摘要
将NtGGPPS1-T质粒和spCAMBIA1300载体分别双酶切(SalⅠ和KpnⅠ)后连接,转化,挑取单克隆,筛选得到植物超量表达载体spCAMBIA1300-NtGGPPS1,并通过农杆菌介导侵染栽培烟草K326,在获得阳性转基因烟草后,进行定量RT-PCR以定量分析烟草牻牛儿基牻牛儿基焦磷酸合成酶基因NtGGPPS1在转基因植株中的转录积累.同时,通过LC-MS和GC-MS检测转基因植株中质体色素和萜类化合物含量的变化.结果显示,与对照组相比,NtGGPPS1基因在编号为Nt1-OE1、2、3、4、7植株中表达上调,阳性转基因烟草中新黄质、紫黄质、叶黄素、叶绿素a、叶绿素b和β-胡萝卜素6种质体色素,以及烟叶中代表性二萜类物质α-西柏三烯二醇(α-CBD)和β-西柏三烯二醇(β-CBD)的含量明显增加.以上结果表明,NtGGPPS1基因参与了烟草中质体色素和二萜类化合物的生物合成.
NtGGPPS1-T and spCAMBIA1300 were similarly digested with SalⅠand KpnⅠ,ligated with T4 DNA ligase and then transformed into competent cells to construct overexpression vector of spCAMBIA1300-NtGGPPS1.The vector was transferred into K326 by agrobacterium-mediated transformation method.After transgenic tobacco plants had been obtained,quantitative RT-PCR was conducted to quantify the transcript accumulation of NtGGPPS1 in the plants.The contents ofα-andβ-cembrenediol and plastid pigments in NtGGPPS1 up-regulated plants were detected by LC-MS and GC-MS.The results clearly show that the expression level of NtGGPPS1 is increased in the leaves of transgenic lines numbered as Nt1-OE1,2,3,4 and 7.Compared with the control,the contents ofα-andβ-cembrenediol,neoxanthin,violaxanthin,lutein,chlorophyll a,chlorophyll b andβ-carotene increase significantly in transgenic plants.This indicates that NtGGPPS1 is involved in the biosynthesis ofα-andβ-cembrenediol and carotenoids in tobacco.
引文
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