Fig 1.
Adding 35S enhancer broadens the tissue specificity of NtAGIP1 in inflorescence.
(A) Schematic diagram of constructs based on binary vector pBI121 for the synthetic promoter assays. (B-E) Expression patterns of GUS gene driven by different promoters in (A). Representative GUS patterns are shown for NtAGIP1::GUS (B), 35SNtAGIP1::GUS (C), CaMV35S::GUS (D), and Promoterless::GUS (E) in transgenic reporter lines. Independent transgenic lines (n > 20 for each construct) were assayed for GUS expression in inflorescences, leaves, stems and roots. Scale bars, 1 mm.
Fig 2.
Adding 35S enhancer increased the promoter activity.
(A) Examples of strong, weak, intermediate, and no GUS staining in inflorescence of NtAGIP1::GUS and 35SNtAGIP1::GUS lines. Scale bars, 1 mm. (B) Statistics of lines showing strong, intermediate, weak, or no GUS staining in inflorescence of NtAGIP1::GUS and 35SNtAGIP1::GUS transgenic tobaccos.
Fig 3.
Adding 35S enhancer enhanced the GUS activity in the inflorescence.
GUS activity in the inflorescence (A), and in inner whorls of the flower (B). Four lines of NtAGIP1::GUS and 35SNtAGIP1::GUS transgenic tobaccos with strong GUS expression were selected randomly for GUS activity assays. Error bars represent standard deviations of three biological replicates. *, P < 0.001; NS, not significant (P > 0.05) relative to #6 line of NtAGIP1::GUS tobacco; two-tailed Student’s t-test.
Fig 4.
Dissection analysis of the NtAG intron to dig potential repressive elements.
(A) Schematic diagram of constructs of serial deletions of NtAGIP1 (left), and GUS expression frequency in leaves of transgenic tobacco lines for each construct (right). (B) Further serial deletions of fragment between -2835 and -2365 for precise localization of potential repressive elements (left), and GUS expression frequency in leaves of transgenic tobacco lines for each construct (right).
Fig 5.
The 100-bp repressive fragment of NtAGI-1 is able to suppress 35S enhancer activity in leaves.
(A) Schematic diagram of GUS reporter constructs. (B) GUS expression frequency driven by the chimeric promoter containing 35S enhancer and the 100-bp repressive fragment and by two control promoters. (C) Quantification of GUS activity in the three transgenic groups. Statistical significance was determined by two-tailed Student’s t-test. The horizontal lines represent the medians.
Fig 6.
The 100-bp repressive fragment (orange line) and GAGA motif (red triangles) of NtAGI-1 are conserved in the species of Solanaceae family.
Fig 7.
Structure of AG homologous genes in different plant species.
Grey boxes indicate non-coding regions, black boxes represent coding regions, and lines indicate introns.
Table 1.
Comparison of N.tabacum AG second intron with those from other plants.
Fig 8.
ChIP assay for H3K27me3 levels in NtAGI-1 intron and chimeric promoters.
(A) ChIP assay for H3K27me3 levels at the NtAGI-1 intron in leaves of wild-type tobacco. (B) ChIP assay for H3K27me3 levels in leaves of the -2835 and -2735 lines. The genomic fragment from immunoprecipitation for qRT-PCR detection is depicted as black horizontal line. Enrichment was represented as percentage of Input (% Input). Error bars represent the standard deviation of three biological replicates.