叶面喷施尿素对葡萄氮代谢相关基因表达的影响

本研究利用VitisEST数据库中EST序列片段结合RT-PCR方法克隆了与氮代谢相关的硝酸盐还原酶（NR）,亚硝酸还原酶（NiR）,谷氨酰胺合成酶（GS）,谷氨酸脱氢酶（GDH）和天冬酰胺合成酶（AS）的基因,并对它们进行亚细胞定位分析。同时,采用半定量RT-PCR和荧光定量PCR法研究葡萄叶面喷施不同浓度尿素后5个基因的表达变化。结果表明,5个基因在葡萄幼叶中的表达水平显著高于老叶,说明叶面喷施尿素以喷布到幼叶对5个基因的表达水平影响显著;5个基因在不同时间段的表达水平差异不一致,但表达水平均高于对照,以0.3%和0.5%浓度的尿素对其影响明显;适当提高尿素水平既能提高氮代谢基因的表达水平,又能提高果实大小等果实指标,使其达到同步增加。喷施尿素6 h后,GS的表达量上升幅度明显高于其它基因,而NR在喷施尿素后48 h内一直保持着比较高的表达水平;NiR、AS表达量的变化趋势分别与NR、GS相一致,并且NR﹥NiR,GS﹥AS。

Effects of foliar applied urea on expression of genes related to nitrogen metabolism in Fujiminori grapevine

Five important genes coding for nitrate reductase(NR),nitrite reductase(NiR),glutamine synthetase(GS),glutamate dehydrogenase(GDH) and asparagine synthetase(AS),were cloned from the related EST sequences deposited in VitisEST database and amplified using RT-PCR.Sub-cellular localization was employed to characterize their functional position,while semi-quantitative RT-PCR and fluorescent quantitative PCR were both used to analyze their expression in leaves after treatment with urea at different concentrations.Results show that the five genes had significantly higher expression in younger leaves compared to old leaves.The differences in expression of these five genes at different times were inconsistent,but generally expression was increased by application of foliar urea,with application of 0.3% and 0.5% urea causing significant increases in gene expression compared to control plants.The expression of the five genes related to foliar fertilizer application was corroborated by the significant increases in related fruit indicators.Expression of GS in grape leaves had the most significant increase at 6 h after spraying of urea,while NR maintained a relatively high level of expression even at 48h after urea application compared to the other genes.NR and GS expression in various stages was higher than NiR and AS respectively,with the trends in expression of NiR and AS being consistent with those of NR and GS respectively.This study provides a theoretical basis for further studies on the mechanisms of nitrogen metabolic pathways and scientifically informed foliar fertilizer application.