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. 2023 May 31;12(11):2179. doi: 10.3390/plants12112179

Figure 2.

Figure 2

Construction of an expression vector for artificial miRNA encoded in exon (AMIE) and in intron (AMIN). (A) Structure of dual miRNA cluster. Orange and green boxes represent MIR171 and MIR164 precursor backbones, respectively. Gray box represents connector sequences. Brown and green triangles represent mature artificial miRNA a and b, while the open triangles represent the corresponding miRNA*. (B) Structures of exon-encoded (AMIE, top) and intron-encoded (AMIN, bottom) dual miRNA clusters. Red boxes represent indicated enzyme restriction sites. Brown boxes represent dual miRNA cluster shown in A. Yellow box represents fragments of GUS-coding sequences. Gray boxes represent intron sequences from PDS gene while blue boxes represent short exon sequences flanking each intron. (C) Structure of expression vector for a dual miRNA cluster. Red box represents 35S promoter sequence. Blue box represents AMIE or AMIN targeting 7 TYLCV proteins, the structure of which is shown in B. Gray box represents 35S terminator sequence. (D) Method to clone eight-miRNA expression vectors pAMIN8 and pAMIE8. (E) Method for cloning six-miRNA expression vectors pAMIN6 and pAMIE6. (F) Method for cloning fourteen-miRNA expression vectors pAMIN14 and pAMIE14. At each cloning step, vector DNA was digested with XbaI and XhoI, while insert DNA was digested with SpeI and XhoI.