花生多胺氧化酶基因的克隆与鉴定(英文)

Molecular Cloning and Characterization of a Putative Polyamine Oxidase Gene from Arachis hypogaea

多胺作为一类新型植物生理活性物质,对植物生长发育起着重要作用,在细胞工程与农业生产中具有广阔的应用价值。多胺氧化酶(Polyamine oxidase,PAO)是催化多胺降解的关键酶之一,对调控植物细胞中多胺浓度非常重要,影响植物的生长发育、形态建成过程。根据国际DNA数据库中已报道的PAO蛋白的保守结构域,设计简并引物(degenerate pri mer),通过RT-PCR方法首次从花生(仲恺花1号)中克隆PAO基因cDNA序列,并利用BLASTn、BLASTp及多重序列排比(Multiple Sequence Alignment)等生物信息学方法对所克隆的cDNA进行序列分析与鉴定,初步确定已从花生中克隆到PAO基因的同系物(homolog)。根据克隆的花生PAOcDNA序列(AhPAO1)设计特异引物,通过半定量RT-PCR方法检测花生胚根、胚芽和幼叶中AhPAO1基因的表达,结果发现胚根中AhPAO1基因表达最强,胚芽和幼叶中的表达无明显差异。本研究为进一步了解花生种子萌发和幼苗生长发育过程中多胺氧化酶基因表达的时空特异性奠定实验基础,并对进一步利用植物基因工程技术综合调控花生体内多胺水平有重要实践意义。

Polyamines are being considered as novel active substances with great significance to plant growth and development and play important potential role in application to cell engineering and agriculture production. Polyamine oxidase （PAO） is one of the key enzymes which catalyze the degradation of polyamines. PAO is of great importance to regulation of polyamines concentration in plant cells and influences growth, development and morphogenesis of plants. In the present study, based on the conserved regions of reported PAO proteins in GenBank database, two degenerate primers were designed to amplify PAO cDNA homolog through reverse transcription polymerase chain reaction （RT-PCR） from peanut （Arachis hypogaea cv Zhongkaihua No. 1）. The cloned PAO cDNA, designated as AhPAO1, was characterized and confirmed by BLASTn, BLASTp and Multiple Sequence Alignment analysis bioinformatically, showing that AhPAO1 is the cDNA encoding a putative PAO of peanut plants. Semi-quantitative RT-PCR with two gene-specific primers was performed to investigate the organ specific expression pattern of AhPAO1 gene in radicles, plumules and young leaves of peanut seedlings. The results showed that the expression of AhPAO1 gene in peanut radicles was significantly stronger than that in plumules and young leaves. This investigation provides experimental foundations for further insight into the temporal and spatial expression patterns of AhPAO1 gene during seed germination and seedling development of peanut plants, and for regulation of polyamines level in peanut plants through transgenic plant engineering.