Data from: Study on expression modes and cleavage role of miR156b/c/d and its target gene Vv-SPL9 during the whole growth stage of grapevine

miR156 regulates the expression of its target SPL (PROMOTER BINDING-LIKE) genes during flower and fruit development, diverse developmental stage transitions, especially from vegetative to reproductive growth phases, by cleaving the target mRNA SPL of one plant-specific transcription factor. However, systematic reports on grapevine have yet to be presented. Here, the precise sequence of miR156 (vvi-miR156b/c/d) in grapevine “Takatsuma” was cloned with a previously cloned grapevine SPL (Vv-SPL9). Expression profiles in 18 grapevine tissues were identified through stem-loop RT-PCR. The interaction mode between vvi-miR156b/c/d and Vv-SPL9 was further validated by detecting the cleavage site and cleavage products of 3′- and 5′-ends via an integrated approach of 5′-RLM-RACE (RNA ligase-mediated 5′-rapid amplification of cDNA ends), 3′-PPM-RACE (poly(A) polymerase-mediated 3′-rapid amplification of cDNA ends), and qRT-PCR (real time reverse transcriptase-polymerase chain reaction). The variation in their cleavage roles in the whole growth stage of grapevine was also systematically investigated. Results showed that vvi-miR156b/c/d exhibited typical temporal–spatial-specific expression levels. The expression levels were higher in vegetative organs, such as leaf, than in reproductive organs, such as tendrils, flowers, and berries. A significant variation was observed during vegetative-to-reproductive transition. The expression patterns of Vv-SPL9 showed the opposite trends with those of vvi-miR156b. We confirmed that the cleavage site was at the 10th site of vvi-miR156b/c/d complementary to Vv-SPL9 in “Takatsuma” grapevine. We also identified the temporal–spatial variation of the cleavage products. This variation can indicate the regulatory function of miR156 on SPL in grapevines. Our findings provide further insights into the functions of vvi-miR156b/c/d and its target Vv-SPL9, and also help enrich our knowledge of small RNA-mediated regulation in grapevine.

miR156可在植物花与果实发育、多种发育阶段转变过程中，尤其是从营养生长向生殖生长阶段转换时，通过切割一类植物特异性转录因子的靶mRNA SPL（SQUAMOSA启动子结合蛋白样，SQUAMOSA PROMOTER BINDING-LIKE）基因，调控其靶标SPL基因的表达。然而，目前尚未有针对葡萄的系统性研究报道。本研究以葡萄品种“高妻（Takatsuma）”为材料，克隆得到该品种中miR156的精确序列（vvi-miR156b/c/d），并结合此前已克隆获得的葡萄SPL基因Vv-SPL9开展后续实验。通过茎环反转录聚合酶链式反应（stem-loop RT-PCR），本研究检测并分析了18种葡萄组织的表达谱。本研究通过整合5'-RLM-RACE（RNA连接酶介导的5'cDNA末端快速扩增，RNA ligase-mediated 5′-rapid amplification of cDNA ends）、3'-PPM-RACE（poly(A)聚合酶介导的3'cDNA末端快速扩增，poly(A) polymerase-mediated 3′-rapid amplification of cDNA ends）以及qRT-PCR（实时反转录聚合酶链式反应，real time reverse transcriptase-polymerase chain reaction）的技术手段，检测靶mRNA的3'和5'端切割位点及切割产物，进一步验证了vvi-miR156b/c/d与Vv-SPL9之间的互作模式。本研究还系统探究了二者在葡萄整个生长周期中的切割作用变化规律。结果显示，vvi-miR156b/c/d呈现典型的时空特异性表达特征，其在叶片等营养器官中的表达量显著高于卷须、花与浆果等生殖器官，且在营养生长向生殖生长的转变阶段存在显著差异。Vv-SPL9的表达模式与vvi-miR156b呈现完全相反的趋势。本研究证实，在“高妻”葡萄中，vvi-miR156b/c/d与Vv-SPL9的互补结合位点的第10位即为切割位点。本研究同时明确了切割产物的时空分布变化，该变化可直观反映miR156对葡萄SPL基因的调控功能。本研究结果不仅为深入解析vvi-miR156b/c/d及其靶基因Vv-SPL9的功能提供了新的理论参考，也为丰富葡萄中小RNA介导的基因调控机制研究提供了重要依据。