Figure 1.
a. Determination of the precise miRNA sequence by 5′ and 3′ miR-RACE. (a) miRNA cDNA library construction. (b) Analysis of miRNA 5′RACE and 3′RACE. Sequences for the 5′RNA adaptor, dT(30)RT primer, MirRacer 3′ Primer, MirRacer 5′ Primer, GSP1, and GSP2 are listed in Table 2.
Figure 2.
Predicted fold-back structures of the identified ptr-miRNAs.
The mature miRNA sequences are shaded. The miRNA precursors may be slightly longer than the sequences shown in this figure. Predicted fold-back structures of the identified ptr-miRNAs. The mature miRNA sequences are shaded. The miRNA precursors may be slightly longer than the sequences shown in this figure.
Table 1.
Summarized results of the validation of the P. trifoliate miRNA sequences.
Table 2.
Primers used for miR-5′ RACE, miR-3′ RACE, and real-time PCR.
Figure 3.
The 3′ RACE and 5′ RACE products of ptr-miRNAs amplified by PCR are shown in an ethidium bromide-stained agarose gel.
The sizes of the molecular weight markers of the bottom and the second from bottom bands are 50 bp and 100bp, respectively. Lanes 1–9 are 3′RACE products of ptr-miR156, ptr-miR164, ptr-miR167, ptr-miR171, ptr-miR319, ptr-miR482a, ptr-miR482b, ptr-miR435, and ptr-miR1446, respectively, and lanes 10–18 are the 5′RACE products of them.
Table 3.
Alignment between ptr-miRNAs and their orthologs in Arabidopsis.
Figure 4.
Relative expression levels of trifoliate orange miRNAs in different the trifoliate orange tisues of root, stem, leaf, flower, and fruit.
Each reaction was repeated three times and the template amount was corrected by 5.8 s rRNAs.
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).
Table 4.
Primers used for modified 5′ RLM-RACE mapping of the miRNA cleavage sites and putative ptr-miRNA target genes.