Figure 5. Transgenic B. napus plants overexpressing miR1885.
(A) Construct with bra-MiR1885 under control of AA6 promoter. pCaMV35S, CaMV35S promoter; Hyg R, hygromycin-resistance gene; pAA, AA6 promoter; LB, left border of T-DNA; t35S, CaMV35S terminator; tAA6, AA6 terminator. Eco RI, Nco I and Bst EII are restriction sites. (B) Plants of T1 transgenic lines and wild type in the field. Red scale bar = 10 cm. X2–X16, transgenic plants. (C) Detection of transgene in rapeseed transgenic lines by PCR using specific primer spanning AA6 promoter andMiR1885 fragment. M, DNA marker. (D) Real-time PCR showing expression of pri-miR1885 in transgenic lines. ACT7 was used as endogenous reference gene. (E) Relative abundance of mature miR1885 in transgenic lines. U6 was used as endogenous reference gene. (F) Northern blot analysis of accumulation of mature miR1885 in MIR1885 -OE lines. U6 was used as endogenous reference gene. (G-H) Relative transcript levels of Bn.TIR.A09 (G) and Bn.TNL.A03(H) in miR1885-OE lines as compared to WT. ACT7 was used as endogenous reference gene. Error bar represents mean ± SE; n = 3. * indicates P < 0.05; ** indicates P< 0.01; Student’s t-test.