Identification of a Novel Jasmonate-Responsive Element in the AtJMT Promoter and Its Binding Protein for AtJMT Repression
Figure 5
Identification of sequence element in the BcNTR1 promoter region to which AtBBD1 binds.
(A) Structures of reporter and activator genes used in Y1H assays. The promoter region of BcNTR1, −3518 to −3390, was divided into 3 segments and each segment was used as bait for Y1H assays. The control does not contain any of those segments. AtBBD1 was fused with the GAL4 activating domain (AD) as an activator. The position of the putative JARE is shown (▾). (B) The segment a was divided further into 8 subsegments (6 nt each) and each subsegment, a1 to a8, was mutated into 6 adenines. Each mutant segment was tested as bait in Y1H assays. (C) Subsegments a6 and a7 to which AtBBD1 bound, were dissected further by mutation in overlapping frames. In each mutant, 6 nucleotides were mutated into 6 adenines. Each mutant subsegment, M0–M5, was tested by Y1H assays. The sequence motif to which AtBBD1 binds is shown in bold. (D) Mutation analysis of the AtBBD1 binding element. Mutant series (CM1 to CMR) of JARE was created by changing a single nucleotide from purine to pyrimidine, or vice versa, in the fragment −2305 to −2278 as shown in Fig. 4A as a bait and Y1H assays were carried out with AD-AtBBD1. CMR is a JARE in reverse orientation.