Figure 3.

(I) Expression analysis of Tdc gene in the two C. roseus accessions, Kew1 and Prabal having (CT)₈ and (CT)₂₁ motifs respectively in the 5′UTR of Tdc gene (a) Northern blot analysis. 10 μg of total RNA was probed with cDNA. Ribosomal RNA was used as loading control (b) Semi quantitative PCR analysis: endogenous actin gene was used as positive control and (c) Real Time quantification of Tdc mRNA. It revealed approximately 8 times higher expression in Prabal (CT) ₂₁ than Kew1 (CT)_8. (II). Schematic representation of cloning of 5′-UTRs of Tdc harboring (CT)_n repeat motifs into pBI121 expression vector using Xba1 and Xma1 restriction enzymes. The sequences of recombinant constructs were confirmed by sequencing (a) Sequence of 5′-UTR (129 bp from Kew1 accession) harboring (CT)₈ motifs in pBI121:(CT)₈ construct and 5′-UTR (155 bp from Prabal accession) harboring (CT)₂₁ motifs in pBI121:(CT)₂₁ construct. Analysis of transiently expressed GusA mRNA, driven by respective recombinant pBI121 vectors, by (b) semi-quantitative PCR. Tobacco actin gene was used as a control and (c) Real time PCR expression analysis of transiently expressed GusA gene transcript, wherein pBI121 empty vector was used as a control. Relative expression profile of recombinant vectors pBI121:(CT) ₈ and pBI121:(CT)₂₁. (The original figure from which Fig. 3.I (a) was cropped and derived is present as Supplementary Figure 1).