Laboratory of Cell & Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou 310058, China.
Cell Mol Biol Lett. 2013 Sep;18(3):416-32. doi: 10.2478/s11658-013-0097-9. Epub 2013 Jul 17.
microRNAs (miRNAs) are a class of newly identified, noncoding, small RNA molecules that negatively regulate gene expression. Many miRNAs are reportedly involved in plant growth, development and stress response processes. However, their roles in the sexual reproduction mechanisms in flowering plants remain unknown. Pollen development is an important process in the life cycle of a flowering plant, and it is closely related to the yield and quality of crop seeds. This study aimed to identify miRNAs involved in pollen development. A microarray assay was conducted using the known complementary sequences of plant miRNAs as probes on inflorescences of a sterile male line (Bcajh97-01A) and a fertile male line (Bcajh97-01B) of the Brassica campestris ssp. chinensis cv. 'Aijiaohuang' genic male sterility sister line system (Bcajh97-01A/B). The results showed that 44 miRNAs were differently expressed in the two lines. Of these, 15 had over 1.5-fold changes in their transcript levels, with 9 upregulated and 6 downregulated miRNAs in inflorescences of 'Bcajh97-01A' sterile line plants. We then focused on 3 of these 15 miRNAs (miR158, miR168 and miR172). Through computational methods, 13 family members were predicted for these 3 miRNAs and 22 genes were predicted to be their candidate target genes. By using 5' modified RACE, 2 target genes of miR168 and 5 target genes of miR172 were identified. Then, qRT-PCR was applied to verify the existence and expression patterns of the 3 miRNAs in the flower buds at five developmental stages. The results were generally consistent with those of the microarray. Thus, this study may give a valuable clue for further exploring the miRNA group that may function during pollen development.
microRNAs (miRNAs) 是一类新发现的非编码、小 RNA 分子，它们通过负调控基因表达来发挥作用。许多 miRNA 被报道参与植物的生长、发育和应激反应过程。然而，它们在有花植物的有性生殖机制中的作用尚不清楚。花粉发育是有花植物生命周期中的一个重要过程，它与作物种子的产量和质量密切相关。本研究旨在鉴定参与花粉发育的 miRNA。使用植物 miRNA 的已知互补序列作为探针，通过 microarray 分析，对甘蓝型油菜无功能雄性不育系（Bcajh97-01A）和可育系（Bcajh97-01B）的花序进行检测。结果表明，在这两个系中，有 44 个 miRNA 表达水平存在差异。其中，15 个 miRNA 的转录水平变化超过 1.5 倍，在‘Bcajh97-01A’不育系植物的花序中，有 9 个上调和 6 个下调的 miRNA。然后，我们将重点放在这 15 个 miRNA 中的 3 个（miR158、miR168 和 miR172）上。通过计算方法，预测到这 3 个 miRNA 的 13 个家族成员和 22 个候选靶基因。通过使用 5' 修饰 RACE，鉴定到 miR168 的 2 个靶基因和 miR172 的 5 个靶基因。然后，通过 qRT-PCR 验证了这 3 个 miRNA 在五个发育阶段的花蕾中的存在和表达模式。结果与 microarray 的结果基本一致。因此，本研究可能为进一步探索可能在花粉发育过程中发挥作用的 miRNA 组提供有价值的线索。
