High-level gene expression in differentiating xylem of tobacco driven by a 2.0 kb Poplar COMT2 promoter and a 4×35S enhancer

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Promoter constructs with high levels of xylem specific expression are needed to obtain efficient expression of candidate genes, microRNAs (miRNAs) and artificial microRNAs (amiRNAs) for the genetic modification of wood properties. The gene for caffeic acid O-methytransferase (PtrCOMT2) has the second most abundant transcript level of all the genes in monolignol biosynthesis in Populus trichocarpa and a high level of specificity in differentiating xylem. To characterize the PtrCOMT2 promoter, we cloned a short (2.0 kb) and a long (3.3 kb) promoter segment and compared their expression using GUS as a reporter gene in the differentiating xylem of Nicotiana tabacum. Both the 2.0 kb and the 3.3 kb promoter segments showed high specificity for differentiating xylem in this heterologous system. GUS activity increased as much as 5 times when the 4×35S enhancer was inserted in front of the 2.0 kb promoter, but GUS activity was only increased 2 times when the enhancer was inserted behind the promoter. The enhancer inserted upstream reduced the expression of the 3.3 kb promoter. While expression of some of the enhancer-plus-promoter constructs increased expression, there was a loss of specificity.