烟草牻牛儿基牻牛儿基焦磷酸合成酶基因NtGGPPS1的克隆和功能分析

Cloning and functional analysis of geranylgeranyl pyrophosphate synthase gene NtGGPPS1 from Nicotiana tabacum

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。

To further study the function of geranylgeranyl pyrophosphate synthase（GGPPS） gene from Nicotiana tabacum, NtGGPPS1 was successfully cloned from tobacco cultivar K326 using gene-specific primers which were designed according to the coding DNA sequence of GGPPS1（Gen Bank： GQ911583.1） from Nicotiana tabacum. Fluorescent expression vector of NtGGPPS1（PFF19-NtGGPPS1-GFP） was constructed for subcellular localization in tobacco. After treated with different phytohormones, the expression level of NtGGPPS1 in K326 was studied by real-time PCR. RNAi vector p Hellsgate2-NtGGPPS1 was introduced into K326 viaAgrobacterium-mediated transformation and the contents of plastid pigments in NtGGPPS1 down-regulated tobacco plants were detected. The results showed that green fluorescence protein NtGGPPS1-GFP fusion was distributed in the plastids of transgenic BY-2 cells, suggesting that NtGGPPS1 located in plastids. The expression level of NtGGPPS1 in K326 was significantly increased after Me JA treatment, while it was contrary to IAA treatment. Comparing with the control, the contents of plastid pigments significantly decreased in NtGGPPS1 down-regulated plants, which indicated that NtGGPPS1 was involved in the biosynthesis ofβ-carotene in Nicotiana tabacum.