Effect of T-DNA configuration on transgene expression.
T-DNA vectors were constructed which carry a beta-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3' end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for beta-glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3'-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.
Breyne
P
P
Laboratorium voor Genetica, Universiteit Gent, Belgium.
Gheysen
G
G
Jacobs
A
A
Van Montagu
M
M
Depicker
A
A
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics*; D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000704:Analysis of Variance; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014026:Nicotiana; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; C021903:D-octopine dehydrogenase; D005966:Glucuronidase