Figure 5.

Inhibition of rutin biosynthesis in an AtMYB12-expressing transgenic line by amiFLS. Artificial miRNA was designed and cloned in plant expression vector under the control of the cauliflower mosaic virus 35S promoter. Part of the phenylpropanoid pathway synthesizing flavonols and anthocyanin as well as associated enzymes is represented. The hairpin structure represents amiFLS to inhibit NtFLS expression in AtMYB12-expressing tobacco lines. The constructs expressing three different amiFLS proteins under the control of the cauliflower mosaic virus 35S promoter were agroinfiltrated in tobacco leaves. Relative expression of NtFLS was studied through semiquantitative RT-PCR analysis to select the most potent construct inhibiting NtFLS expression. Expression of ubiquitin was used for normalization. Bars C, 1, 2, and 3 represent the control and three different amiFLS-expressing constructs. Construct 1 reduced expression of NtFLS approximately 3-fold and was further used for stable transformation. F3′H, Flavonoid 3′-hydroxylase; GT, glucosyltransferase; LDOX, leucoanthocyanidin dioxygenase; RT, rhamnosyltransferase.