芒果核苷二磷酸激酶(NDPK)基因克隆及表达分析

Molecular Cloning of Mango NDPK1 Gene and Its Expression Analysis

采用3′RACE和RT-PCR技术克隆芒果核苷二磷酸激酶基因全长c DNA序列,并用实时荧光定量PCR分析该基因的表达模式。结果表明:Mi NDPK1基因c DNA全长1 019 bp,开放阅读框为447 bp,编码148个氨基酸。Mi NDPK1蛋白定位于细胞质,无信号肽和跨膜区域,具有典型的核苷二磷酸激酶活性结构域和其他磷酸化活性位点;与麻疯树、橡胶树同源性最高为89%,与菠菜同源性最低为82%。Mi NDPK1在芒果各组织中均有表达,以叶片中表达最高,其次是花和果皮;在芒果进入生殖时期的花芽分化期表达量最高,此后在芒果果实发育进程中逐渐降低并趋于平稳。结合病害处理转录组样本中的差异性表达结果,推测芒果Mi NDPK1基因在花芽分化进程、抗病性诱导及免疫应答通路上发挥重要作用。

In this study, the full leng th of NDPK1 cDNA was obtained by 3′ RACE and RT-PCR, and its expression was analyzed by quantitative real-time PCR. The results showed that MiNDPK1 was consisted of 1 019 bp by with an open reading frame of 447 bp, encoding a polypeptide of 148 amino acids. The MiNDPK1 protein located in the cytoplasm and no signal peptide and transmembrane regions. Furthermore, MiNDPK1% contained a typical nucleoside diphosphate kinase activity domain and other phosphorylation activity sites. Sequences and alignment showed that the homology of amino acid sequence between mango and Jatropha curcas or Hevea brasiliensis NDPK1 was as high as 89%, however the homology with Spinacia oleracea was the lowest with 82%.MiNDPK1 was expressed in all tested tissues, with the highest expression in leaves, followed by flowers and fruits.There were the highest expression in the period of flower bud differentiation stage, after gradually reduced and leveled off in the process of mango fruit development. Combined with the result of transcriptome expression by the disease treatment, we speculate that the MiNDPK1 gene play an important role in the process of flower bud differentiation, induced disease resistance and immune response pathways.