Cloning, Subcellular Localization and Expression Analysis of Genes Related to the Synthesis of Gibberellin from Grapevine

【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.