Sub-cellular localization and expression analysis of genes involved in grapevine floral development | 葡萄花发育基因的亚细胞定位和表达分析

Chino. 【目的】分离和克隆葡萄品种‘香悦’Vitis vinifera AGAMOUS (VvAG)、Vitis vinifera APETALA 3 (VvAP3)、Vitis vinifera FLOWERING LOCUS C (VvFLC)、Vitis vinifera FRUITFUL (VvFUL)、Vitis vinifera FLOWERING LOCUS T (VvFT)、Vitis vinifera APETALA2 (VvAP2)和Vitis vinifera SUPPRESSOR OF OVER EXPRESSION OF CO 1 (VvSOC1)7个重要花发育相关基因的ORF序列,并对其进行亚细胞定位与表达分析。【方法】采用特异引物RT-PCR方法克隆基因,并用半定量PCR法研究各基因在不同组织的表达,构建亚细胞定位载体,基因枪转化洋葱表皮细胞,25℃暗培养24 h后激光共聚焦显微镜下观察。【结果】对上述基因在葡萄不同组织器官中的表达水平进行分析发现,它们的表达不存在组织器官的特异性,但有表达强弱差异。其中,VvFT、VvFUL、VvAP3在葡萄幼果中表达量最高,VvAG与VvAP2在花中表达量最高,而VvSOC1与VvFLC在幼叶中表现出最高的表达水平。VvSOC1、VvFT、VvFLC和VvAP2与GFP融合蛋白仅在核内产生绿色荧光,表现出转录因子的典型特点;而VvAG、VvFUL和VvAP3与GFP融合蛋白在细胞质膜和细胞核上均产生绿色荧光信号。【结论】这7个基因与葡萄的花与果实以及营养器官的发育都存在相关性,7个葡萄花发育相关的转录因子都呈现出细胞核内作用的特点,但VvAG、VvFUL和VvAP3也表现出在细胞膜区域定位的现象。

Inglés. 【Objective】The aim of this study was to isolate the open reading frame sequence of Vitis vinifera AGAMOUS (VvAG), Vitis vinifera APETALA 3 (VvAP3), Vitis vinifera FLOWERING LOCUS C (VvFLC), Vitis vinifera FRUITFUL (VvFUL), Vitis vinifera FLOWERING LOCUS T (VvFT), Vitis vinifera APETALA2 (VvAP2) and Vitis vinifera SUPPRESSOR OF OVER EXPRESSION OF CO 1 (VvSOC1) from ‘Xiangyue’, one of the most popular table grape cultivars, and for some preliminary study on their functions. 【Method】 Specific primers RT-PCR method was used to clone genes, and semi-quantitative PCR was used to analyze the expression of genes in different organs and tissues. Recombinant plasmid was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 25℃ in the dark and green fluorescence was monitored under a Laser scanning confocal microscope. 【Result】The expression results of the genes in different tissues showed that they were expressed ubiquitously in all the organs and tissues, but the expression levels were some different. VvFT, VvFUL and VvAP3 were expressed highest in young fruit, VvAG and VvAP2 highest in flower, and VvSOC1 and VvFLC highest in young leaves. VvSOC1, VvFT, VvFLC and VvAP2 combined with GFP were only located in nucleus of onion epidermal cell, which showed typical characteristics of transcription factor; however, VvAG, VvFUL and VvAP3 combined with GFP were located in both the plasma membrane and nucleus. 【Conclusion】All these genes were involved in the developments both of reproductive and vegetative organs, and showed the nucleus location phenomena by combining with GFP except that VvAG, VvFUL, and VvAP3 showed signal at plasma membrane.