Fig 1.
Validation of the leader intron on high activity of the GmScreamM8 promoter.
(a) Schematic of intron deletion and translocation constructs. (b) Transient expression profiles for intron deletion and translocation constructs in lima bean cotyledons. The order of constructs in the figure caption key reflects the highest to lowest activity in the curves. (c) GFP intensity value (±SEM) in soybean hairy roots transformed with intron deletion and translocation constructs. Images of root tips on top of columns represent the average intensity of GFP expression driven by the corresponding promoter construct. Overlapping columns show no significant differences between constructs by the t test (LSD) at p<0.05.
Fig 2.
Partial deletion analysis of the leader intron in the GmScreamM8 promoter.
(a) Schematic of intron partial deletion constructs in the GmScreamM8 promoter. (b) Transient expression profiles for intron partial deletion constructs in lima bean cotyledons. The order of constructs in the figure caption key reflects the highest to lowest activity in the curves. (c) GFP intensity value (±SEM) in soybean hairy roots regulated by different intron partial deletion constructs. Images of root tips on top of columns represent the average intensity of GFP expression driven by the corresponding promoter construct. Overlapping columns show no significant differences between constructs by the t test (LSD) at p<0.05. (d) Left: RT-PCR. Right: conventional PCR using plasmid DNA of each construct as template.
Fig 3.
Validation of synthetic promoters containing the tetrameric element, the core promoter, and the leader intron.
(a) Schematic of synthetic promoter constructs. (b) Transient expression profiles for different synthetic promoter constructs in lima bean cotyledons. The order of constructs in the figure caption key reflects the highest to lowest activity in the curves. GFP expression is presented as “GFP Expression” rather than percent of a control because expression values for two different core promoters are presented. (c) GFP intensity value (±SEM) in soybean hairy roots transformed with different synthetic promoter constructs. Images of root tips on top of columns represent the average intensity of GFP expression driven by the corresponding promoter construct. Overlapping columns show no significant differences between constructs by the t test (LSD) at p<0.05.
Fig 4.
Partial deletion analysis of the leader intron using synthetic promoters.
(a) Schematic of intron partial deletion constructs in synthetic promoters. (b) Transient expression profiles for intron deletion constructs in synthetic promoters in lima bean cotyledons. The order of constructs in the figure caption key reflects the highest to lowest activity in the curves. (c) GFP intensity value (±SEM) in soybean hairy roots transformed with intron deletion variants in synthetic promoters. Images of root tips on top of columns represent the average intensity of GFP expression driven by the corresponding promoter construct. Overlapping columns show no significant differences between constructs by the t test (LSD) at p<0.05.
Fig 5.
Cluster deletion analysis of the leader intron in synthetic promoters.
(a) Schematic of all intron cluster deletion constructs. (b) Transient expression for different intron cluster deletion variants in lima bean cotyledons. The order of constructs in the figure caption key reflects the highest to lowest activity in the curves. (c) GFP intensity value (±SEM) in soybean hairy roots transformed with different intron cluster deletion variants in synthetic promoters. Images of root tips on top of columns represent the average intensity of GFP expression driven by the corresponding promoter construct. Overlapping columns show no significant differences between constructs by the t test (LSD) at p<0.05.
Fig 6.
Validation of intronic regulatory sequences using synthetic introns.
(a) Schematic of synthetic intron constructs in manipulation of the copy number and sequences of intron regions. (b) Transient GFP expression for various synthetic intron constructs in lima bean cotyledons. The order of constructs in the figure caption key reflects the highest to lowest activity in the curves. (c) GFP intensity value (±SEM) in soybean hairy roots transformed with various synthetic intron constructs. Images of root tips on top of columns represent the average intensity of GFP expression driven by the corresponding promoter construct. Overlapping columns show no significant differences between constructs by the t test (LSD) at p<0.05.
Fig 7.
Validation of the repeated sequences in GmScreamM8 leader intron.
(a) DNA sequence and alignment of the three imperfect repeated sequences (* indicated mismatches in alignment). (b) Schematic of partial deletion constructs of 4xEF4-M8CIN3. (c) Transient expression profiles for different deletion constructs of the repeated sequences in lima bean cotyledon tissues. The order of constructs in the figure caption key reflects the highest to lowest activity in the curves. (d) GFP intensity value (±SEM) in soybean hairy roots transformed with different deletion constructs of 4xEF4-M8CIN3. Images of root tips on top of columns represent the average intensity of GFP expression driven by the corresponding promoter construct. Overlapping columns show no significant differences between constructs by the t test (LSD) at p<0.05.