Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/14/2007
Publication Date: 8/14/2007
Citation: Bassett, C.L., Callahan, A.M., Artlip, T.S., Scorza, R., Srinivasan, C. 2007. A minimal peach type II chlorophyll a/b-binding protein promoter retains tissue-specificity and light regulation in tomato. BioMed Central (BMC)Biotechnology. 7:47. Interpretive Summary: The first generation of vectors used in the genetic engineering of agricultural crops promoted expression of the foreign gene in all plant tissues and during most stages of plant development. For the second generation vectors, it is desirable to regulate expression of the inserted gene more precisely in specific tissues/organs and/or during specific stages of development. To obtain precise tissue and developmental expression of a foreign gene, regions of the DNA (promoters) which specify precision expression must be identified, isolated, and used in the new vectors. We describe the isolation of a photosynthetic gene promoter (Cab promoter) which allows expression of attached genes in green tissues, like leaves, stems and young fruit, but which drastically reduces expression in roots and mature fruit. The expression of this promoter from peach was tested in tomato using a reporter gene whose expression was easy to follow. Reporter gene activity was highest in leaves and young (green) fruit, and lowest in roots and mature (red) tomato fruit. Therefore, genes expressed under the control of the peach Cab promoter can be used to express genes in leaves conferring resistance to certain diseases and pests without having these genes expressed in the edible roots or fruits of the engineered plant.
Technical Abstract: A minimal peach chlorophyll a/b-binding protein gene (Lhcb2*Pp1) promoter (Cab19) was isolated and fused to a uidA (GUS) gene containing the PIV intron. A control construct carrying an enhanced mas35S CaMV promoter fused to uidA was also constructed. Two different orientations of the Cab19GUS fusion, relative to the right and left borders of the binary vector, were transformed into tomato cv Ailsa Craig and selected on kanamycin. Regenerated transformants testing positive for the presence of GUS or the peach Cab19 promoter were characterized as to their expression patterns in different tomato organs. Ten lines of each construct and an untransformed control line were assessed both qualitatively and quantitatively for GUS expression in leaves and flowers, and quantitatively in roots. Both types of assays indicated higher GUS expression from the mas35S promoter than the Cab19 promoter. Although GUS levels were readily detectable in flowers and roots of mas35S transgenic plants, little activity was observed in plants carrying the Cab19 promoter constructs. GUS activity in transgenic green fruit was relatively high for all promoter constructs; however, in red, ripe fruit, activities were on the whole lower for the Cab19 promoter constructs than the mas35S. GUS activity from transgenic mas35SGUS seedlings exposed to light or kept in the dark was similar among eight lines tested, whereas, activity in dark-grown Cab19GUS seedlings was undetectable. These results indicate that the minimal Cab19 promoter retains both tissue-specificity and light regulation.