• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

龙眼DlAGOMEL1基因的克隆表达及亚细胞定位分析

Cloning, Expression and Subcellular Localization of DlAGOMEL1 from Longan

  • 摘要:
      目的  探究DlAGOMEL1基因在龙眼体胚发生过程中的调控作用,并明确该基因在亚细胞中的定位,为深入研究DlAGOMEL1基因的功能提供理论依据。
      方法  采用RT-PCR技术,基于龙眼基因组数据库,克隆DlAGOMEL1基因的CDS及该基因的5′端上游启动子序列,采用生物信息学方法分析该基因,通过实时荧光定量PCR技术检测其在龙眼体胚不同发育阶段的相对表达量,同时,构建该基因的瞬时融合表达载体,得到重组质粒,并将其瞬时转入洋葱内表皮细胞中,用荧光共聚焦显微镜观察细胞绿色荧光信号。
      结果  获得DlAGOMEL1基因CDS全长序列及5′端上游启动子序列长度分别为2655 bp、1512 bp,该基因编码884个氨基酸;系统进化树分析表明,龙眼DlAGOMEL1蛋白与胡杨亲缘关系最近,与单子叶植物水稻、玉米等亲缘关系最远;实时荧光定量分析表明,该基因在龙眼体胚不同阶段均表达,在子叶胚时期(CE)的相对表达量较高,而在球形胚时期(GE)的相对表达量最低。亚细胞定位结果发现,DlAGOMEL1基因定位于细胞质中。
      结论  DlAGOMEL1基因在龙眼体胚阶段的相对表达量呈“V”字型,可能主要参与龙眼晚期体胚发生的转录调控过程,在细胞质中发挥主要的功能作用。

     

    Abstract:
      Objective  Still unclear regulatory functions of DlAGOMEL1 on the nonembryonic callus and different embryogenic cultures of longan (Dimocarpus longan) were investigated.
      Method  From the longan genome database, the full-length cDNA sequences (CDS) of DlAGOMEL1 and its promoter were obtained using RT-PCR. Bioinformatics of this gene was analyzed and the relative expressions of this gene in embryogenic cultures were detected by real-time quantitative PCR. The recombinant plasmid was constructed from the transient fusion expression vector, and the onion inner epidermal cells transformed into transient onion cells. Green fluorescence signal of the cells was searched under a fluorescence confocal microscope.
      Result  The CDS of DlAGOMEL1 was 2,655 bp which encoded 884 amino acids and the promoter was 1,512 bp. The DlAGOMEL1 of longan and other plants were highly conserved as shown in a multiple sequences analysis. Phylogenetic tree of the protein indicated its close relation with Populars euphonium but not with monocotyledonous plants, such as rice and maize. The gene expressed differently in somatic embryo development stages—most strongly at cotyledon embryo stage and least at spherical embryo stage. The subcellular localization of DlAGOMEL1 was found in cytoplasm.
      Conclusion  Since the relative expression of DlAGOMEL1 was shown to be high at the late stage of longan somatic embryo, the gene might be functionally active in the late stage of the development. The revealed subcellular localization of the gene seemed to predispose its involvement in the cytoplasm of longan.

     

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