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Title: A COMPARISON OF CONSTITUTIVE PROMOTERS FOR EXPRESSION OF TRANSGENES IN ALFALFA (MEDICAGO SATIVA)

Author
item Samac, Deborah - Debby
item DORNBUSCH, MELINDA - UNIVERSITY OF MINNESOTA
item Gebeyaw, Mesfin
item PUREV, SARUUL - UNIVERSITY OF MINNESOTA
item TEMPLE, STEPHEN - UNIVERSITY OF MINNESOTA

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2004
Publication Date: 8/1/2004
Citation: Samac, D.A., Dornbusch, M., Gebeyaw, M.T., Purev, S., Temple, S.J. 2004. A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa). Transgenic Research. 13:349-361.

Interpretive Summary: To obtain transgenic plants with a desired characteristic, it is often necessary to fuse together a gene controlling region (promoter) and a gene for the trait of interest from different sources to create a "chimeric gene." For many characteristics the promoter needs to be active in all plant cells so that all cells express the new gene of interest. The most commonly used promoter for such whole plant expression is the 35S promoter that originates from the cauliflower mosaic virus. In this study we compared the level of activity of five whole plant promoters in transgenic alfalfa plants and determined where the promoters were expressed in the plants. Activity of the promoters was compared using genes for two different traits, a marker gene and an antifungal gene. The promoter with the highest activity in leaves, stems, and roots was the promoter from the cassava vein mosaic virus. This promoter was highly active in all alfalfa cells. In contrast, the 35S promoter had lower activity and was not active in all cells of the stem, root, and root nodule. A third promoter from sugarcane bacilliform virus had the lowest activity. The high expression obtained with the cassava vein mosaic virus promoter demonstrated that this promoter is useful for expressing genes throughout all parts of transgenic alfalfa plants. This promoter will be useful in generating alfalfa plants with improved characteristics such as greater tolerance to environmental stresses or better forage quality, which can increase crop yields and reduce production costs.

Technical Abstract: Activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus, the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the b-glucuronidase (GUS) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter, and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves the CsVMV promoter had approximately 24-fold greater activity than the 35S promoter and 38-fold greater activity that the ScBV promoter. Four promoters, the 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, and alfalfa small subunit rubisco (RbcS) promoter, were used to control expression of ech42, a cDNA from Trichoderma atroviride encoding an endochitinase. Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV::ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic lines was 50- to 2,650-fold greater than activity in control lines, none of the transgenic lines showed a consistent increase in disease resistance compared with control lines in either whole plant or detached leaf assays. The high constitutive levels of both GUS and endochtiinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.