葡萄赤霉素合成相关基因克隆、亚细胞定位和表达分析

doi:10.3864/j.issn.0578-1752.2012.11.011

摘要/Abstract

摘要： 【目的】分离和克隆‘藤稔’葡萄内根-贝壳杉烯氧化酶基因（VvKO）、GA2-氧化酶基因（VvGA2ox2和VvGA2ox4）、GA3β-羟化酶基因（VvGA3ox4）和GA20-氧化酶基因（VvGA20ox1）等5个重要赤霉素合成相关基因的ORF序列，并对其进行亚细胞定位与表达分析。【方法】采用电子克隆方法克隆相关基因，构建亚细胞定位表达载体，基因枪转化洋葱表皮细胞后激光共聚焦显微镜下观察。并用半定量和荧光定量RT-PCR方法研究各基因的时空表达。【结果】成功克隆了5个基因的完整ORF序列，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，它们在组织器官间的表达有强弱差异。其中，VvGA2ox2主要在果实中表达，VvGA2ox4主要在叶片和果实中表达，而VvKO、VvGA3ox4和VvGA20ox1则在花、叶片和果实中均有一定的表达。VvKO、VvGA2ox2、VvGA2ox4和VvGA3ox4与GFP融合蛋白仅在核内产生绿色荧光；而VvGA20ox1与GFP融合蛋白在细胞核和细胞质膜上均产生绿色荧光信号。【结论】克隆的5个基因与葡萄的花、果实以及营养器官的发育存在不同程度的联系，其中4个基因仅呈现出细胞核内作用的特点，而VvGA20ox1则表现出细胞核和细胞质膜定位的现象。

关键词: 葡萄, 赤霉素, 亚细胞定位, 荧光定量RT-PCR, 基因表达分析

Abstract: 【Objective】The aim of this study was to isolate the open reading frame sequence of Vitis vinifera Ent-Kaurene oxidase gene (VvKO), Vitis vinifera GA2-oxidase genes (VvGA2ox2 and VvGA2ox4), Vitis vinifera GA3β-hydroxylase gene (VvGA3ox4) and Vitis vinifera GA20-oxidase gene (VvGA20ox1) from ‘Fujiminori’, and for some preliminary study on their functions and expression level. 【Method】Silico cloning was used to clone genes. Recombinant plasmid was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Green fluorescence was monitored under a laser scanning confocal microscope. The semi-quantitative and fluorescent quantitative RT-PCR methods were used to detect the spatial-temporal expression pattern of the 5 genes. 【Result】 Five genes related to the synthesis of gibberellins were successfully cloned. The expression results of the genes showed that there were some different expression levels in different tissues. VvGA2ox2 was only expressed in the fruit, VvGA2ox4 in leaf and fruit, and VvKO, VvGA3ox4 and VvGA20ox1 in flower, leaf and fruit. VvKO, VvGA2ox2, VvGA2ox4 and VvGA3ox4 combined with GFP were only located in nucleus of onion epidermal cell; however, VvGA20ox1 combined with GFP was located in both the plasma membrane and nucleus. 【Conclusion】All the 5 genes were involved in the development of both of reproductive and vegetative organs. Four of them only showed the nucleus location phenomena by combining with GFP, but the VvGA20ox1 showed a signal at nucleus and plasma membrane.

Key words: grapevine, gibberellin, sub-cellular localization, fluorescent quantitative RT-PCR, expression analysis of gene

王西成, 任国慧, 房经贵, 李阿英, 刘洪, 吴伟民, 赵密珍. 葡萄赤霉素合成相关基因克隆、亚细胞定位和表达分析[J]. 中国农业科学, 2012, 45(11): 2224-2231.

WANG Xi-Cheng, REN Guo-Hui, FANG Jing-Gui, LI A-Ying, LIU Hong, WU Wei-Min, ZHAO Mi-Zhen. Cloning, Subcellular Localization and Expression Analysis of Genes Related to the Synthesis of Gibberellin from Grapevine[J]. Scientia Agricultura Sinica, 2012, 45(11): 2224-2231.

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