Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes.
The constitutive promoter of cauliflower mosaic virus 35S (CaMV 35S) is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5' end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA (beta-glucuronidase, GUS) and gfp (green fluorescent protein, GFP), were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco (Nicotiana tabacum L.) was visualized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from 8 to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 microg/mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.
Zhang
Chunxiao
C
College of Bioscience and Bioengineering, Hebei University of Science & Technology, Shijiazhuang 050018, China.
Gai
Ying
Y
Wang
Wenqi
W
Zhu
Yanyan
Y
Chen
Xuemei
X
Jiang
Xiangning
X
D016960:Agrobacterium tumefaciens / Q000235:genetics; D019677:Artificial Gene Fusion; D017930:Genes, Reporter / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014170:Transformation, Genetic
D049452:Green Fluorescent Proteins; D005966:Glucuronidase