 to 25 days after pollination, but continued to increase slightly from 25 days after pollination to mature and stored for one year expression. This experiment initially confirmed that Ac LOX1 can promote seed development of onion and is related to seed aging, providing a theoretical basis for elucidating the mechanism of short life of onion seeds.