利用同源比对或RACE克隆了5个落叶松(Larix leptolepis) miRNA前体。结果显示, 在各物种miRNA前体间, 成熟序列高度相似, 但其它序列相似度差异大, 序列相似度与亲缘关系有关。采用qRT-PCR分析了5个miRNA、前体和靶基因在落叶松体胚8个发育阶段的表达变化。结果显示, miRNA表达最高峰出现在后期子叶胚, 暗示与促进胚胎休眠有关; 表达次高峰出现时期不同, 表现为miR397和miR408在PEMIII, miR398在早期单胚, miR156和miR166在早期子叶胚, 表明其与保持薄细胞壁、质子传递、顶端分生组织形成等调控有关。miRNA成熟体表达与前体含量不呈线性相关, 可能受多重调控。研究结果对于阐明MIR基因进化、表达调控及在体胚发育中的调控功能具有重要理论意义。
We used the homolog search strategy or RACE to obtain 5 precursor microRNAs (miRNAs) of larch, Larix leptolepis. Mature miRNAs shared high similarity among plant species, and non-mature sequences showed distinct similarity related to genetic relationships. Expression profiles of the 5 precursor and mature miRNAs were examined during 8 developmental stages with quantitative RT-PCR. The expression of precursor and mature miRNAs changed dynamically during developmental stages, with major peaks in expression for mature miRNAs in late cotyledonous embryos, which might be associated with promoting embryo dormancy. However, miR397 and miR408 showed minor peaks in expression at PEMIII, miR398 at the early single embryo stage, and miR156 and miR166 at the early cotyledonous embryo stage, which might be related to regulation to maintain a thin cell wall, proton transfer between photo systems, and formation of shoot meristem. However, the expression of mature miRNAs was not linearly associated with that of precursor miRNAs, which might be regulated at several levels. Our study sheds light on miRNA evolution, expression regulation of mature miRNAs, and regulation of somatic embryogenesis in larch.
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