Fig. 4: Yeast one-hybrid assay (Y1H) and real-time PCR analysis of flavonoid biosynthetic genes in AaYABBY5-OE plants, AaYABBY5 AnT. plants, pHB vector-containing (35S) plants, and wild-type A. annua plants.
From: Transcriptional regulation of flavonoid biosynthesis in Artemisia annua by AaYABBY5
a Sketch map of prey and bait constructs used to perform the Y1H assay. The coding sequence of the prey protein AaYABBY5 was cloned into the pB42AD vector under the GAL1 inducible promoter sequence as a fusion to the NLS; nuclear localization sequence, AD; activation domain, and HA (hemagglutinin) epitope tag, whereas promoters of PAL, CHS, CHI, DFR, FLS, FSII, LDOX, and UFGT were cloned into the placZ2µ vector as fusions with the lacZ reporter gene to form the bait strains. b AaYABBY5 directly bound to full-length PAL, CHS, CHI, and UFGT promoters in yeast cells cotransformed with these bait strains along with pB42AD-AaYABBY5, as shown by the blue-colored phenotype of yeast clones, but not with empty pB42AD. FLS, FSII, and LDOX did not show positive results. c–j show the relative expression of the PAL, CHS, CHI, DFR, FLS, FSII, LDOX, and UFGT genes in selected AaYABBY5 OE, AaYABBY5 AnT, 35S, and wild-type (W) A. annua plants. Gene expression was found to increase in AaYABBY5-OE plants, whereas in AaYABBY5 AnT. plants, a decrease in the expression of PAL, CHS, CHI, DFR, FLS, FSII, LDOX, and UFGT was found. β-Actin was used as an internal control. The graph shows the mean values ± SD of three experimental replicates. Error bars show the standard deviation for the sample, n = 3. Statistical significance was determined using the Student’s t test. **P < 0.01, *P < 0.05
