Figure 7.

Identification of transcriptional regulators binding to the promoter of NfdnaK2. A, Verification of interactions between the cis-acting element Promoter2-1 (pro2-1) and putative transcription factors NfRre1 or NfPedR by Y1H assay. The transformed yeast cells (10 μL) with DNA fragments and regulator proteins at OD₆₀₀ 0.1, 0.01, and 0.001 (indicated by the slope of the black triangles from left to right) were tested on SD/-Leu medium with or without 250 μg L⁻¹ AbA. B, Interaction analyses between Promoter2-1 and NfRre1 or NfPedR by EMSA. Biotin-labeled Promoter2-1 (5 ng) was incubated with increasing concentrations of purified GST-tagged NfRre1 or NfPedR (lane 1, 0 ng; lane 2, 100 ng; lane 3, 200 ng; lane 4, 400 ng; and lane 5, 600 ng). The unlabeled Promoter2-1 (lane 6, 500 ng) and poly(dI-dC) (lane 7, 2,000 ng) were used as specific competitor (SC) and nonspecific competitor (NSC) inhibitors, respectively. GST-tag (lane 8, 5 ng Biotin-labeled Promoter2-1 was incubated with 200 ng GST protein, which was the equal amount as GST-tagged NfRre1 or NfPedR used in lanes 6 and 7) was used as a negative control to exclude the nonspecific binding of NfRre1/NfPedR with promoter2-1. The shift probe indicates the biotin-labeled promoter2-1 interacting with NfRre1 or NfPedR, and the free probe is the biotin-labeled promoter2-1 without interacting with NfRre1 or NfPedR. C and D, Relative transcriptional levels of Nfrre1 (C) or NfpedR (D) during rehydration and subsequent dehydration. The dried field-collected N. flagelliforme samples were rehydrated (marked by hatching) for 0, 1, 3, 9, and 24 h and subsequently dehydrated for 0.5, 2, and 4 h. Each treatment was repeated three times independently, and data are shown as the mean ± sd of three independent replicates.