龙眼果实发育过程中果糖激酶活性及其基因表达分析
Analyses of the fructokinase activity and its gene expression during the development of longan fruits
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摘要:目的 以石硖龙眼Dimocarpus longan cv.shixia为材料, 研究了龙眼果实在发育过程中假种皮蔗糖、葡萄糖和果糖含量的变化规律, 以及果糖激酶活性及其基因的表达变化情况.方法 利用高效液相色谱测定果实糖含量变化; RT-qPCR测定基因表达变化量; 构建原核表达载体, 并在大肠埃希菌Escherichia coli Rosetta(DE3)中进行表达.结果和结论 随着龙眼果实的发育, 假种皮中蔗糖、葡萄糖和果糖的含量逐渐增加, 但进入成熟期后, 糖含量水平趋于稳定; 在果实成熟前, 果糖激酶活性随着果实发育逐渐降低, 由发育最初的25.97 μmol·g-1 ·h-1下降到13.18 μmol·g-1·h-1, 而在成熟时增大到22.44 μmol·g-1·h-1; 荧光定量分析显示, 龙眼果糖激酶基因的表达量在果实发育前期变化不大, 而在假种皮迅速膨大、含糖量迅速上升时期则呈下降趋势; SDS-PAGE结果表明, 成功构建了pET-32a-DlFRK原核表达载体, 经IPTG诱导能够在大肠埃希菌中得到高效表达.Abstract:Objective Changes in the contents of sucrose, glucose and fructose, the activity of fructokinase (FRK) and its gene expression during the development of longan (Dimocarpus longan Lour.cv. Shixia) fruit aril were studied.Method Sugar contents were determinated using HPLC, and the FRK activity was analyzed using colorimetric method.Real-time qPCR was used to analyze the expression of DlFRK gene.Prokaryotic expression vector was constructed using in-fusion method to express DlFRK gene in Escherichia coli Rosetta (DE3).Result and conclusion The results showed that the contents of sucrose, glucose and fructose in the aril increased gradually with the development of longan fruits, and they tended to become stable at the mature stage.The fructokinase activity decreased gradually from 25.97 μmol·g-1·h-1to 13.18 μmol·g-1·h-1 at the early stages before maturation, and increased during maturation to 22.44 μmol·g-1·h-1 at full maturity.Quantitative RT-PCR analyses showed that the expression of fructokinase gene was relatively stable at the early stages, but it declined during the aril expanding period with a rapid sugar accumulation.The result of SDS-PAGE showed that pET-32a-DlFRK prokaryotic expression vector has been constructed.Gene expression has been successfully induced by IPTG in E.coil BL21.
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