Fig 1.
FAD3 gene expression level of T5 homozygous RNAi lines.
Data are averages of biological triplicates ± SD normalized to CONS7 mRNA. Independent-Samples T Test was used to test the significance. Asterisks indicate significant differences in relative expression between transgenic lines and control. (*, p < 0.05; **, p < 0.01). S24-4D, S-24-13, S-24-15 are T5 homozygous for the pMUFAD transgene. Soybean cultivar Jack is used as wild-type control. (A) to (C) Normalized gene expression level of GmFAD3A, GmFAD3B and GmFAD3C, respectively. The values of wild-type plants were arbitrarily fixed to 1.0. (D) Percentage of mismatches between the 318-bp IR and corresponding regions in GmFAD3A, GmFAD3B and GmFAD3C, respectively.
Fig 2.
hpRNA and bar transcript level of pMUFAD homozygous lines.
Data are averages of biological triplicates ± SD normalized to CONS7 mRNA. Independent-Samples T Test was used to test the significance. Asterisks indicate significant differences in relative expression (*p < 0.05, **p < 0.01). S-24-4D, S-24-13, and S-24-15 are T5 homozygous for the pMUFAD transgene. Soybean cultivar Jack is used as wild-type control. (A) and (B) Normalized transcript level of hpRNA and bar, respectively. The values of S-24-4D plants were arbitrarily fixed to 1.0.
Fig 3.
DNA methylation analysis of T5 homozygous RNAi lines.
(A) Schematic presentation of the T-DNA region of the plant transformation vector, pMUFAD. The expression cassette for the RNAi of GmFAD3 is highlighted in grey. Note: LB and RB, T-DNA left and right borders, respectively; Tvsp, soybean vegetative storage protein gene terminator; bar, bialaphos resistance gene; TEV, tobacco etch virus translational enhancer; CaMV35S, cauliflower mosaic virus 35S promoter; OCS 3’, octopine synthase gene terminator; IR-R and IR-F, the 318-bp inverted repeats of GmFAD3 target sequence in reverse and forward directions, respectively; Rice Waxy-a Intron, rice Waxy-a gene intron; GlyP, soybean glycinin gene promoter. Lines beneath the schematic represent region I, II, III and IV examined by bisulfite sequencing. (B) Schematic of the Glycinin gene (GenBank: AB113349.1). The darkened rectangle represents exon and horizontal line represents intron. The black arrow indicates transcription starting site. Lines beneath the schematic represent region V and VI examined by bisulfite sequencing, with numbers indicating the corresponding position. (C) Methylation status of a 338 bp region I (294bp Glycinin promoter, 32bp vector backbone, 12bp forward inverted-repeat of the FAD3 hairpin), a 276bp region II, a 281bp region III and a 352bp region IV within the plant transformation vector pMUFAD. (D) Methylation status of a 297 bp region V (-295 bp to 2bp) and a 255 bp region VI (1105 bp to 1359bp) within soybean Glycinin gene (GenBank: AB113349.1). Bar heights represent the percentage of methylation at each CGN, CHG and CHH (where N = A, T, G or C; H = A, T, or C) cytosines of 10 clones analyzed by bisulfite sequencing. Two biological replications were performed and similar results were obtained.
Fig 4.
The size of small RNA from each sample was plotted versus frequency (0.0–1.0) among distinct sequences (A) or total sequences (B) to eliminate the bias of different sequencing depth. (C) and (D) Size profiles of the 318-bp IR small RNAs for distinct sequences and total sequences, respectively. Data are average of three replications.
Fig 5.
Comparison of 318-bp IR-derived siRNAs in the three RNAi lines.
(A) Small RNAs matching the 318-bp IR were plotted versus the average of their normalized abundance from three replications. Plus Y-axis labels represent siRNAs from the sense strand of 318-bp region, while minus Y-axis indicate siRNAs found on the opposite strand. For visual clarity, the Y-axis of each diagram is adjusted based on the corresponding small RNA abundance. (B) and (C) Venn diagram represents common and specific reads from total and top 1000 abundant small RNAs in S-24-4D, S-24-13 and S-24-15, respectively.
Fig 6.
318-bp IR-derived siRNAs targeting FAD3B and FAD3C.
(A) and (B) Small RNAs generated from the 318-bp IR matching the corresponding FAD3B and FAD3C target regions were plotted versus the average of their normalized abundance from three replications, respectively. For visual clarity, Y-axis of each diagram is adjusted according to the small RNA abundance.
Fig 7.
(A)—(D) Small RNAs matching the GmFAD3A, GmFAD3B, and GmFAD3C transcript sequences and the rice waxy-a intron were plotted versus the average of their normalized abundance from three replications, respectively. The 318-bp siRNA generating IR on GmFAD3A and corresponding target regions on GmFAD3B and GmFAD3C are indicated with black lines. For visual clarity, Y-axis of each diagram is adjusted according to the small RNA abundance.
Fig 8.
5’RACE on GmFAD3A and GmFAD3C mRNAs in T5 RNAi lines.
Arrows indicate the inferred cleavage sites and numbers above represent the fractions of cloned 5’ RACE PCR products terminating at this position. Degradation sites detected with high frequency are highlighted in red, and those present across the three RNAi lines are highlighted with asterisks. (A) Summary of the 5’ RACE analysis performed on the 318-bp region of GmFAD3A mRNA. (B) and (C) Summary of the 5’ RACE analysis performed on the corresponding region of GmFAD3C mRNA.
Table 1.
Putative functional siRNAs.