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 Cullin1genes(F-box-SKP1-Cullin1).The pattern of S-RNase being ubiquitously labelled by the ...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 Cullin1genes(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 q PCR 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 indepth exploration of the molecular mechanism of the SCF complex and its wide application in the self-incompatibility of plants.展开更多
采用人子宫内膜癌Ishikawa细胞作为研究模型,通过CCK-8检测、克隆形成实验和流式细胞术评估季陵菜酸(PA)对细胞生长和凋亡的影响.JC-1染色及线粒体分离蛋白免疫印迹用于分析线粒体功能变化及细胞色素C氧化酶(cytochrome C oxidase, Cyt...采用人子宫内膜癌Ishikawa细胞作为研究模型,通过CCK-8检测、克隆形成实验和流式细胞术评估季陵菜酸(PA)对细胞生长和凋亡的影响.JC-1染色及线粒体分离蛋白免疫印迹用于分析线粒体功能变化及细胞色素C氧化酶(cytochrome C oxidase, Cyt C)的释放情况.免疫印迹检测用于分析SKP2及其下游调控因子p27、p21的表达变化.进一步利用SKP2过表达质粒验证其在PA作用中的功能.结果显示,PA处理可显著抑制Ishikawa细胞的增殖并降低克隆形成能力.FCM和JC-1染色提示PA能诱导线粒体膜电位丧失并促进凋亡.蛋白检测结果表明,PA处理导致胞质中Cyt C表达升高,同时线粒体中Cyt C含量下降,提示线粒体依赖性凋亡被激活.此外,PA明显下调了SKP2的表达,同时伴随p27与p21的上调.功能实验显示,SKP2的过表达可部分逆转PA的抑制作用,提示SKP2在其中发挥关键作用.研究结果表明,PA能够有效抑制EC细胞的增殖,并诱导线粒体途径介导的细胞凋亡.此外,发现PA能够下调S期的SKP2表达,提示其可能是PA发挥作用的新靶点.进一步研究显示,PA通过抑制SKP2,导致EC细胞周期阻滞并增强细胞凋亡.综上所述,这些结果证明了PA作为EC潜在新型治疗药物的重要性,并提示其可能通过调控线粒体凋亡和SKP2发挥作用,为临床提供新的分子靶向治疗思路.展开更多
基金supported by grants from the National Natural Science Foundation of China(Grant No.31960585)Science and Technology Major Project of Guangxi(Grant No.Guike AA22068092)+1 种基金Guangxi Science and Technology Vanguard Special Action Project(Grant No.202204)State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources(Grant Nos.SKLCUSA-a201906,SKLCU-SA-c201901)。
文摘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 Cullin1genes(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 q PCR 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 indepth exploration of the molecular mechanism of the SCF complex and its wide application in the self-incompatibility of plants.
