Transient activity of the CAMV 35S promoter lacking the mythylation targets in rice and efficient selection of transformed rice cells using the bar gene
1996
Aldemita, R.R. (Philippine Rice Research Inst., Maligaya, Munoz, Nueva Ecija (Philippines))
Gene silencing is usually caused by promoter or gene methylation. An attempt was made to study the activity of a cauliflower mosaic virus (CaMV) 35S promoter with lesser number of methylatable cytosine on gene expression. A plasmid (pSan9) with the CaMV35S promoter mutated at the TGACG motifs (methylation target free (MTF35S)) and CaMV35S plasmid (pSan2) driving the gusA gene were used. The activity of the MTF35S and CaMV35S promoters to drive the expression of gusA reporter gene was determined transiently in rice variety Radon using the polyethylene glycol-mediated transformation. Transient GUS activity of two-day DNA-treated protoplasts showed an expected lower activity in the protoplasts treated with pSan9. Radon protoplasts were co-transformed with the selectable marker bar gene and with either pSan2 or pSan9. Plants were regenerated from the DNA-treated protoplasts at a high selection and co-transformation efficiencies. Two transgenic plants produced with minimum integration events shown in the Southern blot analysis exhibited high GUS activity using a flourimetric assay. However, most of the plants regenerated contained high multiple integration events that resulted to the inability to determine the activity of the MTF35S promoter in stable conditions. Possible reasons and alternative to approach the problem are discussed
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