Expression of a microRNA-Resistant Target Transgene Misrepresents the Functional Significance of the Endogenous microRNA: Target Gene Relationship Junyan.

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Expression of a microRNA-Resistant Target Transgene Misrepresents the Functional Significance of the Endogenous microRNA: Target Gene Relationship Junyan Li, Anthony A. Millar Molecular Plant Volume 6, Issue 2, Pages 577-580 (March 2013) DOI: 10.1093/mp/sss136 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Generation and Analysis of MYB120 and mMYB120 Plants. (A) MYB120 mRNA levels in rosettes of wild-type (Wt) and mir159ab (159ab) measured using qRT–PCR with primers that span the miR159 binding site, so that only un-cleaved MYB120 mRNA will be detected. (B) The MYB120 and mMYB120 transgenes. The entire MYB120 coding sequence (blue box), a 1487-bp upstream region containing all sequences to the adjacent gene (At5g55040, shown as a white box), and a 636-bp region downstream of the stop codon were amplified and cloned. Seven synonymous nucleotide substitutions were made in the miR159 target site of MYB120 to generate mMYB120 as has been previously reported (Allen et al., 2010). The native and mutated miR159 target sites are aligned with the miR159a sequence, with the nucleotide changes indicated in red. MYB120 and mMYB120 encode identical proteins. Figure is not to scale. (C–F) Aerial views of rosettes of 45-day-old plants grown side by side under short-day conditions. (C) Wild-type. (D) Independent MYB120 transformants. (E) mir159ab. (F) Independent mMYB120 transformants. Scale bar = 10 mm. (G) MYB120 mRNA levels in the rosettes of wild-type (Wt), and different T2 transgenic MYB120 (M1–6) and mMYB120 (m1–8) lines. Relative fold changes compared to wild-type are shown below the names of the transgenic lines. (H) Average rosette size of 42-day-old wild-type (Wt) and MYB120 (M1–M6) plants. Unit of measurement shown is relative pixel. Error bars stand for the SD. (I) CP1 mRNA levels in the rosettes of wild-type (Wt), and different MYB120 (M1, M5, and M6) and mMYB120 lines (m1, m5, and m7). Relative fold changes compared to wild-type are shown below the names of the transgenic lines. All qRT–PCR measurements were done as described (Allen et al., 2010), and were normalized with CYCLOPHILIN and are the average of three technical replicates with error bars representing the SEM. Molecular Plant 2013 6, 577-580DOI: (10.1093/mp/sss136) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions