Figure 2

Detection of cleaved PPO gene mRNAs by 5′ RACE-PCR. (A) Schematic diagram of a strategy for detecting the truncated but not the 5′-capped PPO gene mRNAs in amiRNA-expressed transgenic plants. (B) RACE-PCR (1st-round PCR). Lane 1, DNA ladder (NEB, Cat# N0474S); lane 2, 1st-round PCR result from the enriched young leaf poly(A)⁺ RNA of transgenic line amiRPPO1-12. The line with an arrowed end indicates the band of the expected size. (C) Nested PCR round-1 (using the RACE-PCR product as the template). Lane 1, DNA ladder; lane 2, nested PCR-1 result; the line with an arrowed end indicates the band of the expected size. (D) Nested PCR round-2 (using nested PCR-1 product as template). Lane 1, DNA ladder; lane 2, PCR for detection of truncated StuPPO1 gene mRNA; amplified band size as indicated. Lane 3, PCR for detection of truncated StuPPO2 gene mRNA; the faint bands were non-specific amplification. Lane 4, PCR for detection of truncated StuPPO3 gene mRNA; the faint bands were non-specific amplification. Lane 5, PCR for detection of truncated StuPPO4 gene mRNA; the faint bands were non-specific amplification. (E) Determination of the target cleavage site of amiRPPO1 by sequencing the 253 bp of the nested-PCR-1 product (Figure 3C). The target sequences are aligned with the amiRPPO1 complementarily. The arrowed line indicates that 7 of the 7 clones (7/7) were the products cleaved at the expected site. See Additional file 3: Figure S2 and Additional file 4: Figure S3 for the full sequence analysis.