Kalosaka K, Chrysanthis G, Rojas-Gill A-P, Theodoraki M, Gourzi P, Kyriakopoulos A, Tatari M, Zacharopoulou A, Mintzas A C
Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, 26 500 Patras, Greece.
Insect Mol Biol. 2006 Jun;15(3):373-82. doi: 10.1111/j.1365-2583.2006.00650.x.
The promoter of the hsp70 gene of Drosophila melanogaster has been widely used for the expression of foreign genes in other insects. It has been generally assumed that because this gene is highly conserved, its promoter will function efficiently in other species. We report the results of a quantitative comparison of the activities of the medfly and D. melanogaster hsp70 promoters in vivo in transformed medflies. We constructed transformed lines containing the lacZ reporter gene under the control of the two promoters by using Minos-mediated germ-line transformation. The activity of each promoter was evaluated in 15 transformed lines by beta-galactosidase quantitative assays. The heat-inducible activity of the medfly promoter was found several times higher than the respective activity of the heterologous D. melanogaster promoter. These results were confirmed by northern blot analysis and indicate that the D. melanogaster promoter does not work efficiently in medfly. The -263/+105 medfly promoter region that was used in this study was found able to drive heat shock expression of the lacZ reporter gene in all stages of medfly, except early embryonic stages, in a similar fashion to the endogenous hsp70 genes. However the heat inducible RNA levels driven from this promoter region were significantly lower than the endogenous hsp70 RNA levels, suggesting that additional upstream and/or downstream sequences to the -263/+105 region may be necessary for optimum function of the medfly hsp70 promoter in vivo.
果蝇热休克蛋白70(hsp70)基因的启动子已被广泛用于在其他昆虫中表达外源基因。一般认为,由于该基因高度保守,其启动子在其他物种中也能高效发挥作用。我们报告了在转化的地中海实蝇体内对地中海实蝇和果蝇hsp70启动子活性进行定量比较的结果。我们通过minos介导的种系转化构建了在这两种启动子控制下含有lacZ报告基因的转化品系。通过β-半乳糖苷酶定量测定法在15个转化品系中评估了每个启动子的活性。发现地中海实蝇启动子的热诱导活性比异源果蝇启动子的相应活性高几倍。这些结果通过Northern印迹分析得到证实,表明果蝇启动子在地中海实蝇中不能有效地发挥作用。本研究中使用的-263/+105地中海实蝇启动子区域能够以与内源性hsp70基因相似的方式驱动lacZ报告基因在除早期胚胎阶段外的地中海实蝇所有阶段的热休克表达。然而,由该启动子区域驱动的热诱导RNA水平明显低于内源性hsp70 RNA水平,这表明对于地中海实蝇hsp70启动子在体内的最佳功能,可能需要-263/+105区域上游和/或下游的其他序列。