The SKP1 gene is an important component of the SCF (SKP1-Cullin1-F-box) complex and serves as a bridge connecting the F-box and Cullin1 genes (F-box-SKP1-Cullin1). The pattern of S-RNase being ubiquitously labelled by the SCF complex and degraded by the 26S protease accounts for the bulk of the available self-incompatibility studies. In this study, 15 ClSKP1s from the ‘Xiangshui’ lemon genome and ubiquitome exist in the same SKP1 conserved domain (CD) as SKP1s in other species. The qPCR results showed that SKP1-6 and SKP1-14 have tissue expression patterns specific for expression in pollen. In addition, SKP1-6 and SKP1-14 in the stigma, style and ovary were significantly upregulated after self-pollination compared to those after cross-pollination. A subcellular location showed that SKP1-6 and SKP1-14 were located in the nucleus. In addition, yeast two-hybrid (Y2H) assays, bimolecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI) assays showed that SKP1-6 interacted with F-box1, F-box33, F-box34, F-box17, F-box19, Cullin1-2 and 26S proteasome subunit 4 homolog A (26S PS4HA). SKP1-14 interacted with F-box17, F-box19, F-box35, Cullin1-2 and 26S PS4HA. The interaction of Cullin1-2 and the F-box with SKP1 as a bridge was verified by a yeast three-hybrid experiment. The ability of S3-RNase to inhibit pollen and pollen tube growth and development was assessed using in vitro pollen co-culture experiments with recombinant S3-RNase proteins. Overall, this study provides important experimental evidence and theoretical basis for understanding the mechanism of self-incompatibility in plants by revealing the key role of the SCF complex in ‘Xiangshui’ lemon, which is bridged by ClSKP1-6, in self-incompatibility. The results of this study are of great significance for the future in-depth exploration of the molecular mechanism of the SCF complex and its wide application in the self-incompatibility of plants.

中文翻译：

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S 期激酶结合蛋白 1 （ClSKP1） 家族的全基因组分析以及 SCF （Cullin1-SKP1-F-box） 复合物靶向 S-RNase 在 '香水' 柠檬自交不亲和中的作用


SKP1 基因是 SCF （SKP1-Cullin1-F-box） 复合体的重要组成部分，是连接 F-box 和 Cullin1 基因 （F-box-SKP1-Cullin1） 的桥梁。S-RNase 普遍被 SCF 复合物标记并被 26S 蛋白酶降解的模式占可用自交不亲和研究的大部分。在本研究中，来自'香水' 柠檬基因组和泛素组的 15 个 ClSKP1 与其他物种的 SKP1 存在于同一个 SKP1 保守结构域 （CD） 中。qPCR 结果显示 SKP1-6 和 SKP1-14 具有对花粉表达具有特异性的组织表达模式。此外，与异花授粉后相比，自花授粉后柱头、样式和子房中的 SKP1-6 和 SKP1-14 显著上调。亚细胞位置显示 SKP1-6 和 SKP1-14 位于细胞核中。此外，酵母双杂交 （Y2H） 测定、双分子荧光互补 （BiFC） 和荧光素酶互补成像 （LCI） 测定显示，SKP1-6 与 F-box1、F-box33、F-box34、F-box17、F-box19、Cullin1-2 和 26S 蛋白酶体亚基 4 同源物 A （26S PS4HA） 相互作用。SKP1-14 与 F-box17、F-box19、F-box35、Cullin1-2 和 26S PS4HA 交互。通过酵母三杂交实验验证了 Cullin1-2 和 F-box 与 SKP1 作为桥的相互作用。使用 重组 S3-RNase 蛋白的体外花粉共培养实验评估 S3-RNase 抑制花粉和花粉管生长和发育的能力。综上所述，本研究揭示了 '香水' 柠檬中由 ClSKP1-6 桥接的 SCF 复合物在自亲和中的关键作用，为理解植物自交不亲和机制提供了重要的实验证据和理论依据。 本研究结果对未来深入探索 SCF 复合物的分子机制及其在植物自亲和不亲和性中的广泛应用具有重要意义。