CaMV35S启动子在菊花中驱动GUS外源基因的表达分析

[目的]由于CaMV 35S启动子转化菊花存在外源基因表达量低、基因沉默等问题,本研究对35S启动子、2×35S启动子在菊花中驱动GUS外源基因的表达进行了分析,以期为高效菊花转化提供理论指导。[方法]通过设计引物,利用PCR从pCAMBIA1301 Vector中克隆了35S启动子、2×35S启动子的序列并插入pORE R2载体中,分别获得了重组载体pORE R2-35S:GUS和pORE R2-2×35S:GUS,通过农杆菌介导法将构建的遗传转化载体分别导入切花菊品种‘神马’中。[结果]试验共转化2 296个菊花叶盘,经过筛选共得到16个过量表达的转基因株系。经PCR验证,目标片段已经成功插入转基因株系基因组。荧光定量PCR分析和染色分析显示,在转基因菊花中2×35S比35S启动子表现出驱动GUS基因高量表达的特性。[结论]在选择合适菊花品种的基础上,2×35S比35S启动子可以更加高效地促进基因表达。
[Objectives]Due to the low expression or silencing of gene driven by CaMV 35S promoter in chrysanthemum,the 35S promoter,and 2×35S promoter-driven expression of GUS exogenous genes were analyzed in chrysanthemums in this study to provide theoretical basis for the efficient transformation of chrysanthemum. [Methods]The sequence of 35S promoter and 2×35S promoter were cloned from pCAMBIA1301 vector by using PCR,and then inserted into pORE R2 vector,and the following two recombinant vectors were obtained:pORE R2-35S:GUS and pORE R2-2×35S:GUS. The two constructs were introduced into the cut chrysanthemum‘Jinba’by the agrobacterium-mediated genetic transformation system,respectively. [Results]We have transformed 2 296 pieces of leaf-disc,and got 16 over-expressed transgenic lines. The target gene has been inserted into the chrysanthemum genome of the 16 transgenic lines as successfully detected by PCR,respectively. Real-time quantitative PCR(qRT-PCR)analysis was used to detect the function of promoter,and the results indicated that 2×35S promoter exhibited more obvious advantage for exogenous gene expression than 35S promoter in chrysanthemum. Also we can obtain the same conclusion in the former transgenic lines by histechemical evaluation of GUS expression. [Conclusions]All these data indicated that 2×35S promoter might drive gene expression more efficiently than 35S promoter in some chrysanthemum cultivars