A simple method for construction of artificial microRNA vector in plant

Yang Li§·Yang Li§·Sunping Zhao·Sheng Zhong·Zhaohai Wang·Bo Ding·Yangsheng Li*

§:Both authors contributed equally to this work. *: The corresponding author.Email:

Supplementary Table 1A list of all primers used in this study. Primers used for qRT-PCR analysis in tobacco were came from Yan et al (Yan, et al. 2012)

Primer Name / Primer Sequence (5’->3’) / Primer Function
ami-gusF / cagtggtctctgcagTAATAACATACGGCGTGACATcaggagattcagtt / For ami-gus vector construction
ami-M / caggagattcagtttgaagctggacttcacttttgcctctct / For all amiRNA vectors construction
ami-gusR / cagtggtctcaggaaTAATAACATACCGCGAGACATagagaggcaaaag / For ami-gus vector construction
ami-PALF / cagtggtctctgcagTAAGACGAATTCGAATCACAGcaggagattcagtt / For ami-PAL vector construction
ami-PALR / cagtggtctcaggaaTAAGACGAATTGGAAACACAGagagaggcaaaag / For ami-PAL vector construction
P28 / aatccatcgcagcgtaatgc / For GUS RT-PCR
P29 / accacctgccagtcaacaga / For GUS RT-PCR
P32 / taacccaaaggctaatcgtg / For NbActin qRT-PCR
P33 / tccaacacaataccggtggt / For NbActin qRT-PCR
Hyg+ / acggtgtcgtccatcacagtttgcc / For positive rice plants detection
Hyg- / ttccggaagtgcttgacattgggga / For positive rice plants detection
qPAL-F / AGGTCAACTCCGTGAACGAC / For OsPAL1 RT-PCR
qPAL-R / AATGGCAATGGCGATGGGAT / For OsPAL1 RT-PCR
Actin1F / ggaagtacagtgtctggattggag / For OsActin RT-PCR
Actin1R / tcttggcttagcattcttgggt / For OsActin RT-PCR
e-F / cagtcgtctctgcagTACTGATGGTCTCAACTGCCTcaggagattcagttt / For a target sequence containing a BsaI site
e-R / cagtcgtctcaggaaTACTGATGGTCACAAGTGCCTagagaggcaaaagtg / For a target sequence containing a BsaI site

Supplementary Table 2 The annealing system of three primers

Component / Amount
5*PS Buffer (Takara) / 5ul
dNTP Mix (Takara) / 1ul
ami-F (10uM/ul) / 1ul
ami-M (10uM/ul) / 1ul
ami-R (10uM/ul) / 1ul
PrimeSTAR HS DNA Polymerase (Takara) / 0.5ul
Nuclease-free water / 15.5ul
Total / 25ul

Supplementary Figure 1 How to decide the specific primers when you silence a target gene. The results returned from the “Oligo” tool in the WMD3 online software ( will show you four primers thatwould be used in the originalconstructing amiRNA vector method (Schwab, et al. 2006). Using the capital letters “I miR-s” and “IV miR*a”respectively instead ofthe capital letter ofthedesignedami-F and ami-R,which could gain the primers you need

CaMV35S·osa-miR528 precursor 1·BsaI﹣·ccdB·BsaI﹢·osa-miR528 precursor 2·Nos ter

CATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGGACTTGATCCCAGCAGCAGCCACAGCAAAATTTGGTTTGGGATAGGTAGGTGTTATGTTAGGTCTGGTTTTTTGGCTGTAGCAGCAGCAGAGAGACCAATTCTCGACTAAGTTGGCAGCATCACCCGACGCACTTTGCGCCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAACTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCTGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTATCCGTTATCGGGGAAGAAGTGGCTGGTCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGGTCGACGGACTCAACGAGCCCTTGGTCTCATTTCCTGCTGCTAGGCTGTTCTGTGGAAGTTTGCAGAGTTTATATTATGGGTTTAATCGTCCATGGCATCAGCATCAGCAGCGGTACCCTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGCCATCCGCACTGTAGCGGATGGCCTAAAAAAAAAAC

Supplementary Figure 2 Detailed schematic diagram and sequences of the gene cassette in the pTAC vector. The gene cassette, consist of osa-miR528 precursor 1 (blue) and ccdB gene (red) and osa-miR528 precursor 2 (purple), which isbetween a CaMV 35S promoter (yellow) and a NOS terminator (green), the italic letters are two BsaI sites inthe opposite direction

Supplementary Figure 3 Agarose gel electrophoresis of the annealed adaptor with 116 bp length

Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121-1133

Yan P, Shen W, Gao X, Li X, Zhou P, Duan J (2012) High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants. PLoS One 7:e38186

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