Fig 1.
Promoter activity of miR394 and LCR in Arabidopsis flowers and developing siliques.
A: GUS staining of the transgenic Arabidopsis carrying pLCR::GUS (Pro::AtLCR), pMIR394a::GUS (Pro::miR394a) and pMIR394b::GUS (Pro::miR394b). GUS staining in flowers or siliques was identified with days after flowering (DAF). B: The relative expression of GUS in the transgenic Arabidopsis plants. The intensity was quantified using Adobe Photoshop CS software. Vertical bars represent SD of the mean of treatments (n = 3). Asterisks indicate that mean values are significantly different between the GUS intensity of Pro::AtLCR and Pro::miR394a or Pro::miR394b (P<0.05).
Fig 2.
Expression of Brassica napus precursor miR394a/b and its target BnLCR in wild type (WT) and the transgenic plants.
Total RNA from two week-old seedlings was extracted and transcripts were analyzed. A: transcripts of pre-miR394a/b and BnLCR were analyzed using semi-quantitative RT-PCR. B: Base paring of miR394 with the corresponding complementary site of BnLCR and a miR394-resistant form of BnLCR (Bnm6LCR). C: Transcripts of BnLCR in miR394-resistant (35S::Bnm6LCR) and miR394 non-resistant (35S::BnLCR) transgenic lines. Actin was used for cDNA normalization. The number below the band indicates relative abundance of the corresponding gene with respect to the loading controls Actin. or 35S:m5LCR plants and WT (p < 0.05).
Fig 3.
Phenotypes in various transgenic plants including (a) WT; (b) 35S::BnMIR394a; (c) 35S::BnMIR394b; (d) 35S::antiBnLCR (transgenic plant over-expressing antisense mRNA of BnLCR); (e) 35S::BnLCR; (f) 35S::Bnm6LCR.
(A, B) Cotyledon and leaf phenotypes of seven-day-old seedlings. (C) Flowering stage. (D) Plant height phenotype of the transgenic plants at flowering stage. (E) Phenotypes of pods. (F) Seed shapes of the transgenic plants. The scale bar in the graph indicates 5 mm. (G) Seed weight of different transgenic Brassica napus plants. Vertical bars represent SD of the mean. Asterisks indicate that mean values are significantly different between the transgenic plants and wild-type (WT) (P<0.05).
Table 1.
Over-expression of miR394 modified phenotypes of Brassica napus.
Fig 4.
Fruit and seed development in transgenic Arabidopsis plants.
A-C: Morphology of wild-type (A), 35S::MIR394a (B) and 35S::m5LCR (C) plant siliques. D-F: Close-up view of images showing the valve and replum tissues of Arabidopsis fruit (stage 17b) of the wild type (D), 35S::MIR394a (E), and 35S::m5LCR (F) plant siliques by a scanning electron microscope (SEM). G-I: The seed phenotypes of wild-type (G), 35S::MIR394a (H) and 35S::m5LCR (I) plants. Bars, 1 mm (A, B, C); 200μm (D, E, F); 300μm (G, H, I).
Table 2.
Total oil content of Brassica napus transgenic seeds.
Table 3.
Fatty acid (FA) compositions in Brassica napus transgenic and wild-type (WT) seeds (mol %).
Table 4.
Protein and GLS content in Brassica napus transgenic seeds.
Fig 5.
Real-time PCR analysis of Brassica napus genes LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3) involved in oil synthesis regulation and ALTERED TRYPTOPHAN REGULATION1 (ATR1) involved in glucosinolate synthesis.
The expression levels were measured in developing seeds 30 days after pollination (DAP). The relative expression level of each gene was normalized using BnACTIN2 as an internal control. Vertical bars represent the standard deviation of the mean treatments (n = 3). Asterisks indicate that mean values are significantly different between the 35S:MIR394 or 35S:m6LCR plants and WT (p < 0.05).
Fig 6.
DR5-GUS expression in Arabidopsis wild type (Col), 35S::MIR394a and 35S::m5LCR fruits.
A: Visualization of DR5-GUS reporter in stage 17 fruits of pDR5::GUS/Col, pDR5::GUS/MIR394a and pDR5::GUS/m5LCR transgenic plants. B: Relative quantification of GUS intensity in stage 17 fruits of wild type (Col), 35S::MIR394a and 35S::m5LCR. The intensity of GUS coloration was statistically quantified using Adobe Photoshop CS software. Twenty fruits were measured. The relative intensity of WT is considered as 100%. Vertical bars represent the standard deviation of the mean treatments (n = 3). Asterisks indicate that mean values are significantly different between the 35S:MIR394a or 35S:m5LCR plants and WT (p < 0.05).