MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
Figure 5
Mapping of the mRNA cleavage sites by RNA ligase-mediated 5′ RACE.
Each top strand (black) depicts a miRNA complementary site, and each bottom strand depicts the miRNA (red). Watson-Crick pairing (vertical dashes) and G∶U wobble pairing (circles) are indicated. The arrows indicate the 5′ termini of mRNA fragments isolated from citrus, as identified by cloned 5′RACE products, with the frequency of clones shown. Only the cloned sequences that matched the correct gene and had 5′ ends within a 100 nt window centered on the miRNA validation are included (Table 1). The miRNA sequence shown corre (1 out of 4 PCR clones) is indicated in lower case and corresponds to the most common miRNA supported by the miRNA PCR. RNA ligase-mediated 5′RACE was used to map the cleavage sites. The partial mRNA sequences from the target genes were aligned with the miRNAs. The numbers indicate the fraction of cloned PCR products terminating at different positions. Pt-SPL9 (accession FJ502237), Pt-SPL13 (accession FJ502238), Pt-NAC1 (accession FJ619349), Pt-ARF8 (UC46-16450), Pt-SCL6 (accession GQ505957), Pt-TCP4 (GQ505958), Pt-GRAS (accession FC901464). Pt-SPL9 (accession FJ502237) was similar to AT2G42200 (NM_129782) SPL9 (squamosa promoter-binding protein 9); Pt-SPL13 (accession FJ502238) was similar to AT5G50670 (NM_124445) SPL (squamosa promoter-binding protein); Pt-NAC1 (accession FJ619349) was similar to AT5G61430 ( NM_125536) ANAC100 (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 100); Pt-ARF8 (UC46-16450) was similar to AT5G37020 (NM_001085203) ARF8 (AUXIN RESPONSE FACTOR 8); Pt-SCL6 (accession GQ505957) was similar to AT4G00150 (NM_116232) SCL6 (scarecrow-like transcription factor 6); Pt-TCP4 (GQ505958) was similar to AT3G15030 (NM_180258) TCP4 (TCP family transcription factor 4); UC46-36616 was similar to AT1G12220 (NM_101094) RPS5 (RESISTANT TO P. SYRINGAE 5); Pt-GRAS (accession GU072592) was similar to IPR005202 (XM_002318667) GRAS71 (GRAS family transcription factor).