Creation and validation of two AS5ox lines. (A) Diagram of the native tobacco chloroplast rrn operon (WT) with the transgene insertion site. The plastid transformation vector (pAS5OX) is shown below (T-psbA terminator; P-psbA promoter; T2-rps16 terminator). Positions of the XhoI and HindIII restriction sites, and the DNA probe (solid line) used in (B) are indicated. (B) DNA gel blot of WT and two AS5ox lines digested with XhoI and HindIII. A PCR fragment spanning the trnI and trnA region was used as a probe. Relevant sizes of hybridizing bands are shown at the right. (C) Quantitative RT-PCR analysis of AS5 transcripts using primers AS5 qPCR 5' and 3'. Expression levels are an average of three biological and at least two technical replicates of each sample, with error bars representing the standard deviation. The WT expression level was set to 1, and samples were normalized to 18S rRNA and GAPDH mRNA.