*3.3. miR156g, miR858 Silence PeMYB7, and PeMYB11*

MiRNAs can influence tissue anthocyanin formation in previous studies. In petunia, the sequence-specific degradation of CHS RNA is the primary cause of the formation of white sectors in 'Red Star' flowers [23]. In potato (*Solanum tuberosum* L.), small RNAs of miR828 and TAS4 D4(−) can decrease the expression levels of MYB12 and R2R3-MYB genes in purple skin and flesh, which caused to anthocyanin accumulation [31]. In our research, miR156 and miR858 were predicted as the main interference RNAs for PeMYB7 and PeMYB11 (Table 3). In order to confirm *PeMYB7* and *PeMYB11* are the direct targets of the small RNAs, we download the complete cDNAs of *PeMYB7* (GenBank: KF769472.1) and *PeMYB11* (GenBank: MH675649.1), and analyzed the target sites of miR156 and miR858 in these two genes (Figure 8B). This finding confirms that miR156g and miR858 can silence *PeMYB7* and *PeMYB11*.

**Figure 8.** Identification of *PeMYB7*, *PeMYB11* and their target sites by miR156g, miR858. (**A**) Phylogenetic tree inferred from the MYB sequences of *Phalaenopsis equestris* and *Oryza sativa*; (**B**) Identification of target sites of miR156 and miR858 in *PeMYB7* and *PeMYB11.*

In *Arabidopsis thaliana*, miR156 negatively regulates the anthocyanin accumulation by regulating the expression level of target gene *MYB113* indirectly [32]. High miR156 level promotes anthocyanin biosynthesis through the negative regulation of *SPL9* gene because SPL9 can destabilize the MYB-bHLH-WD40 transcriptional activation complex. However, in this study, *SPL9* gene in *Phalaenopsis* doesn't express differently, which indicated that mtr-miR156g-3p may be able to directly silence *PeMYB7* and caused the anthocyanin lacking on the white sector.

The biological functions of miR858 has not been fully explored, however, small RNA sequencing in *Arabidopsis thaliana* [24] and *Vitis vinifera* L. [33] verified that its target gene is *MYB12*, which is a positive transcript factor for the anthocyanin synthesis. Chitwood et al. transferred the constructed binary vector into wild-type tomato plants to reduced MIR858 expression level, and found the expression levels of *SLY-Myb12* in transgenic plants were lower than wild type, with upregulations of the related structure gene *PAL*, *DFR*, *ANS* and *CHS* involved in anthocyanin biosynthesis, and increasing of anthocyanin content [34]. Ballester et al. [35] used VIGS technology silenced *MYB12* and then found the tomato fruits turned pink. In our study the targeted genes of miR858 were predicted to be both *MYB12* and *MYB11* (Table 3), however, the unigene of *PeMYB12* didn't show significantly different expression levels between the spot and non-spot areas, suggesting that miR858 explored higher effect on the unigene of *PeMYB11* in 'Panda'.
