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Bioactive Constituents, Metabolites, and Functions
miRNA and mRNA expression profiles reveal insight into the chitosan-mediated regulation of plant growth Xiaoqian Zhang, Kecheng Li, Ronge Xing, Song Liu, Xiaolin Chen, Haoyue Yang, and Pengcheng Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06081 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018
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Journal of Agricultural and Food Chemistry
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miRNA and mRNA expression profiles reveal insight
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into the chitosan-mediated regulation of plant growth
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Xiaoqian Zhang†,§, Kecheng Li*,†,‡, Ronge Xing†,‡, Song Liu†,‡, Xiaolin Chen†,‡,
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Haoyue Yang†,‡, Pengcheng Li*,†,‡
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†
Key Laborotory of Experimental Marine Biology, Institute of Oceanology,
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Chinese Academy of Sciences, Qingdao 266071, China
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‡
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Laboratory for Marine Science and Technology, Qingdao 266237, China
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§
Laboratory for Marine Drugs and Bioproducts of Qingdao National
University of Chinese Academy of Sciences, Beijing 100049, China
11 12 13 14 15
*Corresponding author.
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Pengcheng Li
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Tel.: +86 532 82898707; fax: +86 532 82968951.
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E-mail address:
[email protected].
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Kecheng Li
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Tel.: +86 532 82898641; fax: +86 532 82968780.
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E-mail address:
[email protected].
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ABSTRACT
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Chitosan has been numerously studied as a plant growth regulator and stress
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tolerance inducer. To investigate the roles of chitosan as bio-regulator on
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plant and unravel its possible metabolic responses mechanisms, we
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simultaneously investigated mRNAs and microRNAs (miRNAs) expression
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profiles of wheat seedlings in response to chitosan heptamer. We found 400
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chitosan-responsive
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up-regulated and 132 down-regulated mRNAs, many of which were related to
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photosynthesis, primary carbon and nitrogen metabolism, defense responses
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and
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chitosan-mediated regulation on plant growth. We identified 87 known and 21
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novel miRNAs, among which 56 miRNAs were induced or repressed by
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chitosan
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miRNA171a, miRNA319 and miRNA1127. The integrative analysis of miRNA
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and mRNA expression profiles in this case provides fundamental information
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for further investigation of regulation mechanisms of chitosan on plant growth
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and will facilitate its application in agriculture.
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KEYWORDS : Chitosan; mRNA; miRNA; Transcription factor; Wheat
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seedlings
transcription
heptamer,
differentially
factors.
such
expressed
Moreover,
as
genes,
miRNAs
miRNA156,
also
including
participate
miRNA159a,
42
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in
miRNA164,
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INTRODUCTION
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On account of various problems such as growing population, worsening
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environmental pollution and decreased soil fertility, conventional crop
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production is increasingly being challenged.1 In order to realize the
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sustainable increase in crop yield, it is crucial to develop techniques to
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promote crop growth and increase crop yield. One current agricultural
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practice to increase crop yield is the application of exogenous biostimulants,
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which are able to increase crop productivity and alleviate the negative effect
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of biotic or abiotic stress. Mounting studies have revealed that biostimulants,
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such as protein hydrolysates, seaweed extracts and microbial fermentation,
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greatly enhanced the plant growth and plant productivity over past
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decades.2,3
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Recently various carbohydrates have also attracted increasing attention
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for their possible roles as resistance inducers. Chitosan is the second most
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abundant carbohydrate biopolymers in the world, which is mainly consisting
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of
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N-acetyl-D-glucosamine.4 Actually, chitin and chitosan derivatives have been
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assigned as one kind of biostimulants.5,6 In the last decades, chitosan had
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been numerously studied as a plant growth regulator and stress tolerance
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inducer. The physiological responses to chitosan in plants have been largely
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investigated in lab, greenhouse or field conditions, such as stimulating seed
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germination,7 improving chlorophyll contents,8 inducing salt and drought
β-1,4-linked
D-glucosamine
and
partially
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β-1,4-linked
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tolerance,8, 9 and activating plant antimicrobial activity.10 In large-scale field
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production, it had been also reported that chitosan oligosaccharides could
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significantly increase the yield of wheat.11 Moreover, cDNA microarray
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analyses of gene expression in rice and Brassica napus treated with chitosan
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oligosaccharides elicitor were conducted by Tomiyama et al. and Yin et al.,
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respectively, which suggested that the differentially expressed genes induced
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by chitosan were involved in different biological processes including defense,
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primary metabolism, transcription, and signal transduction.12, 13 Actually, the
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activities of chitosan are closely related with its degree of polymerization (DP).
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14,15
Based on our prior studies, chitosan heptamer (GlcN)7 was efficient in
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promoting the morphological growth parameters of plant under stress or
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non-stress conditions.16,17 However, the exactly molecular mechanism and
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regulatory networks still remain unknown.
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Recently, high-throughput transcripts profiling greatly facilitated to
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explore the system change of complex biological processes with the
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advantages of high resolution, deep coverage and dynamic landscapes,
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which
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Unquestionably, RNA sequencing (RNA-Seq) would be a powerful tool to
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reveal the global genetic and molecular responses of plants to chitosan. On
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the other hand, microRNAs (miRNAs) are a class of endogenous non-coding,
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short
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post-transcriptional levels by targeting mRNAs for cleavage or translational
is
considerably
small
RNAs
more
(~24
nt)
efficient
that
than
regulate
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microarray
gene
analysis.18
expression
at
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repression.19 Functionally, miRNAs could regulate the expression of many
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important genes, and a majority of these genes are transcriptional factors
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(TFs).20 Numerous studies have suggested that miRNAs are associated with
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diverse plant biological processes, including leaf and root development,
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signal transduction, biotic or abiotic stress responses.21,
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expression analysis of miRNA would also be a valuable tool to demonstrate
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the potential action mode of chitosan in plant system.
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Therefore, the
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Wheat is one of the most important crops in the world because of its high
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production, wide geographical range and high proportion of human
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consumption.1 In the present experiment, chitosan heptamer was further
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used as a representative to investigate the mode of action of chitosan on
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plant growth. We simultaneously profiled mRNA and miRNA expression in
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chitosan-treated
wheat
seedlings
using
high-throughput
sequencing
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technology,and further performed an integrative analysis of mRNAs and
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miRNA expression in order to reveal the complicated network of metabolic
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and signaling pathways involved in chitosan-mediated regulation on plant
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growth. To our best knowledge, this is the first study to investigate the
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regulation mechanism of chitosan on wheat seedlings at mRNA and miRNA
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expression level via deep sequencing analysis. Our results are expected to
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provide a new insight into the molecular mechanisms of chitosan as a growth
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regulator and resistance inducer, which will provide and significant guidance
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for its application in agriculture. 5
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MATERIALS AND METHODS
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Plant growth conditions and treatments. (GlcN)7 (≥93%) was prepared
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following the method described by Li et al. (2013).23 Seeds of wheat (Triticum
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aestivum L. Jimai 22) was used in the present study, which has some
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excellent characteristics with high yield, strong resistance to lodging and
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disease. It has become the most popular wheat cultivar in China since
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2010.24 Seeds were germinated at 25℃ for 24 h in the dark, and then
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transplanted into Petri dishes (11.5 cm in diameter) with Hoagland solution in
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a light growth chamber with 25℃/20℃ and 14-h/10-h light/dark photoperiod.
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After the second leaf was fully developed, the wheat seedlings were divided
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into two experimental groups randomly with three replicates each, including
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the control group (CK, sprayed with distilled water) and (GlcN)7 treatment
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group (sprayed with 15 mg/L (GlcN)7). After forty-eight hours, the second
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functional leaves of twenty randomly-selected plants from two treatment
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groups were collected and immediately frozen with liquid nitrogen and stored
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in -80℃ for further physiological and molecular analyses. For the analysis of
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the biomass, ten plants from each 3 replications in the control and
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(GlcN)7-treated groups were selected randomly and their shoot fractions were
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used to determine fresh weight and dry weight.
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Determination carbohydrates, and total antioxidant activity. The total
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antioxidant activity was measured according to Ertani et al. (2017).25 The
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sucrose was extracted from 0.1g fresh leaves of wheat seedlings with 2M 6
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NaOH, boiled for 5 min and determined based on resorcinol hydrochloric acid
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method. The content of starch was determined according to the method
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described by Zrenner et al. (1995).26
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RNA isolation, library preparation and sequencing. Total RNA was
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isolated using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). The RNA
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quality was monitored on 1% agarose gel and the concentration was
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determined using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life
138
Technologies, CA, USA). mRNA and small RNA libraries were generated
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using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) and
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NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.)
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following manufacturer’s instructions, and the library quality was assessed on
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the Agilent Bioanalyzer 2100 system. Then each sequencing library was
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performed using an Illumina Genome Analyzer (Illumina, San Diego, CA,
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USA).
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Expression analysis of mRNAs. For the transcriptome libraries, after
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removing adaptors and low-quality reads from raw reads, clean reads were
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mapped to the reference genome of wheat released by NCBI wheat EST
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collection
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(http://ftp.ncbi.nih.gov/repository/UniGene/Triticum_aestivum/Ta.seq.uniq.gz)
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and
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(http://plants.ensembl.org/Triticum_aestivum) to searched for CATG sites. All
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the possible CATG + 17 nt sequences were used as a reference tag library for
IWGSC
wheat
genomic
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DNA
sequences
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following clean tags mapping. All clean tags that were uniquely mapped to the
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reference sequences (with ≤1 mismatch) were used for differential expression
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analysis. The gene expression level of mRNA was calculated by the RPKM
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(reads per kb per million reads) method. Differential gene expression analysis
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between two mRNA libraries was performed using the R packages of DESeq
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and those genes with an adjusted P-value of 1 were considered to be differentially expressed.
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Validation of mRNA and miRNA expression by qRT-PCR. To validate the
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sequencing data, we randomly choose 15 differentially-expressed mRNAs
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and 8 miRNAs for quantitative real-time RT-PCR (qRT-PCR) analysis. The
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primers for the mRNA and miRNA RT-PCR were designed by primer premier
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5.0 and shown in supplementary Table S1. For mRNA quantification, total
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RNA was extracted with Takara MiniBEST Plant RNA Extraction Kit (Takara,
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Dalian, China) according to the manufacturer’s instructions. Then total RNA
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was used as template to synthesis complementary DNA (cDNA) by
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PrimeScriptTM RT reagent Kit (Takara, Dalian, China), which contains 5 ×
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gDNA Eraser Buffer, gDNA Eraser, PrimeScript RT Enzyme Mix, RT Primer
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Mix and 5 × PrimeScript Buffer 2. Then, RT-PCR was performed with SYBR®
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Premix Ex TaqTM (Tli RNaseH Plus) (Takara, Dalian, China). In brief, the
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qRT-PCR were conducted in a total volume of 20 µL as follows: 10 µL SYBR
the
using
miRNA
qvalue.
prediction
Small
software
miRNA
RNAs
target
with
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Mireap
prediction
qvalue