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为进一步明确烟草牻牛儿基牻牛儿基焦磷酸合成酶基因NtGGPPS1的功能,根据已公布的普通烟草(Nicotiana tabacum)GGPPS1基因编码区序列设计特异性引物,从普通烟草K326中克隆到NtGGPPS1基因。构建荧光表达载体PFF19-Nt GGPPS1-GFP,对NtGGPPS1基因进行亚细胞定位研究。通过荧光定量PCR分析了普通烟草K326在不同激素处理下NtGGPPS1基因的表达量变化。构建NtGGPPS1基因的RNAi载体pHellsgate2-Nt GGPPS1,通过农杆菌浸染烟草K326后获得NtGGPPS1基因显著下调的烟株,检测了烟株中质体色素含量的变化情况。结果表明:Nt GGPPS1融合蛋白基因的绿色荧光分布在烟草BY-2细胞的质体上,表明该基因定位于质体。用茉莉酸甲酯(MeJA)处理烟草后,NtGGPPS1基因的表达量显著升高,而用生长素(IAA)处理后,该基因的表达量下降。与对照相比,在NtGGPPS1基因下调的烟株中其质体色素含量均显著降低,预示NtGGPPS1参与了烟草β-胡萝卜素的生物合成。
In order to further clarify the function of tobacco geranylgeranyl diphosphate synthase gene NtGGPPS1, a specific primer was designed according to the published Nicotiana tabacum GGPPS1 gene coding region sequence, and the NtGGPPS1 gene was cloned from Nicotiana tabacum K326 . A fluorescent expression vector PFF19-Nt GGPPS1-GFP was constructed to sub-locate the NtGGPPS1 gene. The expression of NtGGPPS1 gene in Nicotiana tabacum K326 was analyzed by fluorescence quantitative PCR. The RNAi vector pHellsgate2-Nt GGPPS1 of NtGGPPS1 gene was constructed. After tobacco K326 was infected with Agrobacterium tumefaciens, the tobacco plants significantly down-regulated in NtGGPPS1 gene were obtained, and the change of plastid pigment content in tobacco plants was detected. The results showed that the green fluorescence of Nt GGPPS1 fusion protein was distributed on the plastids of tobacco BY-2 cells, indicating that the gene was located on the plastid. The expression of NtGGPPS1 gene was significantly increased after treated with methyl jasmonate (MeJA), while decreased with IAA treatment. Compared with the control, the plastid pigment content of the tobacco plants down-regulated by the NtGGPPS1 gene was significantly reduced, indicating that NtGGPPS1 is involved in the β-carotene biosynthesis.