Bi-directional Duplex Promoters with Duplicated Enhancers Significantly Increase Transgene Expression in Grape and Tobacco

Abstract

Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding beta-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.

Bidirectionalization of a methyl jasmonate-inducible plant promoter

“…This strategy was based on the hypothesis that, for many promoters, only the core promoter sequence needs to be oriented with respect to the coding sequence of the gene to ensure transcription in the correct direction, while the upstream promoter elements can be present in either orientation (Peremarti et al 2010). Previously, several studies also employed the same or a similar strategy to produce chimeric bidirectional promoters from unidirectional ones (Barfield and Pua 1991;Xie et al 2001;Li et al 2004;Chaturvedi et al 2006;Lv et al 2009), which provides precedence for the biotechnological application of this hypothesis.…”

“…In addition to the inducible activity of the selected promoter, it is also desirable to design a regulatory module that would express multigene traits in a coordinately regulated manner (Halpin 2005). One strategy is to modify a unidirectional promoter into a bidirectional one, as shown by several key studies (Barfield and Pua 1991;Xie et al 2001;Li et al 2004;Chaturvedi et al 2006;Lv et al 2009). …”

The intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes functions as a bidirectional promoter

“…Xie et al (2001) have demonstrated the essence and utility of an artiWcial bidirectional promoter derived from CaMV35S. Subsequently several researchers developed bidirectional promoters from existing unidirectional promoters or from artiWcially designed synthetic sequences using a similar approach (Frey et al 2001;Li et al 2004;Chaturvedi et al 2006;Zhang et al 2008).…”

Identification and Characterization of a T-DNA Promoter Trap Line of Arabidopsis thaliana Uncovers an Embryo Sac-Specific Bi-directional Promoter

“…Use of bi-directional promoter would ensure simultaneous expression of two genes and also halve the number of promoters needed for expressing transgenes (Li et al 2004) and thus minimize the amount of foreign DNA in transgenic plants. Further, homology-dependent gene silencing due to the repeated use of a promoter can be avoided through the use of bi-directional promoter.…”