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. 2016 Jan 23;14(7):1633–1645. doi: 10.1111/pbi.12526

Figure 6.

Figure 6

The Interaction with other MdbHLHs proteins affects the function of MdbHLH104 in the activation of MdAHA8 promoter. (a) Transcription activation assay of GUS reporter gene in yeast cells. A series of transformant yeast cells containing different plasmid combinations, indicated as 1–8, were used. 1, pAD/pBD‐GUS; 2, pAD/pBD‐PM d AHA 8 ::GUS; 3, pAD/pBD‐MdbHLH104‐PM d AHA 8 ::GUS; 4–8, transformant yeast cells containing pBD‐MdbHLH104‐PM d AHA 8 ::GUS plus pAD‐MdbHLHs plasmids, including 4, pAD‐MdbHLH105; 5, pAD‐MdbHLH115; 6, pAD‐MdbHLH11; 7, pAD‐MdbHLH121; 8, pAD‐MdPYE. (b) The interaction enhances the binding of the MdbHLH104 protein to the promoter fragment of the MdAHA8 gene. The immunoprecipitated DNAs were quantified through qPCR using specific primers of candidate fragments containing the E‐box cis element. The results were quantified as the percentage of total input DNA by qPCR. (c) The interaction enhances the transcriptional activity of the MdbHLH104 to the MdAHA8 promoter, as indicated by GUS staining and the activity test. The WT calli were labelled as 1; the transgenic calli, 2. After the expression vector 35S::MdbHLH104‐GFP was cotransformed into the calli 2, transgenic calli PM d AHA 8 ::GUS+35S::MdbHLH104‐GFP were obtained and labelled as 3. The transgenic calli 3 were used as the background for transient co‐expression with 5 viral vectors pIR‐MdbHLHs containing other IVc subgroup bHLH genes, labelled as 4–8. 4, pIR‐MdbHLH105; 5, pIR‐MdbHLH115; 6, pIR‐MdbHLH11; 7, pIR‐MdbHLH121; 8, pIR‐MdPYE. (d) Expression levels of MdAHA8 gene in the WT, 35S::MdbHLH104‐GFP and co‐expression of bHLHs transgenic apple calli, as determined with qPCR.