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Engineering artificial microRNAs for multiplex gene silencing and
simplified transgenic screen
Nannan Zhang1,†, Dandan Zhang1, †, §, Samuel L. Chen2, Ben-Qiang Gong1, Yanjun Guo1,
Lahong Xu1, Xiao-Ning Zhang2, Jian-Feng Li1,*
1Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of
Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, School of Life
Sciences, Sun Yat-sen University, Guangzhou 510275, China.
2Department of Biology, St Bonaventure University, New York 14778, USA
†These authors contributed equally to this work as first authors.
§Present address: Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA
*To whom correspondence should be addressed. Tel: +86 20 39943513; Fax: +86 20
39943513; Email: [email protected].
Supplemental Data
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Supplemental Figure S1. Sequence alignment between GLK1 and GLK2 identifies the most conserved region (in red frame) for amiRNA design.
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Supplemental Figure S2. Comparison of the activities of amiRNA candidates designed by two different strategies for silencing Arabidopsis APK2 or SERK family using the ETPamir assay. A, amiR-APK2-m1 (red) designed by the new strategy is the most efficient amiRNA for silencing APK2A/B. APK2B is labeled by an asterisk in the blot. B, amiR-SERK-m1 (red) designed by the new strategy is the most efficient amiRNA for silencing SERK1-5. Target genes encoding FLAG-tagged proteins and GFP-FLAG (transfection control) were co-expressed with indicated amiRNAs in Arabidopsis protoplasts for 36 h. At least three independent repeats were conducted with similar results. Ctrl, control without amiRNA co-expression.
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Supplemental Figure S3. ETPamir screens identify the most effective amiRNA for silencing Arabidopsis EFR, CERK1, PEPR1 or RLP23. Optimal candidates amiR-EFR-m3, amiR-CERK1-m3, amiR-PEPR1-m2 and amiR-RLP23-m2 are highlighted in red. Target genes encoding FLAG-tagged proteins and GFP-FLAG (transfection control) were co-expressed with indicated amiRNAs in Arabidopsis protoplasts for 36 h. At least three independent repeats were conducted with similar results. Ctrl, control without amiRNA co-expression.
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AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATCGAGCAAACACACGCTCGGACGCATATTACACATGTTCATACACTTAATACTCGCTGTTTTGAATTCATGTTTTAGGAATATATATGTAGAGGCATTTCCAACGTCCCTTTTTCACAGGTCGTGATATGATTCAATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGCGGTAGACAAATTGGATCATTGATTCTCTTTGATAAAGGGTCGTTGGAAATACCTCTCTCTTTTGTATTCCAATTTTCTAGATTAATCTTTCCTGCACAAAAACATGCTTGATCCACTAAGTGACATATATGCTGCCTTCGTATATATAGTTCTGGTAAAATTAACATTTTGGGTTTATCTTTATTTAAGGCATCGCCATGACTAGTCTGCATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATCGAGCAAACACACGCTCGGACGCATATTACACATGTTCATACACTTAATACTCGCTGTTTTGAATTCATGTTTTAGGAATATATATGTAGAGCCATAATCTTCTGGAAGGATTCACAGGTCGTGATATGATTCAATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGCGGTAGACAAATTGGATCATTGATTCTCTTTGATTCCTTCGAGAAGATTATAGCTCTCTCTTTTGTATTCCAATTTTCTAGATTAATCTTTCCTGCACAAAAACATGCTTGATCCACTAAGTGACATATATGCTGCCTTCGTATATATAGTTCTGGTAAAATTAACATTTTGGGTTTATCTTTATTTAAGGCATCGCCATGACTAGTCTGCATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATCGAGCAAACACACGCTCGGACGCATATTACACATGTTCATACACTTAATACTCGCTGTTTTGAATTCATGTTTTAGGAATATATATGTAGACACTTCTTCAATCCCGCCTTTTCACAGGTCGTGATATGATTCAATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGCGGTAGACAAATTGGATCATTGATTCTCTTTGATAAGGCGCGATTGAAGAATTGTCTCTCTTTTGTATTCCAATTTTCTAGATTAATCTTTCCTGCACAAAAACATGCTTGATCCACTAAGTGACATATATGCTGCCTTCGTATATATAGTTCTGGTAAAATTAACATTTTGGGTTTATCTTTATTTAAGGCATCGCCATGACTAGTCTGCATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATCGAGCAAACACACGCTCGGACGCATATTACACATGTTCATACACTTAATACTCGCTGTTTTGAATTCATGTTTTAGGAATATATATGTAGATCCCTTGTGACGAATCTAAGTTCACAGGTCGTGATATGATTCAATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGCGGTAGACAAATTGGATCATTGATTCTCTTTGATCTTAGAATCGTCACAAGTGATCTCTCTTTTGTATTCCAATTTTCTAGATTAATCTTTCCTGCACAAAAACATGCTTGATCCACTAAGTGACATATATGCTGCCTTCGTATATATAGTTCTGGTAAAATTAACATTTTGGGTTTATCTTTATTTAAGGCATCGCCATGACTAGTCTGCATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATCGAGCAAACACACGCTCGGACGCATATTACACATGTTCATACACTTAATACTCGCTGTTTTGAATTCATGTTTTAGGAATATATATGTAGACTAAATTAGACCTATCTTATTTCACAGGTCGTGATATGATTCAATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGCGGTAGACAAATTGGATCATTGATTCTCTTTGATATAAGAAAGGTCTAATTGAGTCTCTCTTTTGTATTCCAATTTTCTAGATTAATCTTTCCTGCACAAAAACATGCTTGATCCACTAAGTGACATATATGCTGCCTTCGTATATATAGTTCTGGTAAAATTAACATTTTGGGTTTATCTTTATTTAAGGCATCGCCATGACTAGT
Supplemental Figure S4. Sequence of the tRNA-pre-amiRNA tandem repeats for co-silencing five Arabidopsis immune receptor genes. The tRNAGly is in orange. Each Arabidopsis pre-miR319a-based pre-amiRNA is underlined with amiRNA and amiRNA* highlighted in magenta and blue, respectively.
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Supplemental Figure S5. Growth inhibition of the fls2 mutant is comparable to that of the wild-type in the presence of a cocktail of five immune ligands. A, The fls2 null mutant remains sensitive to the mixture of flg22 (10 or 100 nM), elf18 (10 or 100 nM), chitin (20 or 200 µg/ml), pep3 (10 or 100 nM) and nlp20 (10 or 100 nM). Seedlings were germinated without immune ligands for 5 d and then treated with immune ligands or mock for 7 d. Scale bar = 1 cm. B, Quantification of seedling growth inhibition. Relative inhibition was measured based on fresh weights of seedlings and is presented as the mean value ± s.d. of three biological replicates, each with 4 seedlings. WT, wild-type; NS, not significant.
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Supplemental Figure S6. amiRNA-mediated silencing of five immune receptor genes is largely due to translational inhibition instead of transcript degradation. A, Transcript levels of five target genes are reduced to different extents in transgenic Arabidopsis lines overexpressing the tRNA-pre-amiRNA tandem repeats. Transcript levels of target genes were quantified by RT-qPCR with the level in wild-type (WT) plants arbitrarily set as 1. Data are shown as mean ± s.d. (n = 3). **P < 0.01 (Student’s t-test). NS, not significant. B, FLS2 is barely detectable in these transgenic silencing lines.
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Supplemental Figure S7. Intronic amiR-CNGC2-m1 is active for silencing Arabidopsis CNGC2 in ETPamir assay. A, ETPamir screen identifies amiR-CNGC2-m1 as the optimal amiRNA (highlighted in red) for silencing CNGC2. B, Sequence of the GFP reporter gene containing the intronic amiR-CNGC2-m1. GFP is in green. The IV2 intron is in orange. The NotI site for pre-amiRNA insertion is underlined. The Arabidopsis pre-miR319a-based pre-amiR-CNGC2 is in black with amiRNA and amiRNA* highlighted in magenta and blue, respectively. C, Comparable silencing activities between normal amiR-CNGC2-m1 and intronic amiR-CNGC2-m1 (GFPamiR-CNGC2-m1) in ETPamir assay. Constructs encoding HA- or FLAG-tagged CNGC2 protein and the same tagged GFP (transfection control) were co-expressed with indicated amiRNAs in Arabidopsis protoplasts for 18 h. At least three independent repeats were conducted with similar results. Ctrl, control without amiRNA co-expression.
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Supplemental Figure S8. Intronic amiRNA-producing GFP could serve as a selectable marker for transgenic plants. A, Transgenic T1 plants overexpressing GFPamiR-CNGC2-m1 (marked by dashed borders) showed a cyan color when irradiated by a blue-light flashlight, whereas surrounding non-transformed plants showed a dark purple color. B, Transgenic T2 plants with GFP fluorescence exhibit an obvious dwarf phenotype. Scale bar = 1 cm.
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Supplemental Figure S9. Intronic amiRNA-producing GFP reporter facilitates the screen of transgenic Arabidopsis plants with optimal silencing of At1g55000. A, ETPamir screen identifies amiR-At1g55000-m5 as the optimal amiRNA (highlighted in red) for silencing At1g55000. Constructs encoding At1g55000-FLAG and LYK5-FLAG (transfection control) were co-expressed with indicated amiRNAs in Arabidopsis protoplasts for 36 h. At least three independent repeats were conducted with similar results. Ctrl, control without amiRNA co-expression. B, Quantified transcript levels of At1g55000 in randomly selected transgenic Arabidopsis plants based on herbicide selection or GFP selection. Transcript levels of At1g55000 were quantified by RT-qPCR with the level in wild-type (WT) plants arbitrarily set as 1. Data are shown as mean ± s.d. (n = 3). **P < 0.01 (Student’s t-test).
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Supplemental Figure S10. Intronic amiR-GFP and normal amiR-GFP are equally active for silencing GFP in ETPamir assay. A, Sequence of the NLS-mCherry-GUS reporter gene containing the intronic amiR-GFP. The nuclear localization sequence is in green. mCherry is in red and GUS is in purple. The IV2 intron is in orange. The NotI site for pre-amiRNA insertion is underlined. The Arabidopsis pre-miR319a-based pre-amiR-GFP is in black with amiRNA and amiRNA* highlighted in magenta and blue, respectively. B, Constructs encoding GFP-HA and LYK5-HA (transfection control) were co-expressed with indicated amiRNAs in Arabidopsis protoplasts for 36 h. The experiment was repeated three times with similar results. Ctrl, control without amiRNA co-expression.
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Supplemental Figure S11. Intronic amiR-CEBiP-m3 for silencing rice CEBiP. A, ETPamir screen identifies amiR-CEBiP-m3 (red) as the most effective amiRNA for silencing rice CEBiP. Constructs encoding HA-tagged CEBiP protein and the same tagged GFP (transfection control) were co-expressed with indicated amiRNAs in rice protoplasts for 36 h. At least three independent repeats were conducted with similar results. Ctrl, control without amiRNA co-expression. B, Sequence of the GFP reporter gene containing the intronic amiR- CEBiP-m3. GFP is in green. The IV2 intron is in orange. The NotI site for pre-amiRNA insertion is underlined. The rice pre-miR528-based pre-amiR-CEBiP is in black with amiRNA and amiRNA* highlighted in magenta and blue, respectively.
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Supplemental Figure S12. Rice amiRNA precursor and intronic amiRNA-producing GFP reporter are both functional in wheat cells. A, Five amiR-CEBiP candidates produced from the rice amiRNA precursor (miR528) are also active in silencing CEBiP in wheat protoplasts. B, Wheat protoplasts expressing GFPamiR-CEBiP-m3 exhibit strong GFP fluorescence. The construct expressing nuclear localized HY5-mCherry was co-transfected to label the transfected cells. C, Intronic amiR-CEBiP-m3 and normal amiR-CEBiP-m3 are equally active for silencing CEBiP in wheat protoplasts. In A and C, constructs encoding HA-tagged CEBiP protein and the same tagged GFP (transfection control) were co-expressed with indicated amiRNAs in wheat protoplasts for 36 h. The experiment was repeated three times with similar results. Ctrl, control without amiRNA co-expression.
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Supplemental Table S1. Summary of amiRNAs tested in this study.
Target
amiRNA
Design Name
Sequence
(5’ to 3’)
Target site
(5’ to 3’)
GFP amiR-GFP TTGATATAGACGTTGTGGCTG 441-461 WMD3
FLS2
(At5g46330.1)
amiR-FLS2-c1
amiR-FLS2-c2
amiR-FLS2-c3
amiR-FLS2-c4
amiR-FLS2-m1
amiR-FLS2-m2
TTTAAATTAGCCAGAGTGCCA
TATATTATTGAACCCCACCGG
TATGGTTTTCTGATAACGCCA
TAACCGAGACTACATGTCCGT
TCGCTAGTGCAATGCCAAAGA
TAAAGGGTCGTTGGAAATACC
702-722
1162-1182
1016-1036
278-298
116-136
184-204
WMD3
WMD3
WMD3
WMD3
new
strategy
new
strategy
GLK1
(At2g20570.1)
amiR-GLK-m1
amiR-GLK-c1
amiR-GLK-c2
amiR-GLK-c3
TAGCAACGTTGTGACGAGTGC
TATAACACCGTCAACCGACGG
TCGTTGTGACGAGTGACACAT
TCGTTGTGACGAGTGACACAA
703-723
1314-1334
698-718
698-718
new
strategy
WMD3
WMD3
WMD3
GLK2
(At5g44190.1)
amiR-GLK-m1
amiR-GLK-c1
amiR-GLK-c2
amiR-GLK-c3
TAGCAACGTTGTGACGAGTGC
TATAACACCGTCAACCGACGG
TCGTTGTGACGAGTGACACAT
TCGTTGTGACGAGTGACACAA
623-643
1159-1179
618-638
618-638
new
strategy
WMD3
WMD3
WMD3
APK2A
(At1g14370.1)
amiR-APK2-c1
amiR-APK2-c2
amiR-APK2-c3
amiR-APK2-c4
amiR-APK2-m1
TTAAGTGGCTGCGCGCCACGT
TTTGCAATGCAAGACTAGCTG
TATACGGTGTTGCCCAGCCTA
TCAGAATTAGCATGGGGGCGA
TTTAAGCATTGCAATGCAAGG
832-852
1305-1325
1206-1226
331-351
1312-1332
WMD3
WMD3
WMD3
WMD3
new
strategy
APK2B
(At2g02800.1)
amiR-APK2-c1
amiR-APK2-c2
amiR-APK2-c3
amiR-APK2-c4
amiR-APK2-m1
TTAAGTGGCTGCGCGCCACGT
TTTGCAATGCAAGACTAGCTG
TATACGGTGTTGCCCAGCCTA
TCAGAATTAGCATGGGGGCGA
TTTAAGCATTGCAATGCAAGG
733-753
1206-1226
1107-1127
241-261
1213-1233
WMD3
WMD3
WMD3
WMD3
new
strategy
SERK1
(At1g71830.1)
amiR-SERK-c1
amiR-SERK-m1
TATGTAGGGATAAACAGGCTA
TGCTCCAAGTACTGCAAGTAT
1534-1554
696-716
WMD3
new
strategy
SERK2
(At1g34210.1)
amiR-SERK-c1
amiR-SERK-m1
TATGTAGGGATAAACAGGCTA
TGCTCCAAGTACTGCAAGTAT
1534-1554
696-716
WMD3
new
strategy
SERK3
(At4g33430.1)
amiR-SERK-c1
amiR-SERK-m1
TATGTAGGGATAAACAGGCTA
TGCTCCAAGTACTGCAAGTAT
1561-1581
759-779
WMD3
new
strategy
15
SERK4
(At2g13790.1)
amiR-SERK-c1
amiR-SERK-m1
TATGTAGGGATAAACAGGCTA
TGCTCCAAGTACTGCAAGTAT
1252-1272
456-476
WMD3
new
strategy
SERK5
(At2g13800.1)
amiR-SERK-c1
amiR-SERK-m1
TATGTAGGGATAAACAGGCTA
TGCTCCAAGTACTGCAAGTAT
1041-1061
287-307
WMD3
new
strategy
SR45.1
(At1g16610.1) amiR-SR45.1-m1 TTTTCCGTTGAGGAGATGTCT 744-764
new
strategy
EFR
(At5g20480.1)
amiR-EFR-m1
amiR-EFR-m2
amiR-EFR-m3
TCTCCTGCGGCCACATGTGAC
TGGTACTTCCAAAAGAGTTGT
TTCCTTCGAGAAGATTATAGC
213-233
334-354
403-423
new
strategy
new
strategy
new
strategy
CERK1
(At3g21630.1)
amiR-CERK1-m1
amiR- CERK1-m2
amiR- CERK1-m3
TCGGAGCGATTAGAGAAATCT
TCCTGCACTTAGATTCCACGG
TAAGGCGCGATTGAAGAATTG
62-82
110-130
204-224
new
strategy
new
strategy
new
strategy
PEPR1
(At1g73080.1)
amiR-PEPR1-m1
amiR-PEPR1-m2
amiR-PEPR1-m3
TATGTGGGTCGATAGAAAGAG
TCTTAGAATCGTCACAAGTGA
TTGACCTGAAACCCTAGAACG
49-69
206-226
250-270
new
strategy
new
strategy
new
strategy
RLP23
(At2g32680.1)
amiR-RLP23-m1
amiR-RLP23-m2
amiR-RLP23-m3
TGGGGACGACAGGCCACAATA
TATAAGAAAGGTCTAATTGAG
TCTCTAATCTATGGAGATTGC
99-119
449-469
650-670
new
strategy
new
strategy
new
strategy
CNGC2
(At5g15410.2)
amiR-CNGC2-m1
amiR-CNGC2-m2
amiR-CNGC2-m3
amiR-CNGC2-m4
amiR-CNGC2-m5
TCGGAAAACAGTCCAATCCAC
TAGCACCGGCGTCTCATCGCT
TGGGACGCCTACTTGTGTGCA
TAAAGCCATCCCACGAGCTAA
TCGAGACGTAGGCCAGTCTGA
86-106
186-206
228-248
444-464
613-633
new
strategy
new
strategy
new
strategy
new
strategy
new
strategy
16
At1g55000.1
amiR- At1g55000-m1
amiR- At1g55000-m2
amiR- At1g55000-m3
amiR- At1g55000-m4
amiR- At1g55000-m5
TATCACGGCAATAAAGCGCCA
TACAGACGCAGCTCGCACGCG
TCCGCCATGGAGCCGTGAAAG
TGACGGCGAGGCTAGTCACAC
TCTTGAGTAAATACCATGGTC
130-150
193-213
259-279
376-396
447-467
new
strategy
new
strategy
new
strategy
new
strategy
new
strategy
CEBiP
(Os03g04110.1)
amiR-CEBiP-m1
amiR-CEBiP-m2
amiR-CEBiP-m3
amiR-CEBiP-m4
amiR-CEBiP-m5
TGGCTGACATTTATCTTGTTA
TATTTCTGGTGAGAAGCGTGG
TCCATAGGTGCCATCCGGGAG
TGGCACTGACGGGCATCCCTT
TAACCACTGTAGGCGCACGTT
532-552
681-701
809-829
905-925
982-1002
new
strategy
new
strategy
new
strategy
new
strategy
new
strategy
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Supplemental Table S2. Primers for RT-qPCR or stem-loop RT-PCR used in this study.
Gene Primer
Name Sequence (5’ to 3’)
- miRNA-RP GTGCAGGGTCCGAGGT
mCherry
mCherry-RT GGTGTAGTCCTCGTTGTGGG
mCherry -QF GACGGCCCCGTAATGCAGAA
mCherry -QR GGTCTTGACCTCAGCGTCGT
amiR-FLS2-m1 amiR-FLS2-m2-RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG
GATACGACGGTATT
amiR-FLS2-m2-FP TCGCGGTAAAGGGTCGTTGGAAA
amiR-EFR-m3 amiR-EFR-m3-RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG
GATACGACGCTATA
amiR-EFR-m3-FP GCGGCGGTTCCTTCGAGAAGATTA
amiR-CERK1-m3 amiR-CERK1-m3-RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG
GATACGACCAATTC
amiR-CERK1-m3-FP GGTACGGTAAGGCGCGATTGAAGA
amiR-PEPR1-m2 amiR-PEPR1-m2-RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG
GATACGACTCACTT
amiR-PEPR1-m2-FP GCGGCGGTCTTAGAATCGTCACAA
amiR-RLP23-m3 amiR-RLP23-m3-RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG
GATACGACCTCAAT
amiR-RLP23-m3-FP GCTCCGGCGGTATAAGAAAGGTCTAAT
amiR-CNGC2-m1 amiR-CNGC2-m1-RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG
GATACGACGTGGAT
amiR-CNGC2-m1-FP TAGGCGGTCGGAAAACAGTCCAAT
SnoR101
(At1g20690.1)
AtSnoR101-FP GGGATACACTTGATCTCTGAACT
AtSnoR101-RP GCATCAGCAGACCAGTAGTTATC
FLS2
(At5g46330.1)
AtFLS2-QF CGAGGTCGAGAAGTTTAGCAG
AtFLS2-QR ACGTCCAAAGTATTCAACCTTCGT
EFR
(At5g20480.1)
AtEFR-QF CGGATGAAGCAGTACGAGAA
AtEFR-QR CCATTCCTGAGGAGAACTTTG
CERK1
(At3g21630.1)
AtCERK1-QF TCGAAACAGTTCTTGGCGGAAC
AtCERK1-QR ACAATATCCAATCAGGCGAACCAG
PEPR1
(At1g73080.1)
AtPEPR1-QF ATTCTCGTGGACGAGCTTCTGG
AtPEPR1-QR TGCCAGTTCCGTCACTTGCATC
RLP23
(At2g32680.1)
AtRLP23-QF GCTCTGTGATGGTGCCTCTT
AtRLP23-QR AAGCTTTGAGCCAGAACGGA
At1g55000.1 At1g55000-QF GCAATCCTGAAATTCTCGCAAACAC
At1g55000-QR AGAACTCAGACAATGCGGATCTAGG
ACT1
(At2g37620.1)
AtACT1-QF GATTCCGTTGTCCTGAGGTTCTTTAC
AtACT1-QR GACTCGTCATACTCTGCCTTTGCG
CEBiP
(Os03g04110.1)
OsCEBiP-QF ATTCTAGATGTCCCGCTCCCTGTG
OsCEBiP-QR TGCAGTTTCCTGCGGTGAATCC
18
Actin-1
(Os03g50885.1)
OsActin-1-QF CCACTATGTTCCCTGGCATT
OsActin-1-QR GTACTCAGCCTTGGCAATCC
19
Supplemental Dataset S1 Gene-specific amiRNA candidates for Arabidopsis nuclear genes. The compressed excel file includes 533,429 gene-specific amiRNA candidates bioinformatically identified to target the CDSs of 27,136 Arabidopsis genes. Comprehensive information is provided for each amiRNA candidate from left to right: amiRNA_ID (column 1), Chromosome (column 2), Target AGI (column 3), Target start site (column 4), Target end site (column 5), amiRNA sequence (column 6), Mismatch (column 7), Hybridization energy (column 8), and Energy ratio (column 9). Note that all amiRNA candidates have either no mismatch (“0” in column 7) or a single mismatch at the position 21 (“1” in column 7). Energy ratio means the hybridization energy of the amiRNA to the target sequence divided by that of a perfect complement to the target sequence. Therefore, if there is no mismatch between the amiRNA and the target sequence (“0” in column 7), the Energy ratio is 100%. The user can use “Find” function to search the gene ID to obtain ready-to-screen amiRNA candidates for the gene of interest.
