The cauliflower mosaic virus (CaMV) 35S promoter is widely used in many plant transformation vectors, and is generally reported to be a strong constitutive promoter, conferring strong transgene expression in all plant tissues. Transformation of alfalfa with constructs containing the CaMV 35S promoter driving the expression of the beta-glucuronidase (GUS) reporter gene does produce a percentage of plants with high GUS activity, most often demonstrated by histochemical staining. However, during the course of genetically engineering alfalfa to produce a foreign phytoalexin (Hipskind and Paiva, 2000), we observed very low levels of transgene mRNA in roots, when using an enhanced ("double") CaMV 35S promoter. There have also been many anecdotal reports of CaMV 35S being an unsatisfactory promoter for many transgene experiments in alfalfa, but also several success stories. Many binary vectors containing CaMV 35S are in widespread use, although these may differ in the exact sequences of the CaMV 35S promoter region driving the transgene (such as GUS), or differ in the 5’ untranslated regions, 3’ untranslated regions, translation start site elements, etc.

Much promoter analysis has relied on indirect inference of promoter strength using histochemical staining or enzyme assays of the GUS reporter gene. The measured GUS activity is actually the net sum of transcription (promoter activity), mRNA stability, translation, and protein stability. While generally accepted to give meaningful relative estimations of promoter strength, GUS quantitation can give over-estimation of promoter activity, due to the extreme stability of the GUS protein. The quantitation of mRNA levels by northern blot analysis, while more tedious, can provide a more direct analysis of promoter strength.

We are in the process of re-examining the relative strength of transgene expression levels conferred by common plant transformation vectors in transgenic alfalfa plants. We are assaying both GUS activity and GUS mRNA, and comparing their levels in roots, stems and leaves of rooted cuttings. We are comparing both the absolute levels of GUS expression, as well as the relative root:stem:leaf ratio of expression, to determine if components other than the promoter are influencing mRNA and protein levels, and to search for vectors conferring higher relative root expression.

Two vectors under study (pBI121 and pKYLX71GUS) contain similar lengths of the CaMV 35S promoter region, but different 3’ untranslated (polyadenylation) regions and possibly other sequence variations. Preliminary histochemical staining indicates that pKYLX71GUS confers much higher GUS activity than pBI121. We are also comparing these vectors with the so-called "Superpromoter" (a fusion of octopine and mannopine synthase mas promoter/activators; Ni et al., 1995). This promoter was reported to drive 25-50 times higher GUS expression than a "double" CAMV 35S vector in tobacco and other species. Preliminary analysis indicates that while the promoter is functional in alfalfa, and the root:leaf ratio of GUS activity may be higher, its expression in mature alfalfa leaves is generally low compared to CaMV 35S promoter vectors.

Hipskind, J.D. and Paiva, N.L. 2000. Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis. Mol. Plant-Microbe Interact. 13: 551-562.

Ni, M., Cui, D., Einstein, J., Narasimhulu, S., Vergara, C.E., Gelvin, S.B. 1995. Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. Plant J. 7: 661-676.

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