Enhanced expression of the GUS reporter gene by stable co-expression of the TBSV-encoded P19 suppressor in transgenic sugarcane.
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(a) Relative abundance of P19 and GUS transcripts was determined by northern blot and quantitative RT-PCR (qRT-PCR) analyses in two representative P19-GUS transgenic sugarcane lines co-expressing GUS and P19 (two plants per line). Lines expressing GUS with no suppressor were used as a control. Blots of RNA (15 µg per sample) were probed with radioactively labeled P19 DNA, stripped and then reprobed with GUS DNA. Normalized qRT-PCR P19 expression levels of the P19-GUS lines are reported as a percentage, relative to that of the highest expressing plant. GUS activity (pmoles of 4-methylumbelliferone/min/µg protein) of the P19-GUS lines is also indicated. Values represent three biological samples and three technical repeats, and are reported with the standard error. (b) Methylation status of the coding region and promoter of the GUS reporter gene in the P19-GUS transgenic sugarcane lines. Southern blot of genomic DNA (10 µg per sample) of two representative P19-GUS lines, one non-silenced (Line 1, plants 4 and 5) and one silenced (Line 3, plants 12 and 16), digested with methylation-sensitive HpaII (H), and methylation-insensitive MspI (M), restriction endonucleases, were probed with the GUS gene or the Ubi promoter. Shifts in DNA hybridization fragments indicate methylation.