Grapevine (Vitis vinifera L.), which has important nutritional values and health benefits, is one of the most economically important fruit crops cultivated worldwide. Several studies showed a large number of microRNAs (VvmiRNAs) involved in the modulation of grape growth and development, and many VvmiRNA families have multiple members. However, the way by which various members from the same miRNA family work is unclear, particularly in grapes. In this study, an important conserved VvmiR172 family (VvmiR172s) and their targets were set as a good example for elucidating the interaction degree, mechanism, and spatio-temporal traits of diverse members from the same miRNA family. miR-RACE and Stem-loop RT-PCR were used to identify the spatio-temporal expressions of various members of VvmiR172s; together with RLM-RACE, PPM-RACE, Western blot, transgenic technologies, their interaction degree, and regulation mechanism were further validated. The expression of VvmiR172c was significantly higher than that of VvmiR172a, b, and d and showed a positive correlation with the abundance of VvAP2 cleavage products. These findings indicated that VvmiR172c might be one of the main action factors of the VvmiR172 family in flower development. The ability of VvmiR172c to cleave target genes differed due to divergence in complementary degree with VvAP2 and expression levels of various members. In VvmiR172 transgenic lines, we observed that 35S::VvmiR172c resulted in the earliest and abundant flowering, indicating the strong function of VvmiR172c. In contrast, the non-significant phenotypic changes were detected in the VvAP2 transgenic lines. The qRT-PCR and Western bolt results demonstrated that VvmiR172c plays a major role in targeting VvAP2. VvmiR172 up-regulated the expression of NtFT and decreased the expression of NtFLC. The up/down regulation of VvmiR172c was the most pronounced. The functions of four VvmiR172 members in grape differed, and miR172c had the strongest regulation on AP2.

中文翻译：

---

葡萄花发育过程中 microRNA172 家族成员调控机制的表征。


葡萄（Vitis vinifera L.）具有重要的营养价值和健康益处，是全世界种植的最具经济重要性的水果作物之一。多项研究表明大量的microRNA（VvmiRNA）参与葡萄生长发育的调节，并且许多VvmiRNA家族有多个成员。然而，来自同一 miRNA 家族的不同成员的工作方式尚不清楚，特别是在葡萄中。在这项研究中，一个重要的保守VvmiR172家族（VvmiR172s）及其靶点被设定为阐明同一miRNA家族不同成员之间相互作用程度、机制和时空特征的一个很好的例子。利用miR-RACE和Stem-loop RT-PCR鉴定VvmiR172s各成员的时空表达；与RLM-RACE、PPM-RACE、Western blot、转基因技术一起进一步验证了它们的相互作用程度和调控机制。 VvmiR172c的表达显着高于VvmiR172a、b和d，并且与VvAP2裂解产物的丰度呈正相关。这些发现表明VvmiR172c可能是VvmiR172家族在花卉发育中的主要作用因子之一。由于与VvAP2的互补程度和各成员表达水平的差异，VvmiR172c切割靶基因的能力有所不同。在 VvmiR172 转基因株系中，我们观察到 35S::VvmiR172c 导致最早且丰富的开花，表明 VvmiR172c 的强大功能。相反，在 VvAP2 转基因品系中检测到不显着的表型变化。 qRT-PCR 和 Western blot 结果表明，VvmiR172c 在靶向 VvAP2 中发挥着重要作用。 VvmiR172上调NtFT的表达并降低NtFLC的表达。 VvmiR172c 的上/下调调节最为明显。葡萄中VvmiR172的4个成员的功能存在差异，其中miR172c对AP2的调节作用最强。