Documentos de Académico
Documentos de Profesional
Documentos de Cultura
PAPER
unique miRNA sequences were veried by quantitative real-time PCR (qRT-PCR), and the results nearly
agreed with the high-throughput sequencing data. The present study increases the number of miRNAs
DOI: 10.1039/c5ra14672k
identied in B. napus and suggests that there are possible dierences in the miRNA expression between
www.rsc.org/advances
Introduction
In eukaryotes, gene expression is regulated and controlled at
the transcriptional and post-transcriptional levels, and one
class of regulators of post-transcriptional gene silencing
(PTGS) is small RNAs (sRNAs).1,2 MicroRNAs (miRNAs), which
constitute a major class of sRNAs that are approximately 2124
nucleotides (nt) in length, are endogenous, noncoding, regulatory sRNA molecules that have been widely identied in
animals, plants and viruses.25 Increasing lines of evidence
have shown that miRNAs play an important role in a wide
range of plant processes, including development, signal
transduction, and various biological and environmental stress
responses.5
In higher plants, miRNA genes are transcribed into primary
miRNA (pri-miRNA) transcripts by RNA polymerase II, and these
transcripts form stable hairpin structures. Dicer-like 1 (DCL 1),
information
(ESI)
available.
See
DOI:
RSC Advances
Paper
High-throughput sequencing
Two sRNA libraries were constructed using the Illumina TruSeq
Small RNA Preparation Kit (LC Sciences, Hangzhou, China). The
total sRNA was ligated to 3p and 5p adapters (ADTs; 3p adapter:
50 -TGGAATTCTCGGGTGCCAAGG-30 ; 5p adapter: 50 -GUUCAGAGUUCUACAGUCCGACGAUC-30 ), and the corresponding cDNA
was obtained by reverse-transcription PCR. Following purication, the cDNA from the two sRNA libraries was sequenced
using an Illumina Genome Analyzer II (LC Sciences, Hangzhou,
China). Aer removing low-quality data and low-copy-number
reads (reads < 10), the raw reads were obtained using the
Illumina Pipeline v1.5 soware (LC Sciences, Hangzhou,
China). The ACGT101-miR program (LC Sciences, Hangzhou,
China) was then used for further data analysis.2833
miRNA identication
Aer ltering out ADTs data, sequences with lengths < 17 and >25
nt, junk data, RFam (http://rfam.janelia.org) and repeats (http://
www.girinst.org/repbase), the remaining reads (clean reads) were
subjected to miRNA identication using the Brassicaceae premiRNAs and miRNAs in miRBase 21.0 (http://www.mirbase.org)
and the B. napus genome database (http://www.genoscope.cns.fr/
brassicanapus). Three mismatches were allowed between the
candidate miRNAs and the known Brassicaceae pre-miRNA and
miRNA sequences.3136 The reads that mapped to known Brassicaceae pre-miRNAs and whose pre-miRNAs or candidate miRNAs
mapped to the B. napus genome were identied as conserved
miRNAs. In addition, the reads that did not map to known Brassicaceae pre-miRNAs but mapped to the B. napus genome were
considered novel miRNAs. Furthermore, the stem-loop structures
(secondary structures) of all of the identied and potential
pre-miRNAs from the B. napus genomic positions were predicted
using the UNAFold soware (http://www.mfold.rna.albany.edu/?
qmfold/RNA-Folding-Form),34,37 and only those with high
minimal folding energy indexes (MFEIs) were considered (MFEI $
0.7 for conserved miRNAs, MFEI $ 0.9 for novel miRNAs).38
Degradome sequencing
Two degradome libraries were constructed based on the
method described by Ma et al.39 poly(A)-enriched RNAs were
obtained and ligated to a 5p adapter harboring the EcoP15 I
recognition site. Through reverse-transcription PCR, the ligated
sequences were converted to cDNA. Following EcoP15 I digestion and purication, the cDNA was subjected to cluster analysis with an Illumina Cluster Station (LC Sciences, Hangzhou,
China) and sequenced using an Illumina Genome Analyzer II
(LC Sciences, Hangzhou, China). Lastly, aer removing lowquality data, the raw reads were obtained using the Illumina
Pipeline v1.5 soware (LC Sciences, Hangzhou, China).
Target identication and annotation
Aer ltering out ADTs data and reads with lengths <15 nt, the
remaining reads were compared with a cDNA library in the B.
napus genome database. Aerward, the mapped-cDNA reads
were compared with the identied miRNAs to perform an
Paper
RSC Advances
Results
Analysis of sRNA libraries
To identify conserved and novel miRNAs in transgenic B.
napus (TG) and its acceptor (B. napus cv. Westar, WA), two
sRNA libraries were constructed, rstly. Through highthroughput sequencing, a total of 15 287 111 (from the WA
library) and 13 084 245 (from the TG library) raw reads were
generated (Additional le 1: Table S1). Aer ltering out the
3ADT data, reads with lengths < 17 and >25 nt, junk data,
RFam and repeats, 11 285 339 and 9 574 008 clean reads
corresponding to 3 073 405 and 2 579 167 unique reads with
lengths of 1725 nt were obtained for the two libraries,
respectively (Additional le 1: Table S1, Additional le 2:
Table S2.1). The distribution of the lengths of the clean
reads was mainly between 21 and 24 nt in the two libraries
RSC Advances
Fig. 2
Paper
Distribution of the families of the conserved unique miRNA sequences in the WA and TG libraries.
Table 1
Conserved unique miRNA sequences that are highly expressed (normalized reads $ 1000) in the WA and TG libraries
Namea
Sequence
WA norm
TG norm
log2 ratiob
bna-miR156e_R+1
bra-miR158-3p
bra-miR158-5p
bna-miR159
bna-MIR159-p5
ath-MIR159a-p5, aly-MIR159a-p5
bra-miR162-3p
bna-miR166a,b,c,d,e
aly-miR166a-5p
bna-miR166f
aly-miR166b-3p_L+1
bna-MIR167c-p3
bna-miR168a
bra-miR168b-5p,c-5p
ath-miR390a-3p_R-1_1ss5AG
bna-miR396a_1ss21TG
bra-miR398-3p
ath-miR399c-3p
bna-miR403
bna-MIR824-p3
bna-miR1140
bna-MIR1140-p5
bra-miR1885b
bra-MIR5719-p3_1ss21AT
bra-MIR9563a-p5_1ss17CT
TGACAGAAGAGAGTGAGCACT
TTTCCAAATGTAGACAAAGCA
CTTTGTCTATCGTTTGGAAAAG
TTTGGATTGAAGGGAGCTCTA
AGCTGCTAAGCTATGAATCCC
AGCTGCTAAGCTATGGATCCC
TCGATAAACCTCTGCATCCAG
TCGGACCAGGCTTCATTCCCC
GGAATGTTGTCTGGCTCGAGG
TCGGACCAGGCTTCATCCCCC
TTCGGACCAGGCTTCATTCCCC
GATCATGTTCGTAGTTTCACC
TCGCTTGGTGCAGGTCGGGAA
TCGCTTGGTGCAGGTCGGGAC
CGCTGTCCATCCTGAGTTTC
TTCCACAGCTTTCTTGAACTG
TGTGTTCTCAGGTCACCCCTG
TGCCAAAGGAGAGTTGCCCTG
TTAGATTCACGCACAAACTCG
CCTTCTCATCGATGGTCTAGA
ACAGCCTAAACCAATCGGAGC
TCCGATTGGCTTTAGGCTGTT
TACATCTTCTCCGCGGAAGCTC
TCGGATTATCATCACAACACT
AGCGGAATATAAGAACTCGTCTCT
3496
18 164
11 274
29 676
1078
1476
2464
131 410
3809
36 772
1859
4435
2131
3475
546
22 401
3207
3183
8430
2537
6607
1526
8990
949
3901
687
26 437
14 289
18 564
472
867
2548
213 973
2522
59 919
2380
3639
1828
3766
1043
24 363
39 219
1257
10 001
4809
3771
671
8240
1608
2617
2.35**
0.54
0.34
0.68
1.19**
0.77
0.05
0.70
0.59
0.70
0.36
0.29
0.22
0.12
0.93
0.12
3.61**
1.34**
0.25
0.92
0.81
1.19**
0.13
0.76
0.58
a
Name regulation following Additional le 3: Table S3; red marked miRNAs were detected in degradome analysis. b log2(WA norm/TG norm); **
indicates highly signicant variation (p-value < 0.01 from both Chi-square 2 2 & Fisher's exact test and |log2(WA/TG)| $ 1) between the WA and TG
libraries.
Paper
RSC Advances
Table 2 Dierentially expressed conserved unique miRNA sequences (p < 0.01 and |log2(WA/TG)| $ 1) with above-average expression levels
(normalized reads $ 100) in the WA and TG libraries
Namea
Sequence
WA norm
TG norm
log2 ratiob
bna-miR156d,e,f,a_R-1
bna-miR156e_R+1
bna-MIR159-p5
bna-miR160a,b,c,d
bna-MIR162a-p5
bra-miR168b-3p,c-3p
bna-MIR169m-p3
ath-MIR169e-p3_L+1
ath-miR171-5p, bna-MIR171g-p5
bna-miR390a,b,c
bra-miR391-3p
bna-miR393_R+1
bra-miR398-3p
bra-miR398-5p
bol-miR398a-3p_R-1
ath-miR399c-3p
bna-MIR1140-p5
bra-miR5654a
bra-MIR5719-p5_1ss16AC
bna-miR6028_R+2
bna-miR6030
bna-miR6031
bra-MIR9557-p3_1ss13GA
TGACAGAAGAGAGTGAGCAC
TGACAGAAGAGAGTGAGCACT
AGCTGCTAAGCTATGAATCCC
TGCCTGGCTCCCTGTATGCCA
GGAGGCAGCGGTTGATCGATC
CCCGCCTTGCATCAACTGAAT
GCAAGTCGACTTTGGCTCTG
GCAAGTTGACTTTGGCTC
TATTGGCCTGGTTCACTCAGA
AAGCTCAGGAGGGATAGCGCC
ACGGTATCTCTCCTACGTAGC
TCCAAAGGGATCGCATTGATCC
TGTGTTCTCAGGTCACCCCTG
GGGTCGACATGAGAACACATG
TGTGTTCTCAGGTCACCCCT
TGCCAAAGGAGAGTTGCCCTG
TCCGATTGGCTTTAGGCTGTT
ATAAATCCCAAGCATCATCCA
TGTTGTGATGATAATCCGACT
TGGAGAGTAAGGACATTCAGATC
TCCACCCATACCATACAGACCC
AAGAGGTTCGGAGCGGTTTGAAGC
AATGAGGGCTGAATTGGAACGC
291
3496
1078
104
241
87
139
116
124
815
77
56
3207
21
35
3183
1526
84
144
176
93
375
170
81
687
472
366
91
229
30
21
19
408
172
143
39 219
143
366
1257
671
192
330
72
370
118
62
1.85
2.35
1.19
1.82
1.41
1.40
2.21
2.47
2.71
1.00
1.16
1.35
3.61
2.77
3.39
1.34
1.19
1.19
1.20
1.29
1.99
1.67
1.46
Name regulation following Additional le 3: Table S3; red marked miRNAs were detected in degradome analysis. b log2(WA norm/TG norm).
RSC Advances
Table 3
Paper
Novel unique miRNA sequences with above-average expression (normalized reads $ 100) in the WA and TG libraries
Namea
Sequence
WA norm
TG norm
log2 ratiob
PC-3p-9
PC-3p-15
PC-3p-20
PC-3p-47
PC-3p-53
PC-3p-72
PC-3p-88
PC-3p-101
PC-3p-128
PC-3p-133
PC-3p-138
PC-5p-21
PC-5p-23
PC-5p-50
PC-5p-92
PC-5p-94
PC-5p-102
PC-5p-110
PC-5p-116
PC-5p-127
PC-5p-137
PC-5p-138
GCTTTCCCCGGACGACTTTAAATT
TATCCCGGACGACCTTAAATT
ATTCCGACAAGAACTCCGCCCT
AAAGATTGTTGTGGTCTTGTGCCT
ATCCATTAGTATCAGACGATC
AGAGTCGGGTTCTTATATTCTGCT
ATATTCCGTTAAGAACTCCACCCT
TGCTTTCCCCGGACGACTTTAAAT
AACTTCCGCTAAGAACTCCATCCT
GGTCATGTCTGACAGCTTCACT
TAAGAACCCACATTAATCATG
TGAACCCAAGATCTCACCCTT
AGCGGAATATAAGAACCTGACTCT
ATCGGAATATAAGAACTCGTCTCT
ATCGGAATATAAGAAACTGTCTCT
AACGGAATATAAGAATTTGACTCT
AGCGGAATATAAGAATTTGTCTCT
GTTTTGAGAGATTGGGAAGCT
ACCGGAATATAAGAACTCGTCTCT
AGCGGAAAATAAGAACTCGTGTCT
GAGTGAGAAATGGAGTGATGAACA
AGCGGAATATAAGAACTCGACTCT
183
209
102
153
156
288
215
46
73
101
53
333
238
353
173
200
137
225
121
139
339
121
473
237
212
169
164
131
360
114
125
66
105
377
130
180
97
122
79
69
72
93
51
64
1.37**
0.18
1.06**
0.14
0.07
1.14**
0.74
1.31**
0.78
0.61
0.99
0.18
0.87
0.97
0.83
0.71
0.79
1.71**
0.75
0.58
2.73**
0.92
a
Name regulation following Additional le 5: Table S5; red marked miRNAs were detected in degradome analysis. b log2(WA norm/TG norm); **
indicates highly signicant variation (p-value < 0.01 from both Chi-square 2 2 & Fisher's exact test and |log2(WA/TG)| $ 1) between the WA and TG
libraries.
Verication of miRNAs
To verify the miRNA expression prole identied by highthroughput sequencing, 11 dierentially expressed unique
miRNA sequences at above-average expression (normalized
reads $ 100) targeting high-condence transcripts (categories
0, 1, 2 and 3) were selected for verication by quantitative realtime PCR (qRT-PCR). The results nearly agreed with the highthroughput sequencing data: bra-miR168b-3p,c-3p, bnamiR390a,b,c, bna-miR393_R+1, bra-miR398-3p, ath-miR399c3p and PC-3p-72, were possible dierentially expressed in the
two libraries (Fig. 5, Additional le 10: Table S10.2). However,
some of them did not exhibit signicant dierences in two lines
in the qRT-PCR verication, such as bna-miR156e_R+1, bnamiR160a,b,c,d and bna-miR6030 (Fig. 5, Additional le 10:
Table S10.2).
RSC Advances
Paper
Fig. 4 Classication and count of Gene Ontology (GO) annotation of the miRNA targets in the WA and TG degradome libraries. There are three
ontologies in GO: biological process, cellular component, molecular function.47. (Con BP) conserved miRNAs' targets in biological process; (Nov
BP) novel miRNAs' targets in biological process; (Con CC) conserved miRNAs' targets in cellular component; (Nov CC) novel miRNAs' targets in
cellular component; (Con MF) conserved miRNAs' targets in molecular function; (Nov MF) novel miRNAs' targets in molecular function.
RSC Advances
Paper
qRT-PCR validation of 11 potential dierentially expressed unique miRNA sequences in the WA and TG lines from high-throughput
sequencing.
Fig. 5
Discussion
Conserved and novel miRNAs
Although high-throughput sequencing is an ecient platform
for miRNA identication, it remains incapable of distinguishing highly homologous miRNAs. For example, bna-miR160a,
bna-miR160b, bna-miR160c and bna-miR160d, which correspond to the same sequence, had identical raw data (Additional
le 3: Table S3.1). However, through pairwise comparisons,
some miRNAs could be recognized. For example, only bnaMIR160c-p3 and bna-MIR160d-p3 were detected, which
demonstrates that the sequence only represents bna-miR160c
and bna-miR160d because these are from dierent arms of
the same pre-miRNA (Additional le 3: Table S3.1).
In miRBase 21.0, there are 92 B. napus miRNAs corresponding to 90 pre-miRNAs belonging to 33 miRNA families. As
expected, 119 B. napus miRNAs corresponding to 74 premiRNAs belonging to 27 miRNA families were identied in
this study. Among these miRNAs, 52, including bna-MIR159-p5,
bna-MIR166d-p5, bna-MIR167c-p3 and bna-MIR824-p3, are
novel strand miRNAs that map to B. napus pre-miRNAs but not
B. napus miRNAs because they are generated from the opposite
pre-miRNA arm (Additional le 3: Table S3.1). Furthermore,
the analysis also revealed 66 miRNAs (including 24 novel strand
miRNAs) corresponding to 47 pre-miRNAs belonging to 33
miRNA families that are included in miRBase 21.0 as detected
in other Brassicaceae species, i.e., Brassica rapa, Brassica oleracea, Arabidopsis thaliana and Arabidopsis lyrata, and 24 of the
miRNA families are not found in the B. napus data included in
Paper
RSC Advances
Conclusions
In summary, through high-throughput sequencing and degradome analysis, the present study identied numerous
conserved and novel unique miRNA sequences and their corresponding targets in transgenic B. napus and its acceptor
(Westar). These results contribute valuable information for
further increases in the number of identied miRNAs and the
identication of their target transcripts in B. napus. Furthermore, our results also suggest that some miRNAs might be
dierentially expressed between transgenic B. napus and its
acceptor. Moreover, dierential miRNA expression might lead
to dierential target degradation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CT, MY, and YY contributed to the experiment design and
management. CT, HZ and GL performed the experiments. CT,
FW, YP and RY participated in the data analyses. CT written
the manuscript. All authors checked and approved the
manuscript.
RSC Advances
Paper
Abbreviations
WA
TG
sRNA
miRNAs
nt
PTGS
pri-miRNA
pre-miRNA
DCL 1
HYL 1
SE
RISC
AGO
ESTs
TCs
ADTs
MFEIs
GO
KEGG
Acknowledgements
This work was nancially supported by the Program for
Changjiang Scholars and Innovative Research Team in University (IRT_14R27) and the National Natural Science Foundation
of China (NSFC). We appreciated Dr Song Chen for providing
the seedlings of the transgenic B. napus and its acceptor (B.
napus cv. Westar).
References
1 D. P. Bartel, MicroRNAs: genomics, biogenesis, mechanism,
and function, Cell, 2004, 116(2), 281297.
2 B. Khraiwesh, M. A. Arif and G. I. Seumel, et al.,
Transcriptional control of gene expression by microRNAs,
Cell, 2010, 140(1), 111122.
3 V. Ambros, microRNAs: tiny regulators with great potential,
Cell, 2001, 107(7), 823826.
4 V. Ambros, B. Bartel and D. P. Bartel, et al., A uniform system
for microRNA annotation, RNA, 2003, 9(3), 277279.
5 M. W. Jones-Rhoades, D. P. Bartel and B. Bartel, MicroRNAs
and their regulatory roles in plants, Annu. Rev. Plant Biol.,
2006, 57, 1953.
6 Y. Kurihara and Y. Watanabe, Arabidopsis micro-RNA
biogenesis through Dicer-like 1 protein functions, Proc.
Natl. Acad. Sci. U. S. A., 2004, 101(34), 1275312758.
7 A. C. Mallory, T. Elmayan and H. Vaucheret, MicroRNA
maturation and actionthe expanding roles of Argonautes,
Curr. Opin. Plant Biol., 2008, 11(5), 560566.
8 S. Griths-Jones, R. J. Grocock and S. van Dongen, et al.,
miRBase: microRNA sequences, targets and gene
nomenclature, Nucleic Acids Res., 2006, 34, D140D144.
9 S. Griths-Jones, H. K. Saini and S. van Dongen, et al.,
miRBase: tools for microRNA genomics, Nucleic Acids Res.,
2008, 36, D154D158.
Paper
RSC Advances
RSC Advances
Paper
60
61
62
63