Identification of soybean herbivoryregulated genes and a transgenic

investigation of their potential in insect

resistance
Yongli Wang, Hui Wang, Yujie Ma,
Wenming Yang, Qing Yang & Deyue Yu

Plant Cell, Tissue and Organ Culture

(PCTOC)
Journal of Plant Biotechnology
ISSN 0167-6857
Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-015-0837-9

1 23

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DOI 10.1007/s11240-015-0837-9

ORIGINAL ARTICLE

Identification of soybean herbivory-regulated genes

and a transgenic investigation of their potential in insect resistance
Yongli Wang1,2 Hui Wang1 Yujie Ma1 Wenming Yang1
Qing Yang3 Deyue Yu1,4

Received: 16 April 2015 / Accepted: 30 July 2015

Springer Science+Business Media Dordrecht 2015

Abstract Soybean (Glycine max) is an important economic crop worldwide that has been affected by common
cutworm (Spodoptera litura Fabricius) in southern China.
In this study, RNA-Seq was applied to extend our understanding of the functional complexity of the soybean
transcriptome in response to insects. Totals of 1004 and
1580 unigenes were differentially expressed in resistant
and susceptible soybean lines, respectively. The functional
classification of these unigenes identified the same significant categories as in the previous microarray results, most
of which were related to stress and defense responses. A
qRT-PCR analysis of 21 selected genes confirmed the
results of the RNA-Seq analysis. The integration of RNASeq, microarray and qRT-PCR data highlighted GmVSPb
and GmN:IFR as two candidate genes that may confer
insect resistance. A functional analysis of GmVSPb and
GmN:IFR revealed a certain degree of resistance to common cutworm in overexpressing tobacco lines, and
GmVSPb was more efficient in this role, which may be

Electronic supplementary material The online version of this

article (doi:10.1007/s11240-015-0837-9) contains supplementary
material, which is available to authorized users.
& Deyue Yu
dyyu@njau.edu.cn
1

National Center for Soybean Improvement, National Key

Laboratory of Crop Genetics and Germplasm Enhancement,
Nanjing Agricultural University, Nanjing 210095, China

Biofuels Institute, School of the Environment, Jiangsu

University, Zhenjiang 212013, China

College of Life Sciences, Nanjing Agricultural University,

Nanjing 210095, China

College of Agriculture, Nanjing Agricultural University,

Nanjing 210095, China

associated with JA signaling regulation and nicotine

biosynthesis. Taken together, our studies provide an overview and survey of the genes that are involved in insect
resistance. Transgenic studies of two herbivory-regulated
genes demonstrated their function in insect resistance and
indicated their potential application to enhance insect
resistance in crops.

Keywords Soybean  Common cutworm  RNA-Seq 

Vegetative storage protein  Transgenic plant  Insect
resistance

Introduction
Plants are sedentary in nature and experience various
environmental stresses, including herbivorous insect
attacks and microbial pathogen infections. Losses caused
by pests and diseases have been estimated at 37 % of the
agricultural production world-wide, of which at least
13 % is lost directly to insects (Gatehouse et al. 1992).
Although the application of chemical insecticides may kill
the pests, these chemicals also harm the environment and
increase costs to farmers. To lower costs of production
and respond to concerns related to insecticide residues,
additional research should be conducted on crop resistance to insect pests. All plants possess a certain degree of
resistance to insects; therefore, only a limited range of
herbivores are capable of feeding on each individual
species. Individual plants within one genus or even one
species vary in their strategies and levels of insect resistance. The defense mechanisms that have evolved in
many plants can be constitutive or inducible and expressed only upon herbivore damage (Zhang et al. 2008). The

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importance of induced defenses in reducing herbivore

damage has been established in both laboratory and field
studies. For example, transgenic tomato plants with an
inhibited ability to induce defense proteins are hypersusceptible to Manduca sexta (Orozco-Cardenas et al.
1993).
Soybean (Glycine max L. Merrill) is an important
economic crop with a wide range of applications in food,
feed and industrial products (Wilson 2008). Common
cutworm (Spodoptera litura Fabricius, CCW) is a major
pest in southern China and has a wide host range and
large appetite, feeding on nearly all vegetables and many
important grains (Cui and Gai 1997). We have initiated
studies to characterize the induced responses in two
different local soybean lines, Wanxianbaidongdou (WX)
and Nannong99-10 (NN), which are constitutively
resistant and susceptible to CCW, respectively (Wang
et al. 2011), and observed that the induced resistance
peaked at different times in the two soybean lines. The
longevity and level of induced resistance in the WX line
were higher than those in the susceptible NN line (Wang
et al. 2014). However, regardless of the difference, there
was always a point when the induced resistance peaked
in both of the lines, indicating that the signaling cascades
and downstream genes mediating the herbivore-induced
resistance that is activated at this time point may be
crucial to the induced defense mechanisms (Wang et al.
2014).
In nature, plants use numerous dynamic defense
strategies for protection against insect attacks (Walling
2000; Kessler et al. 2004). There are certain common
defensive proteins toxic to insects, including alkaloids,
phytoalexins, lectins and protease inhibitors (Broadway
and Duffey 1986; Felton et al. 1992; Steppuhn et al.
2004). These defensive secondary compounds are either
found as constitutive components or synthesized in
response to pest attack in various plant tissues. Genes
encoding the insecticidal proteins have been identified
and isolated from various plant species (Shewry and
Lucas 1997; Blair et al. 2006), including genes that
encode protease inhibitors, a-amylase, lectins, and
enzymes, such as chitinases and lipoxygenases. A number
of these genes conferring insect resistance have been
engineered and incorporated into various plant species
(Gatehouse and Gatehouse 1998; Schuler et al. 1998).
However, there is concern that herbivorous insects may
adapt to plant defenses by evasion and/or detoxification
(Despres et al. 2007). Therefore, continuing research on
novel insect resistance genes is essential for the long-term
control of insect pests.
Considering the impact of CCW on the soybean yield
in southern China, it is important to elucidating potential
insect resistance genes in soybean, which is a long-term

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process including two steps. In the first step, studies

should be performed to characterize transcriptomic,
metabolomic and proteomic changes in herbivore-attacked plants using unbiased omic approaches (Sinha
et al. 2015). Many different plantherbivore interactions
have been investigated with microarrays revealing that
genes involved in numerous aspects of metabolism are
regulated (Alignan et al. 2006; Kant and Baldwin 2007;
Ehlting et al. 2008; Schmidt and Baldwin 2009). As
more transcript profiling data become available, the
second step will be to validate the roles of those
unknown or candidate genes in plant defense against
herbivores by forward or reverse genetic approaches.
The agrobacterium-mediated transformation of Nicotiana
tabacum has been improved to be a model ecological
expression system of dicotyledonous plants with high
frequency of transformation and stability of foreign gene
integration (Bellini et al. 1992; Zhang et al. 2009). Thus
genetic transformation of foreign genes in transgenic
Nicotiana tabacum plants becomes typically an efficient
method to study gene function and transgenic plants with
novel traits.
With the sequencing and publication of the soybean
genome (Schmutz et al. 2010), RNA-Seq as new transcriptomic technique has been used to identify transcript
abundance changes that occur in various tissues or in
response to various treatments in soybean (Vidal et al.
2012; Kang et al. 2012). In this study, the RNA-Seq
technique was used and combined with microarray
results to identify soybean transcript profiles that correlate with CCW resistance. A qRT-PCR analysis of 21
consistently identified genes confirmed the results of the
RNA-Seq analysis. The integration of RNA-Seq and
microarray data provided a list of genes that were significantly regulated in both resistant and susceptible
soybean lines. Among these genes, a vegetative storage
protein (VSP)-encoding gene (GmVSPb), which is
involved in response to water deficit, wounding, and
jasmonic acid (JA) but does not contribute to plant
productivity as normal storage proteins, and a nicotinamide adenine dinucleotide phosphate (NADPH): isoflavone reductase-encoding gene (GmN:IFR), which is
the key enzyme involved in the soybean phytoalexin
biosynthesis pathway were determined to be good candidates for CCW resistance to evaluate the potential
function of these genes in insect resistance. The
expression of two JA-related genes and two nicotinerelated genes were also linked with improved CCW
resistance in transgenic lines. Our study elucidates a
comprehensive understanding of soybean resistance to
CCW and suggests the strong potential of the GmVSPb
gene for engineering transgenic plants with enhanced
insect resistance.

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Materials and methods

Plant materials and RNA collection
Plants of two soybean lines, WX and NN, which are
constitutively resistant and susceptible to CCW (Wang
et al. 2011), were treated with common cutworms
(Spodoptera litura Fabricius, CCW) at the V4 stage for
48 h, after which the larvae were removed. The leaves of
the control and treated plants from the WX and NN
soybean lines were excised at the previous identified
peak induced resistance time points (5 days for WX and
1 day for NN) after feeding by CCW (Wang et al. 2014).
Samplings with three biological replications were double-collected for the RNA-Seq experiment and qRT-PCR
test and stored in a -80 C freezer until RNA extraction.
The total RNA from the frozen soybean leaves was
isolated using the RNeasy Plant Mini kit (QIAGEN Inc.,
Valencia, CA, USA) according to the manufacturers
protocol. One unit of volume of plant RNA Isolation Aid
(Ambion, Austin, TX, USA) was added for each unit of
mass of frozen tissue (ml/g) before the tissue homogenization step to remove common contaminants, such as
polysaccharides and polyphenolics. The total RNA was
treated with RNase-Free DNase (QIAGEN Inc.) to digest
any genomic DNA that might be present. Total RNA was
quantified using an ultraviolet (UV) spectrophotometer
and the RNA quality and integrity were examined using
an RNA Lab-On-A-Chip (Caliper Technologies Corp.,
Mountain View, CA, USA), which was evaluated on an
Agilent Bioanalyzer 2100 (Agilent Technologies, Palo
Alto, CA, USA). After extraction, the total RNA from
three biological replicates of each treatment was pooled
together and immediately stored at -80 C for transportation to BGI (Beijing Genomics Institute, China) for
Illumina sequencing.

Illumina libraries and sequencing

Illumina sequencing was performed at BGI, China
according to the manufacturers instructions (Illumina, San
Diego, CA). First, mRNA with a poly (A) tail was isolated
from 20 lg of total RNA using Sera-Mag Magnetic Oligo
(dT) Beads (Illumina). To avoid priming bias, the purified
mRNA was firstly fragmented into small pieces
[100400 base pair (bp)] using divalent cations at 94 C
for 5 min. With random hexamer primers (Illumina), the
double-stranded cDNA was synthesized using the SuperScript double-stranded cDNA synthesis kit (Invitrogen,
CA). The synthesized cDNA was subjected to end-repair
and phosphorylation, and then the repaired cDNA fragments were 30 adenylated with Klenow Exo-(30 to 50 exo

minus, Illumina). Illumina paired-end adapters were ligated

to the ends of these 30 -adenylated cDNA fragments. To
select the proper templates for downstream enrichment, the
products of ligation reaction were purified on 2 % agarose
gel. The cDNA fragments (about 200 bp) were excised
from the gel. Fifteen rounds of PCR amplification were
carried out to enrich the purified cDNA template using
PCR primer PE 1.0 and 2.0 (Illumina) with phusion DNA
polymerase. Finally, the cDNA library was constructed
with a 200-bp insertion fragment. After validation with an
Agilent Technologies 2100 Bioanalyzer, the library was
sequenced using an Illumina HiSeqTM 2000 (Illumina Inc.,
San Diego, CA, USA). The 90-bp raw paired-end reads
were generated by the Illumina Genome Analyzer II
system.
Data filtering and de novo assembly
The original image data were transferred to sequence data
by base calling, which is defined as raw data or raw reads,
and saved as FASTQ files. The FASTQ data format provides compact storage of quality scores for each base that
may be used to filter individual sequences. Before the
transcriptome assembly, we conducted a stringent filtering
process of the raw sequencing reads. For reads in which
with more than 10 % of the bases have a quality score of
Q \ 20, the non-coding RNA (such as rRNA, tRNA and
miRNA), ambiguous sequences represented as N and
adaptor contaminations were removed. The de novo
assembly was conducted using the short reads assembling
program SOAPdenovo, which applies the de Bruijn graph
algorithm (Li et al. 2010). SOAPdenovo combines high
quality reads into contigs and then connects them into
scaffolds. Subsequently, the sequences without redundancy
that contained the least amount of Ns and were not
extended on either end were defined as unigenes. These
sequences were used for blast searching and annotation
using BLASTx programs (e value \0.00001) against the
NCBI non-redundant (nr) protein database, and the optimal
alignment results were used to determine the sequence
direction of the unigene sequences (Conesa et al. 2005).
When a unigene was unaligned, the software named
ESTScan (Iseli et al. 1999) was introduced to predict its
coding regions and determine its sequence direction.
Differentially expressed gene analysis
The raw digital gene expression data were normalized as
RPKM (Mortazavi et al. 2008). The genes that were differentially expressed between the induced and non-induced
soybean lines were identified with a statistical comparison
using the rigorous algorithm method (Audic and Claverie
1997). A threshold of FDR \ 0.001 was used to judge the

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significance of the gene expression difference. The

expression data were log2-transformed and filtered at a
level of fourfold or greater between the induced and noninduced treatments. Each list of DEGs was functionally
categorized into 10 broad categories based on the putative
function of the genes in the GenBank database (NCBI)
(Supplementary Table S1S5). A number of identical
genes were represented by more than one unigene. Identical probes that represented the same or homologous genes
were merged into one putative identification. It should be
noted that certain functional categorizations might have
been arbitrary, and overlapping might have occurred.
qRT-PCR verification
The total RNA for the qRT-PCR experiment was extracted
from homogenized frozen tissue as described above. The
housekeeping soybean b-tubulin gene (GenBank accession
no. AY907703) was used as a reference gene for relative
quantification (Chen et al. 2009). Target gene-specific
primers were designed for qRT-PCR using the online
software Primer3 version 0.4.0 (Rozen and Skaletsky
2000). Reverse transcription reactions were performed with
an oligo (dT) 18 primer and ReverTra Ace Moloney
murine leukemia virus reverse transcriptase (Toyobo,
Osaka, Japan) according to the manufacturers instructions.
All primers were designed based on the sequences obtained
using the BLASTn search in the NCBI database or soybean
genome sequences database (http://www.phytozome.net).
The primer sets used for qRT-PCR are listed in Supplementary Table S6. qRT-PCR was performed on an ABI
7500 real-time PCR system (Applied Biosystems, Foster
City, CA, USA) using the SYBR Green Real-time Master
Mix (Toyobo). A qRT-PCR analysis was performed on
three biological replicates and the data were analyzed with
the software SDS 2.0 (Applied Biosystems). The consistency of RNA-Seq and previous microarray data with the
qRT-PCR analysis was measured using Pearsons correlation based on log2-fold changes (SPSS Inc., Chicago, IL,
USA).
Gene cloning and plasmid construction of GmVSPb
and GmN:IFR
The full-length GmVSPb (M20038.1) and GmN:IFR
(AJ003245.1) cDNA were amplified based on their
sequence information in the NCBI database by PCR using
high-fidelity Turbo Pfu polymerase and gene-specific primers (Table S8). GmVSPb (Glyma08g21410.1) and
GmN:IFR (Glyma01g37810.1) genomic DNA were
amplified based on their sequence information in the soybean genome sequence database (Table S8) and using total
genomic DNA isolated from WX and NN soybean lines as

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the template, respectively. All the PCR products were

purified and cloned into the pMD19-T easy vector (Takara,
Dalian, China) and sequenced (Invitrogen, Shanghai,
China). The gene structures of GmVSPb and GmN:IFR are
presented based on their cDNA and genomic DNA
sequences using the Gene Structure Display Server (http://
gsds.cbi.pku.edu.cn; Guo et al. 2007). Sequence homology
searches were performed with BLAST 2.0 (Altschul et al.
1997). The SignalP prediction program (http://www.cbs.
dtu.dk/services/SignalP/) and TMHMM prediction program (http://www.cbs.dtu.dk/services/TMHMM/) were
used on the deduced amino acid sequence of GmVSPb and
GmN:IFR.
The expression vector construction used Gateway
technology with the ClonaseTM II Kit (Invitrogen, Carlsbad, CA). For Gateway cloning, two recombination reactions (BP and LR reactions) constitute the basis of the
Gateway technology. The Vector Construction Kit was
used according to the manufacturers protocol. The entire
coding sequence of the GmVSPb and GmN:IFR was
amplified using primers with an attB site (Table S8). The
amplified attB fragment was then subcloned into the
pDONR221 vector, resulting in the entry clone, and the
entry clone was then subcloned again into the expression
vector pMDC83, resulting in the pMDC83-GmVSPb and
pMDC83-GmN:IFR constructs under the control of doubled CaMV35S promoter. The two resulting constructs
were introduced into EHA105 and prepared for transformation via the freezethaw method (Belles-Isles et al.
2001).
Tobacco plant transformation
Tobacco (Nicotiana tabacum L. cv. SamSun) was transformed by the Agrobacterium-mediated leaf disk transformation method (Horsch et al. 1985; Krugel et al. 2002).
Non-transformed tobacco was used as a control.
Agrobacterium-infected and control explants were transferred to solid Murashige and Skoog (MS) medium for cocultivation at 25 C in the dark for 2 days. Then the the
explants were transferred to the agar plates with selection
MS medium (1 mg/l 6-benzylaminopurine, 50 mg/l ceftaroline) and subcultured to fresh selection medium at a
week interval. The regenerated putative transgenic shoots
were further transferred to rooting MS medium (50 mg/l
hygromycin, 200 mg/l Indole acetic acid) and cultured the
shoots at a 16-h photoperiod for 3 weeks. The rooted
plantlets were planted in sterile soil in pots and grown at
25 C under a 16-h light/8-h dark photoperiod. The plant
genomic DNA was extracted from the regenerated putative
transgenic seedlings using a DNeasy Kit (Qiagen, GmbH,
Hilden, Germany). Integration of the expression cassette in
the transgenic genome was confirmed by genomic PCR

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using DNA polymerase (Taq polymerase; Takara, Kyoto,

Japan) and gene-specific primers. Genomic DNA (100 ng)
from each transgenic line was used as a template. Nontransgenic control plant DNA was included as a negative
control and plasmid DNA as a positive control in each
PCR. The PCR was 2 min of 94 C preheating, followed
by a 30 cycle amplification program (1 min at 94 C for
denaturation, 1 min at 58 C for annealing and 1 min at
72 C for extension) and a final extension at 72 C for
5 min. The PCR products were analyzed by electrophoresis
on a 1.0 % agarose gel. One month later, the plants were
transferred from the chamber to a greenhouse, and the
overexpression level of GmVSPb and GmN:IFR in the
selected transgenic and control tobacco plants was tested
by qRT-PCR using reversed cDNA from each transgenic
line as templates.
Assessment of transgene copy numbers
To quantify transgene copy numbers, SYBR Green I realtime PCR was developed to determine exogenous transgene
copy number in five transgenic tobacco lines for each of
GmVSPb and GmN:IFR gene. There is a linear relationship
between the log of the starting amount of a template and its Ct
value during real-time PCR (Yuan et al. 2007). A conserved
Nicotiana tabacum gene, 18S rRNA gene (AJ236016) was
used in a set of PCR reactions to obtain a standard curve of
the cycle threshold (Ct) relative to the log of each initial
template copy as an internal control (Schmidt and Delaney
2010). Five fivefold serial dilution of normal tobacco genome DNA with were used as PCR templates. Specific primers used were 18S-F (GGTGGAGCGATTTGTCTG) and
18S-R (CAGGCTGAGGTCTCGTTCG). Similarly, the
standard curves for the GmVSPb and GmN:IFR transgenes
were generated using five fivefold serially diluted pMDC83GmVSPb and pMDC83-GmN:IFR plasmid DNA solutions
as PCR templates, respectively. Each SYBR Green qRTPCR reaction contained 10 ll of SYBR Green Master Mix
reagent (Applied Biosystems), 5 ll of diluted template and
2 ll each of a gene specific forward and reverse primer in a
final volume of 25 ll. The following standard PCR reaction
conditions were used for all transcripts: 95 C for 2 min (1
cycle); 40 cycles of 95 C for 15 s, 60 C 1 min. All reactions were completed in triplicate and concluded with a
melting curve evaluation. After standard curves were prepared for the transgenes (GmVSPb and GmN:IFR) and the
endogenous 18S gene, the third round PCR reactions were
performed for 18S and GmVSPb genes using genomic DNA
isolated from V6, V13, V12, V11, V1 transgenic and control
plants and for 18S and GmN:IFR genes using genomic DNA
isolated from F6, F3, F13, F19, F7 transgenic and control
plants. The Ct values of 18S and GmVSPb/GmN:IFR gene in
each transgenic and control line was obtained to calculate

their initial amount of template. The transgene copy number

of GmVSPb and GmN:IFR transgenic lines was calculated by
comparing the initial template copy of GmVSPb/GmN:IFR
to 18S gene in each of the same transgenic line (Yuan et al.
2007). Excel was used to calculate means, standard deviations, and construct linear regression with their equations and
R2 values.
Insect bioassay of transgenic tobacco lines
Five PCR-positive transgenic lines for GmVSPb and
GmN:IFR (each) were selected for two insect bioassays. The
third-instar larvae of CCW were fed an artificial diet purchased from the Jiangsu Academy of Agricultural Sciences.
For the dual-choice feeding-preference tests, mature fullgreen leaves from transgenic tobacco (T) and control plants
(C) were placed on moist filter paper in an oblong-shaped
container, and 10 third-instar CCW larvae were released into
the middle of the container. The leaf area was measured
using a LICOR-3000 area meter (LI-COR, Lincoln, NE,
USA). The consumed area of the control (C) and transgenic
(T) plants was used to calculate the feeding preference index
(PI), where PI = 2C/(T ? C) (Kogan 1972). PI = 1 indicated that the larvae fed on the transgenic plants in the same
amount as the control, PI [ 1 indicated a preference for the
control plants and relative resistance of the transgenic lines,
and PI \ 1 indicated a preference for the transgenic plants
and relative susceptibility in of the transgenic lines. For the
whole-plant feeding test, five third-instar larvae were placed
on the in vivo transgenic and control tobacco leaf surfaces.
The consumed leaf area of the control and transgenic plants
were directly compared. In addition, the relative growth rate
(RGR) of CCW insects feeding on the control and transgenic
plants was also calculated using the formula
RGR = (W1 - W2)/W1, where W1 and W2 are the
weights of the five common cutworm larvae before being
placed in the plant and after feeding for 24 h, respectively
(Wu et al. 2008). Statistical analysis of the data was performed using Microsoft Excel 2007. Each experiment was
repeated three times, and the mean values and standard
deviation (SD) were calculated. Analysis of variance (GLM
procedure of SAS; SAS 1989) was used to determine the
significance of differences between transgenic plants and
controls.
Expression analysis in the transgenic tobacco lines
The expression of two JA biosynthesis-related genes
(LOX3, AY254349.1; AOS, AJ295274.1) and two nicotine
biosynthesis-related genes (PMT, D28506.1; A622,
D28505.1) that may be involved in CCW feeding responses
in transgenic and control tobacco lines was examined by
qRT-PCR using an ABI 7500 Real-Time PCR system

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(Applied Biosystems). The target gene-specific primers

were designed based on their sequence information in the
NCBI database and are listed in Table S8. The measurements were obtained using the relative quantification
method (Livak and Schmittgen 2001). The means and
standard deviation were calculated for three replicates per
experiment.

Results
The complexity of plantinsect interactions makes it difficult to determine the metabolites or regulated factors that
effectively limit insect infestation. The time points at
which transcript profiles are monitored is critical, and
should be chosen based upon observations of the plant
insect interaction (Varshney et al. 2009). Our previous
work identified that the peak induced resistance time of
resistant WX and susceptible NN soybean lines (Wang
et al. 2014). In this study, RNA-Seq was applied to the
peak time induced and non-induced samples to identify
unbiased candidate genes that may be useful in the
improvement of soybean resistance against CCW.
RNA-Seq-based transcriptome analysis
In this research, the total RNA extracted from CCW-treated
leaves and non-treated control leaves from two soybean
lines (WX-T, WX-CK, NN-T and NN-CK) were sequenced
with Illumina paired-end sequencing technology. After
filtering the unqualified reads from the raw sequencing
data, approximately 26.6 million and 25.9 million clean
reads with an average length of 100 bp were generated
from the WX-CK and WX-T transcriptomes, respectively and 26.1 million and 25.7 million clean reads were
generated from the NN-CK and NN-T transcriptomes,
respectively (Table 1). The average length of each clean
read was approximately 90 bp, and the Illumina sequence
data were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA)
database under accession number SRP049638. Interestingly, the guanine-cytosine (GC) percentages in the insecttreated transcriptome were 0.54 and 0.29 % higher than
Table 1 Output statistics of
total reads and nucleotides from
RNA-Seq data

Samples

Total reads

Total nucleotides

Q20 percentagea

N percentage

GC percentage

WX-CK

26,644,448

2348,000,280

95.09

0.01

46.51

WX-T

25,911,114

2320,000,200

94.90

0.01

47.05

NN-CK

26,088,892

2398,000,320

94.85

0.00

46.99

NN-T

25,777,780

2332,000,260

94.79

0.00

47.28

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those in the control transcriptome in the two soybean lines

WX and NN, respectively (Table 1). A high GC content is
a feature of genes encoding transcriptional activators or
repressors that regulate gene expression under various
stress conditions (Miyazaki et al. 2009; Risso et al. 2011).
A significant increase in the GC content of stress-regulated
miRNA sequences is a critical parameter for the miRNAs
that regulate gene expression under stress conditions in
Arabidopsis thaliana (Mishra et al. 2009). Thus, the overall
increase in the GC content in the CCW-induced transcriptome may be important for comprehensive defense
interactions in soybean.
According to the overlapping information provided by
the high-quality reads, approximately 460,000 contigs were
generated in each of the four samples with an average
length of more than 130 bp (Table 2). More than 140,000
scaffolds were obtained with an average length of more
than 250 bp. After the reads were successively assembled
into contigs and scaffolds, they were analyzed for unigenes, and approximately 90,000 unigenes with a mean
length of 744 bp were present in each of the four treatments (Table 2). The gap distribution of most of the
assembled unigenes showed \5 % gap regions along the
total length (Fig. 1). These results suggest that our transcriptome sequencing data were effectively assembled and
that the sequence data were highly suitable for further
analysis.
To identify genes that were differentially represented
between the induced and non-induced treatments in the two
soybean lines, the normalized expression of the genes was
calculated by the Reads Per kb per Million reads (RPKM)
method (Mortazavi et al. 2008), and the differentially
expressed unigenes (DEGs) were identified by the false
discovery rate (FDR) method by pairwise comparisons
(Audic and Claverie 1997). After filtering against the
thresholds of (FDR) B 0.001 and |log2 Ratio| C 2, 1004
(781 up-regulated and 213 down-regulated-) and 1580 (874
up-regulated and 706 down-regulated-) unigenes were
detected with significant differential expression levels
between the induced and non-induced treatments of WX
and NN soybean lines, respectively (Fig. 2). Among these
genes, 179 DEGs (132 up-regulated, 32 down-regulated
and 15 oppositely regulated) were overlapped between WX

Q20 percentage: percentage of bases whose quality was larger than 20 in clean reads

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Table 2 Output statistics of the contigs, scaffolds and unigenes from RNA-Seq data
Samples

Cotigs

Scaffolds

Unigenes

Numbers

Mean size (bp)

Numbers

Mean size (bp)

Numbers

Size range (bp)

Mean size (bp)

WX-CK

443,918

134

141,444

253

87,672

1003739

327

WX-T

468,835

133

145,278

256

89,976

1003623

335

NN-CK

468,312

134

144,516

261

89,356

1003414

345

NN-T

470,708

134

148,743

257

92,112

1003506

336

Fig. 1 Gap distribution of all

the unigenes and unigenes
found in the four treatments
[resistant soybean line induced
(WX-T) and non-induced (WXCK); susceptible soybean line
induced (NN-T) and noninduced (NN-CK)]. The gap
distribution (%) represents the
percentage of the number of N
divided by the sequence length
of assembled scaffold or
unigene

genes were co-expressed in the same manner, whereas only

a small number of genes were expressed in an opposite
way.
Functional classification of DEGs from the RNA-Seq
data

Fig. 2 Venn diagram representing genes differentially expressed in

WX and NN soybean lines in treated plants compared with control
plants identified by RNA-Seq analysis

and NN lines. The DEGs from RNA-Seq were further

manually compared with the DEGs from previous
microarray data (NCBI accession number: GSE51823) and
certain unigenes that were obtained in our research may
have multiple hits against the probes in microarray data
(Wang et al. 2014). Overall, 60 identical genes were
identified by both platforms as commonly regulated in the
two soybean lines (Table 3), and the other two groups of 16
genes were identified by both platforms as uniquely regulated in WX and NN lines (Tables 4, 5). Most of these

A functional annotation of all of the assembled unigene

sequences was performed against the non-redundant protein database (NR) with an e value cut-off of 1 9 e-20.
Among the 2405 unigenes, 1826 (75.93 %) were similar to
known protein sequences. The remaining unigenes that
showed no significant similarity using the BLASTx and
tBLASTx programs might be novel transcripts that are
specific to soybean. All of the DEGs from RNA-Seq were
divided into five gene lists according to their regulated
expression patterns in the two soybean lines: (a) common
regulated genes in WX and NN; (b) unique up-regulated
genes in WX; (c) unique up-regulated genes in WX;
(d) unique up-regulated genes in NN; and (e) unique downregulated genes in NN. Each gene list was then consolidated into the same ten categories based on the putative
annotations (Fig. 3). In the common gene list, the genes
that were categorized into defense/stress (18 %), primary
metabolism (14 %), and cell wall modification (13 %)
were overrepresented (Fig. 3a). The unique genes that were

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Table 3 List of common DEGs in WX and NN detected by both RNA-Seq and microarray analysis
Putative identificationsa

Categorizations

Microarray
WX-FC

Signaling

Binding and transport

Defense/stress

Transcriptional regulation

Secondary metabolism

Cell wall modification

123

RNA-Seq
NN-FC

WX-FC

NN-FC

Lipoxygenases

5.36

1.55

7.41

7.38

Allene oxide synthase

2.3

3.12

2.01

30.09

1-aminocyclopropane-1- carboxylate oxidase

4.53

3.61

0.42

6.06

Abscisic acid receptor

2.2

1.31

2.33

14.11

Calnexin

0.87

2.36

3.13

3.16

Receptor protein kinase

3.89

1.02

2.49

2.64

7
8

Purple acid phosphatase 1

Serine/threonine-protein kinase

0.93
7.07

0.41
0.5

0.16
19.87

0.25
0.03

Auxin-induced protein 6B

7.35

0.7

2.17

0.2

10

Ankyrin repeat family protein

2.04

1.02

2.76

0.03

11

UDP-galactose transporter

2.17

1.13

5.49

31.88

12

U-box domain-containing protein

3.4

1.05

2.58

2.1

13

Heat shock factor protein HSF70

1.82

2.2

17.37

3.84

14

Hydroxycinnamoyl transferase

1.1

5.4

14.87

13.15

15

F-box proteins

7.07

1.02

20.97

0.16

16

Vegetative storage protein a

1.47

3.93

3.74

4.46

17

Vegetative storage protein b

4.67

2.03

46.38

37.33

18

Polyphenol oxidase

3.45

16.55

16.32

78.84

19

Protease inhibitor 1

3.48

38.23

2.11

4.99

20

Cysteine proteinase inhibitor

1.63

13

3.27

62.38

21

Lectin

6.24

1.97

18.66

36.7

22
23

Pathogenesis-related proteins
Stress-induced protein

14.32
22.42

2.02
2.54

8.8
11.55

19.66
9.76

24

Disease resistance protein

3.57

2.8

18.97

26.75

25

Patatin group protein

3.7

2.4

30.25

12.87

26

C2 domain-containing protein

2.53

1.14

0.5

11.72

27

Leucine-rich repeat protein

6.29

0.8

5.22

0.05

28

Glutathione S-transferase

2.2

29

Cationic peroxidase

21.66

30

Peroxidases

31

Peroxisomal glycolate oxidase

32

Dehydrin

17.04

2.33

0.91

8.66

33.14

24.42

19.46

36.95

15.79

0.63

0.42

0.58

0.22

0.98

0.45

0.17

0.41

33

Ethylene-responsive transcription factor

9.27

3.36

6.08

14.69

34

BTB/POZ domain-containing protein

2.19

0.46

12.49

0.4

35

Myb family transcription factor

8.29

0.69

7.49

0.33

36

NADPH: isoflavone reductase

4.91

2.99

2.96

3.15

37

Caffeic acid O-methyltransferase

12.28

0.74

2.47

8.74

38
39

Flavonol synthase
chalcone synthase

2.09
3.8

3.58
0.94

2.15
2.46

11.73
4.51

40

Chalcone O-methyltransferase

11.82

None

5.38

0.22

41

Endo-1,3-beta-glucanase

14.32

2.61

4.66

2.32

42

Polygalacturonases

3.42

0.85

8.4

7.45

43

Laccases

40.12

3.82

12.43

44

Hydroxyproline-rich glycoprotein

19.72

2.2

5.11

3.09

45

Myo-inositol oxygenase

1.93

4.85

12.14

14.85

46

Repetitive proline-rich cell wall protein

8.55

12.09

16.32

6.59

47

Cellulose synthase

4.88

0.88

17.98

0.14

81.7

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Table 3 continued
Putative identificationsa

Categorizations

Microarray
WX-FC

Growth and development

Primary metabolic

Others

RNA-Seq
NN-FC

WX-FC

NN-FC

48

Xyloglucan endotransglucosylase

6.83

0.31

13.85

0.11

49

Subtilisin-like protease

6.52

0.91

57.02

5.41

50

LOB domain-containing protein

4.89

3.28

0.5

4.33

51

Nodulin

0.51

2.24

0.18

5.29

52

Sucrose synthase

3.26

0.32

3.49

0.11

53

GDSL esterase/lipases

20

2.56

6.13

2.28

54
55

Esterase
Amino acid permease

2.12
4.62

0.34
0.77

3.84
2.4

0.18
0.13

56

Cysteine protease

3.45

0.81

19.58

35.56

57

Endonuclease

0.88

3.05

7.08

8.35

58

Histones

2.25

1.27

8.58

4.01

59

Nucleoredoxin

2.48

0.63

3.51

0.19

60

Cytochrome P450

28.1

18.03

21.95

Putative identifications: we identified genes whose e values exhibited at least less than 1 9 e-20 with the homology searches in DNA databases

FC, fold changes of genes expressed in control soybean plants against CCW induced soybean plants

Table 4 List of unique DEGs in WX detected by both RNA-Seq and microarray analysis
Categorizations

Putative identifications

WX resistant line
Microarray

Signalling

Phospholipase

Binding and transport

Nitrate and chloride transporter

12.89

9.52

Serine carboxypeptidase

10.11

4.18

Defense/stress
Transcriptional regulation

2.95

RNA-Seq
22.08

With no lysine kinase

6.55

14.09

24 kDa seed coat protein

6.67

3.67

Dehydration-responsive element binding protein

3.49

4.23

Homocysteine s-methyltransferase

12.39

7.87

MYB139

3.15

4.25

MYB73

2.25

4.94

10

WRKY43

2.83

5.1

11

Nuclear transcription factor Y subunits

4.63

6.61

Secondary metabolism

12

4-coumarate: CoA ligases

3.74

32.47

Cell wall modification

13

COBRA-like protein

7.56

12.84

Primary metabolism

14
15

Sugar transport proteins

Epoxide hydrolase 3

2.22
2.09

5.15
4.02

16

Nitrate reductase

2.53

92.89

up-regulated in WX were enriched for the categories of

binding and transport (15 %), transcriptional regulation
(10 %) and signaling (9 %) (Fig. 3b). The small proportion
of genes that were uniquely down-regulated in WX were
enriched for the same categories as in the WX up-regulated
gene list. However, the functional categories of primary
metabolism and secondary metabolism were absent in this

gene list (Fig. 3c). As in the common gene list, defense/

stress (12 %) was also the predominant category in the NN
up-regulated gene list. The least prominent functional
categories were transcriptional regulation (6 %), cell wall
modification (3 %) and others (2 %) (Fig. 3d). In the NN
down-regulated gene list, more than half of the genes were
of unknown function, whereas the categories of defense/

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Table 5 List of unique DEGs
in NN detected by both RNASeq and microarray analysis

Categorizations

Putative identifications

Microarray
Signalling

RNA-Seq

IAA-amino acid hydrolase

2.59

4.92

Major latex protein homolog

5.38

36.13

SPX domain-containing protein

0.43

0.09

Salicylic acid methyl transferase

0.18

0.43

Galactinol galactosyltransferase

0.47

0.14

Metalloendopeptidase

0.5

0.16

Defense/stress

Inositol monophosphatase 3-like

0.33

0.06

Secondary metabolism

Geranyl-diphosphate synthase

2.73

26.83

Leucoanthocyanidin dioxygenase

8.18

16.07

10

Isoflavone synthase 1

0.4

0.5

Cell wall modification

11
12

Polygalacturonase inhibiting protein

Acid beta-fructofuranosidase-like

18.62
2.41

134.69
13.71

Growth and development

13

Carbonic anhydrase

4.59

54.48

14

Chlorophyllase

2.6

14.02

15

2-on-2 hemoglobin

0.25

0.06

16

Arginase

2.72

10.96

Binding and transport

Primary metabolic

Fig. 3 Functional classification of DEG lists commonly or specifically regulated in WX and NN from RNA-Seq data. Pie charts
represent function categories according to putative identifications.

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NN susceptible line

a Commonly regulated genes in WX and NN. b Unique up-regulated

genes in WX. c Unique down-regulated genes in WX. d Unique upregulated genes in NN. e Unique down-regulated genes in NN

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stress (9 %), binding and transport (7 %), and primary

metabolism (6 %) were overrepresented among the
remaining genes (Fig. 3e).
qRT-PCR validation and correlation analysis
An comparison of our RNA-Seq data with previous
microarray data highlighted a series of candidates that were
related to soybean defenses against CCW (Tables 3, 4, 5).
To validate the reliability of the expression profiles that
were obtained from RNA-Seq data and confirm the correlation between RNA-Seq and microarray platforms, we
conducted qRT-PCR analysis for a subset of 21 genes from
various functional categories that may have potential biological significance in plant defenses against insects. An
inspection indicated that although the magnitude of
expression of certain genes from the qRT-PCR results was
different from the RNA-Seq or microarray results, the
direction of most genes expression was the same across
the three platforms (Table S7). Previous studies indicated
that additional emphasis should be placed on the direction
in changes of gene expression rather than the magnitude of
change (Yauk and Berndt 2007). The genes that exhibited
inconsistent gene expression between the RNA-Seq and
microarray platforms were validated by qRT-PCR. The
consistency of each platform with the qRT-PCR analysis
was measured by a Pearsons correlation of the fold
changes. However, the correlations between the qRT-PCR
and the RNA-Seq data were slightly lower in the resistant
line (R = 0.665) than in the susceptible line (R = 0.863),
suggesting that the data obtained from RNA-Seq may
require more stringent evaluation. Overall, the correlations
observed across the three platforms in both of the lines
were within a good range (Fig. 4). These results clearly
confirm that these candidate genes were regulated by CCW
feeding in soybeans.
Identification of CCW candidate resistance genes
The RNA-Seq based transcriptome analyses integrated
with previously established microarray data reduced the
number of potential false positives in the RNA-Seq data
and finalized a list of reliable candidate genes for functional studies (Table 3). Based on the qRT-PCR verification results, six genes encoding soybean vegetative
lectin, cysteine proteinase inhibitor, isoflavone synthase 1,
NADPH: isoflavone reductase, VSPb and peroxisomal
small heat shock protein have been evaluated in a detailed
spatial and temporal expression analysis (Wang et al.
2014). Among these genes, GmVSPb and GmN:IFR were
considered good candidates for CCW resistance because
of their involvement in the systemic CCW-induced
responses throughout the entire plant in both of the

soybean lines. 2623- and 1358-bp genomic DNA

sequences of the GmVSPb and GmN:IFR genes were
cloned and found be consistent in both the WX and NN
soybean lines. A sequence comparison between the isolated fragments and sequences obtained from soybean
genome database demonstrated 100 % identity for
GmVSPb and 95 % identity for GmN:IFR. The full-length
GmVSPb and GmN:IFR cDNA sequences were subsequently obtained. A combined sequence analysis showed
that GmVSPb contains two introns and GmN:IFR contains
four introns (Fig. 5a). Analysis using SignalP and
TMHMM found a signal peptide and transmembrane
domain in the N-terminus of GmVSPb that was absent
from the GmN:IFR protein (Supplementary Fig. S1),
suggesting that GmVSPb is a secretory protein with acid
phosphatase activity that may be related to its molecular
mechanism in defense against insects (Alfano and Collmer 2004).
Generation of transgenic plants
The two candidate genes, GmVSPb and GmN:IFR, were
further introduced into tobacco plants to acquire overexpressing transgenic tobacco lines for functional analyses of
these genes. Constructs containing the GmVSPb and
GmN:IFR open reading frame under the control of double
Cauliflower Mosaic Virus (CaMV) 35S promoters were
used for the transformation of tobacco (Fig. 5b). Twenty and
twenty-five independent hygromycin-resistant GmVSPb and
GmN:IFR transgenic plants were regenerated, respectively.
Among these plants, eighteen GmVSPb transgenic lines and
twenty-two GmN:IFR transgenic lines were confirmed as
positive transformants by PCR analysis (Fig. 5c). Eleven
transgenic lines for each gene were randomly selected for
qRT-PCR analysis to evaluate their expression. Varying
levels of the GmVSPb and GmN:IFR transcripts were
detected among the different transgenic lines, whereas no
GmVSPb or GmN:IFR transcripts were detected in the
controls (Fig. 5d). Overall, a higher level of GmVSPb
expression than that of GmN:IFR was recorded in the
transformants. In addition, transgene copy numbers of
GmVSPb and GmN:IFR in five selected transgenic lines for
each gene and control plants were determined using quantitative real-time PCR assay, respectively. This very sensitive real-time PCR technique permits accurate comparison
of DNA amounts and allows clear discrimination between
variant fold DNA levels (Yang et al. 2005; Casu et al. 2012).
The internal standard curve for tobacco reference 18S gene
was generated as y = -6.9518x ? 8.9131, R2 = 0.9014
and two standard curves for GmVSPb and GmN:IFR transgenes were y = -3.0421x ? 12.666, R2 = 0.9753 and
y = -3.6282x ? 15.003,
R2 = 0.9788,
respectively
(Fig. S2). Then by the comparative calculation using relative

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Fig. 4 Correlation of qRT-PCR results with RNA-Seq (triangle) and

microarray (circle) Log2 expression fold changes (treated vs. control)
on 21 genes, respectively. R1 and R2 represent the Pearson
correlation coefficients of qRT-PCR expression data versus RNASeq and qRT-PCR expression data versus microarray data. a qRT-

PCR validation of two platform expression data from resistant line

(R1 = 0.665, P = 0.001; R2 = 0.859, P = 0.000). b qRT-PCR
validation of two platform expression data from susceptible line
(R1 = 0.863, P = 0.000; R2 = 0.900, P = 0.000)

Ct values, we achieved statistically significant estimates of

58 copies of GmVSPb transgene and 16 copies of
GmN:IFR transgene in the primary transformants, respectively (Table 6).

larvae feeding on GmVSPb and GmN:IFR was generally

smaller than that of larvae feeding on control plants
(Fig. 7b). These results indicate that the growth of CCW is
significantly inhibited by feeding on transgenic tobacco
plants.

Insect-resistance analysis of tobacco plants

overexpressing GmVSPb and GmN:IFR
The five transgenic lines of GmVSPb and GmN:IFR gene
with different expression levels and transgene inserted
copy numbers were used in insect bioassays to test their
function in insect resistance. Two transgenic lines of each
gene (V13, V11, F3 and F19) were selected for a dualchoice assay, and ten third-instar larvae that were starved
were placed in the middle of apallet containing leaves from
transgenic plants on one side and leaves from control plants
on the opposite side in three independent bioassays
(Fig. 6). After 12 h, the PI values of V13 and V11 that
were calculated from the leaf area loss were significantly
[1, indicating that most of the CCW preferred control
leaves to the transgenic tobacco leaves, especially those of
GmVSPb transgenic plants. In addition, the other three
lines of each gene (V6, V12, V1, F6, F13 and F7) were
selected for a whole-plant feeding test to evaluate the
effects of the overexpressed gene on the insect and plant.
Five starved third-instar larvae were weighed and randomly placed on the leaves of each pair of transgenic line
and control line. Leaves from the GmVSPb and GmN:IFR
transgenic tobacco lines were both less damaged than the
control leaves (Fig. 7a), and the relative growth rate (RGR)
of the larvae feeding on transgenic plants was lower than
that of larvae feeding on control plants, and the size of

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Co-expression analyses in GmVSPb and GmN:IFR

overexpressed lines
To further investigate the associated molecular mechanisms that were involved in the improved defense
response of GmVSPb and GmN:IFR overexpressed lines,
the expression of two JA biosynthesis-related genes
(LOX3 and AOS) and two nicotine biosynthesis-related
genes (PMT and A622) was examined by qRT-PCR in
transgenic and control lines (Fig. 8). A significantly
increased transcript accumulation of LOX3 and AOS was
observed in the GmVSPb and GmN:IFR transgenic lines
used in the resistant analysis. These two JA biosynthesis-related genes were also identified in our two sets of
microarray and RNA-Seq transcriptome data in both of
the soybean lines induced by CCW (Table 3). The
overexpression of GmVSPb and GmN:IFR in transgenic
tobacco also enhanced the expression of NtPMT
(Fig. 8), suggesting the involvement of an induced
accumulation of nicotine production in transgenic lines.
However, the transcript level for the nicotine-related
gene A622 was only enhanced in GmVSPb transgenic
lines but virtually unchanged in GmN:IFR transgenic
plants (Fig. 8), which may be explained by the high
homology of GmN:IFR with NtA622 leading to a

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Fig. 5 Characterization and
transformation of GmVSPb and
GmN:IFR. a Gene structure of
GmVSPb and GmN:IFR. Black
filled boxes indicate exons and
lines between boxes indicate
introns. b Schematic structure of
pMDC83-GmVSPb and
pMDC83-GmN:IFR constructs
used for plant transformation.
c Representative picture
showing PCR analysis of
GmVSPb and GmN:IFR
transgenic tobacco lines. M, DL
2000 maker; P, positive control;
CK: non-transgenic control
plant, V, GmVSPb transgenic
plant; F, GmN:IFR transgenic
plant. d qRT-PCR analysis of
GmVSPb and GmN:IFR
expression levels in their
transgenic tobacco lines. Error
bars represent the standard
deviation

redundant function of NtA622 in GmN:IFR transgenic

lines.

Discussion
Plant responses to herbivores are often strongly correlated
with the regulation of gene expression after insect feeding or
attack. Only a small number of genes that are involved in
secondary metabolism and octadecanoid signaling have

been studied in relation to CCW resistance in soybean

(Jouanin et al. 1998; Marchetti et al. 2000; Zhu-Salzman
et al. 2003; Wu et al. 2008), which limits the development of
CCW resistance in soybean. In this study, RNA-Seq-based
transcriptome analysis revealed thousands of DEGs in
response to CCW feeding in both of the soybean lines. Our
previous cDNA microarray analysis has provided a primary
overview of the diverse transcripts that are regulated by
CCW in the two soybean lines (Wang et al. 2014). However,
most of the differentially expressed genes have not been

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Table 6 Relative calculation of copy number of GmVSPb and GmN:IFR gene in each independent transformant
Transgenic lines

Calculation of 18S genea

Calculation of GmVSPbb or GmN:IFRc gene

GmVSPb/18S or GmN:IFR/18S

Copy number

V6

-0.42563

-3.21757

7.56

V13

-0.49701

-3.33102

6.70

V12

-0.6217

-3.56353

5.73

V11

-0.64781

-3.50971

5.42

V1

-0.61759

-3.06227

4.96

CK

N/A

N/A

N/A

N/A

F6

-0.5557

-3.24333

5.84

F3

-0.66203

-2.48266

3.75

F13

-0.64857

-2.36752

3.65

F19

-1.06354

-2.74856

2.58

F7
CK

-2.70002
N/A

-3.03688
N/A

1.12
N/A

1
N/A

The DNA template amount of Nt18S rRNA gene from two sets of transgenic and control samples were calculated according to the standard
curve with formula of y = -6.9518x ? 8.9131
b

The DNA template amount of GmVSPb gene from GmVSPb and controls were calculated according to the standard curve with formula of
y = -3.0421x ? 12.666
c
The DNA template amount of GmN:IFR gene from GmVSPb and control sample were calculated according to the standard curve with formula
of y = -3.6282x ? 15.003. V: GmVSPb transgenic plant; F: GmN:IFR transgenic plant

previously associated with defense responses against insects

or any other functions. Compared with previous microarraybased transcriptome data (Wang et al. 2014), RNA-Seq
identified more DEGs, even using a fold changes threshold of
over four, which was two-fold higher than that used for
microarrays (2405 vs. 1097). In addition, RNA-Seq generally produced fold changes of a larger magnitude than those
from the microarrays, with more genes showing fold changes
C20 or B-20 compared with the fold-change ranges of 210
or -8 to -2 for the microarray data, which indicates that
RNA-Seq was more productive and sensitive in the identification of significant DEGs.
Our RNA-Seq-based transcriptome is more complicated
than that based on the microarray. However, functional
analysis of the RNA-Seq data showed the same significant
function categories similarly to the results of the microarray analysis, such as the defense/stress category, which was
overrepresented in the common and NN up-regulated gene
list; the transcriptional regulation category, which was
overrepresented in the WX up-regulated gene list but
underrepresented in the NN regulated gene list; and the
primary metabolism category, which was overrepresented
in the NN down-regulated gene list. In the RNA-Seq
common gene list, genes encoding lipoxygenase (LOX)
and allene oxide synthase (AOS) that were related to JAsignaling pathways were up-regulated in both of the lines
as in the previous microarray data (Table 3). This result
indicates that the jasmonate signaling cascade may constitutes an important regulatory mechanisms induced by

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CCW in soybean. In the transcriptional regulation category, genes encoding transcription factors, such as
MYB139, MYB73 and WRKY43 were specifically
induced in resistant line. These transcription factors
uniquely regulated in WX suggested a distinct regulation
mechanism in response to CCW in resistant soybean line.
In the stress/defense category, certain well-characterized
anti-insect genes or homologous genes encoding protease
inhibitor 1, cysteine proteinase inhibitor, polyphenol oxidase (PPO), and lectin were induced in both of the soybean
lines (Table 3). The effectiveness of PPO in defense has
been demonstrated by experiments in which purified PPO
inhibited the growth of tomato pests (Constabel et al.
1996), and PPO-overexpressing transgenic plants clearly
showed an increased resistance to CCW (Mahanil et al.
2008). Other genes in this category were mostly related to
pathogen defense and abiotic stresses, represented by genes
that encode pathogenesis-related (PR) proteins, leucinerich repeat proteins, VSP a/b, dehydrin and peroxidases,
which demonstrates an overlap of the conserved response
in insect induction with that of pathogenic, wound and
drought-stress responses. In the secondary metabolism
category, all of the represented genes encoded key enzymes
in the phenylpropanoid pathway, including upstream
enzymes (4-coumarate:CoA ligase and chalcone synthase)
and enzymes in other branch pathways that lead to flavonoids (flavonol synthase and isoflavone synthase 1), phytoalexins (NADPH: isoflavone reductase), anthocyanins
(leucoanthocyanidin dioxygenase-like protein) and lignins

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Fig. 6 Dual-choice assays for insect resistance. a Effects of CCW

larvae on GmVSPb and GmN:IFR transgenic plant leaves compared
with control plant leaves. Leaves from two independent transgenic
lines were analyzed, but images of a representative one are presented.
b Preference indexes (PI) of the control and transgenic plants after 12
h feeding by CCW larvae. Thick line represents a PI of 1 indicating no
preference. PI values[1 indicate resistance and PI values\1 indicate
susceptibility in transgenic lines. Error bars represent the standard
deviation. **Highly significant (P \ 0.01)

(caffeic acid O-methyltransferase). Among them, the isoflavones and phytoalexins pathway has been particularly
well characterized to play a role in legume defense against
insect herbivores (Edens et al. 1995; Zabala et al. 2006;
Hart et al. 1983; Dixon et al. 2002). These results highlight
the importance of the soybean phenylpropanoid pathway in
soybean-CCW interactions.
The three other categories of cell wall modification,
growth and development and primary metabolism were
more closely related to morphological and anatomical
aspects that reflect the ability of plants to compensate for
herbivory damage. The expression of several cell wall
associated genes including cellulose synthase, hydroxyproline-rich glycoproteins, repetitive pro-rich protein and
polygalacturonases were up-regulated in both of the soybean lines. These genes lead to the remodeling of cell
walls, suggesting a physical protection of cell wall as a
barrier against herbivores (Cassab 1998; Somerville et al.
2004). In the primary metabolism category, the most

Fig. 7 Whole-plant feeding tests for insect resistance. a Leaf area

loss and effects of CCW larvae on the control and transgenic whole
tobacco lines after 24 h feeding. Intact seedlings from three
independent transgenic lines were analyzed, but images of a
representative one are presented. b Relative growth rate and
representative size comparison of the CCW larvae on the control,
GmVSPb and GmN:IFR transgenic tobacco lines. Error bars represent the standard deviation. **Highly significant (P \ 0.01); *significant (P \ 0.05)

common gene was sucrose synthase gene, which was upregulated in the resistant line but down-regulated in the
susceptible line. The induction of sucrose synthase is
usually found under conditions of limited ATP supply or

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Plant Cell Tiss Organ Cult

Fig. 8 Expression levels of plant defense-response genes in transgenic and control tobacco lines. LOX3 and AOS are the JA synthesis-related
genes in tobacco. PMT and A622 are nicotine biosynthesis related genes in tobacco. Error bars represent the standard deviation

increased demand for the translocation of carbohydrates

(Heim et al. 1993; Gordon et al. 1997). Other genes that
were associated with lipid and protein metabolism were
also induced in the resistant line but suppressed in the
susceptible line. The reconfiguration of the primary metabolism may reflect changes in the plant growth processes
that are required to recover from herbivore attack, and also
function directly as a defense or indirectly by influencing
an herbivores ability to detoxify secondary metabolites
(Felton et al. 1992; Govenor et al. 1997; Green et al. 2001).
The result of correlation analysis between qRT-PCR and
the RNA-Seq data in the resistant line (Fig. 4) indicated
that the huge amount of information from RNA-Seq data
must be carefully evaluated, and the list of the differentially expressed genes must be narrowed down before
performing the functional analyses that may indicate the
real potential of each gene for insect resistance. The RNASeq based transcriptome analyses integrated with previously established microarray data showed that genes that
were significantly induced by CCW feeding in soybean covered a wide range of functional categories,
including signaling transduction, stress/defense, secondary

123

metabolism and transcriptional regulation. Among them,

GmVSPb from stress/defense category was up-regulated in
both soybean lines with greater and faster levels in the
resistant line, and GmN:IFR from secondary metabolism
category was up-regulated in both soybean lines with
greater and faster levels in the susceptible line (Table 3).
The protein of GmVSPb is an acid phosphatase with
phosphatase activity (DeWald et al. 1992). The expression
of GmVSPb could be induced by mechanical wounding,
methyl jasmonate (MeJA), osmotic and nutritional stresses
and insect herbivory (Mason and Mullet 1990), which is a
common response of many genes encoding anti-insect
proteins (Cheong et al. 2002; Chung et al. 2008; Howe
2008). A previous analysis of antisense GmVSPs transgenic
soybean suggested that VSPs may serve other functions
instead of plant productivity (Staswick et al. 2001). The
other gene encodes NADPH: isoflavone reductase is key
enzymes involved in phytoalexin biosynthesis (Paiva et al.
1991; Tiemann et al. 1991; Barz and Welle 1992). Phytoalexins are a major component in the defense mechanisms of higher plants, including soybean, and they can be
directed against potential pathogens as well as insects

Author's personal copy

Plant Cell Tiss Organ Cult

(Russell et al. 1978; Sutherland et al. 1980; Hart et al.

1983). The gene encoding NADPH: isoflavone reductase,
GmN:IFR, is also homologous to the tobacco nicotine
biosynthesis key gene A622 (DeBoer et al. 2009). Nicotine
is an alkaloid that is highly toxic to many insects because it
interferes with their nerve systems (Self et al. 1964; Parr
and Thurston 1972; van Dam et al. 2000; Harvey et al.
2007). These analyses strongly indicate that GmVSPb and
GmN:IFR may play a role in soybeans defense against
CCW.
Transgenic investigation of potential defense related
proteins has been studied as a means to achieve increased
resistance for many years (Alan and Earle 2012). For
example, the genetic manipulation of JA synthesis pathway
genes LOX and AOS genes in transgenic plants exhibit
enhanced insect resistance compared to non-transgenic
plants (Halitschke and Baldwin 2003; Wu et al. 2008). In
our dual-choice assay, leaves from transgenic tobacco lines
V13, V11 overexpressing GmVSPb were preferred by most
of CCWs and exhibited increased resistance to CCW
compared to wild-type plants. Further in whole-plant
feeding tests, both the GmVSPb and GmN:IFR transgenic
plants showed more resistant to CCW than the controls,
indicating that overexpression of GmVSPb and GmN:IFR
might have comprehensively and systematically activated
plant resistance occur in the whole-plant levels. Although
these results suggest that GmVSPb and GmN:IFR may all
involved in defense against insects, stronger evidences
were pointed to GmVSPb gene to be an important plant
defense gene. Plant transformation with a given transgene
may yield independent transgenic plants with different
phenotypes caused by copy number and transcription
effects of the transgene. The different levels of insect
resistance different transgenic lines can be correlated with
their overexpressed levels and copy number effects (Bhat
and Srinivasan 2002). The transgenic line V6 displayed the
resistance level to CCW with the highest expression level
and inserted copy number of 8. The GmVSPb transgenic
lines showing higher transcript levels with higher inserted
copy numbers were more resistant to CCW than GmN:IFR
transgenic lines. While the GmN:IFR transgenic lines with
relatively low overexpression levels and inserted copy
numbers did not show significant resistance in the dualchoice assay. These results suggest that the observed
variation in insect resistance is caused by transgene insertion and transcript accumulation. Taken together, these
data confirms the defensive role of GmVSPb against insects
in plants and provide new insights into the metabolic
engineering of this gene in transgenic plants.
Plants possess multiple mechanisms that provide protection against insect attack, including signaling, gene
expression regulation and direct secondary metabolite
defenses, which consist of an integrated set of defense

responses. Changes in specific defense mechanisms may

lead to a coordinated induction of other factors (SasakiSekimoto et al. 2005; Chen et al. 2006). For example, the
overexpression of the Bph14 gene in rice not only
enhanced resistance to brown plant hopper insects but also
induced the expression of BowmanBirk trypsin inhibitor
genes and several SA synthesis-related genes in the transgenic plants, suggesting that Bph14-mediated resistance
against the piercing-sucking behavior of brown plant
hopper insects may be associated with trypsin inhibitor
production in a SA-dependent resistance pathway, which is
similar to that of pathogen defenses (Du et al. 2009).
Similarly in our study, the up-regulation of JA biosynthesis
related gene LOX3 and AOS in GmVSPb and GmN:IFR
transgenic lines suggests an important regulatory role for
JA in the defense network against CCW in both soybean
and tobacco systems. In addition, JA family-molecules
play a central role in regulating the biosynthesis of
numerous secondary metabolites, including nicotine in
tobacco (Truman et al. 2007; Shoji et al. 2008). The genes
associated with nicotine biosynthesis including NtPMT and
NtA622 are suggested to play important roles in the defense
against herbivory (Kidd et al. 2006; Gulati et al. 2013).
These results strongly suggest that the relationship between
anti-insect performance in transgenic plants and overexpressed genes, especially GmVSPb, is likely associated
with nicotine production and are defense components primarily through JA signaling-dependent pathways. Additional work will be required to fully understand this
coordination.

Conclusions
In this study, RNA-Seq based transcriptome analysis
revealed a large number of DEGs in resistant and susceptible soybean lines following CCW induction, which provided a better understanding of the complex defense
systems in the two soybean lines. An integrated analysis of
RNA-Seq data with previously established microarray data
highlighted a substantial fraction of confirmed candidate
genes including a VSP-encoding gene (GmVSPb) and a
NADPH: isoflavone reductase-encoding gene (GmN:IFR)
that were up-regulated in both of the lines. Overexpression
of GmVSPb and GmN:IFR in tobacco plants significantly
increased the resistance of five GmVSPb transgenic lines
and three GmN:IFR transgenic lines to CCW. In addition,
co-expression analyses of two JA biosynthesis-related
genes (LOX3 and AOS) and two nicotine biosynthesis-related genes (PMT and A622) in transgenic lines indicated a
relationship between GmVSPb-mediated defense mechanisms with JA signaling and nicotine production. Our study
revealed the potential role of GmVSPb and GmN:IFR gene

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in plant defense against insects and may be useful in the

selection and manipulation of high insect resistant genotypes in the future.
Acknowledgments This work was supported in part by the Key
Transgenic Breeding Program of China (2013ZX08004-003),
National Natural Science Foundation of China (31201230,
31171573), Jiangsu Provincial Support Program (BK2012768,
BE2012328), Program for Changjiang Scholars and Innovative
Research Team in University (PCSIRT13073) and Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP).
Author contribution statement Yongli Wang designed and performed research, analyzed data and wrote the manuscript. Hui Wang
and Qing Yang participated in the improvement of the manuscript.
Yujie Ma participated in the insect bioassay of transgenic tobacco
lines. Wenming Yang participated in the assessment of transgene
copy numbers experiment. Deyue Yu designed the research and
revised the manuscript.
Compliance with ethical standards
Conflict of interest
of interest.

The authors declare that they have no conflict

References
Alan AR, Earle ED (2012) Plant disease resistance enhancement via
transgenic expression of antimicrobial peptide MSI-99 gene in
crop plants. J Biotechnol 161:14
Alfano JR, Collmer A (2004) Type III secretion system effector
proteins: double agents in bacterial disease and plant defense.
Annu Rev Phytopathol 42:385414
Alignan M, Hewezi T, Petitprez M, Dechamp-Guillaume G,
Gentzbittel L (2006) A cDNA microarray approach to decipher
sunflower (Helianthus annuus) responses to the necrotrophic
fungus Phoma macdonaldii. New Phytol 170:523536
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids
Res 25:33893402
Audic S, Claverie JM (1997) The significance of digital gene
expression profiles. Genome Res 7:986995
Barz W, Welle R (1992) Biosynthesis and metabolism of isoflavones
and pterocarpan phytoalexins in chickpea, soybean and phytopathogenic fungi. In: Stafford HA, Ibrahim RK (eds) Phenolic
metabolism in plants. Springer, New York, pp 139164
Belles-Isles J, Dusabenyagasani M, Tremblay FM (2001) An
improved procedure for production of white spruce (Picea
glauca) transgenic plants using Agrobacterium tumefaciens.
J Exp Bot 52:20892095
Bellini C, Giordani C, Lupotto E, Locatelli F, Cuzzoni E, Avogadro
E, Sala F (1992) Stability of a foreign gene in transgenic
Nicotiana tabacum L. plants during a cycle of dedifferentiation/
differentiation. Plant Sci 82(2):193200
Bhat SR, Srinivasan S (2002) Molecular and genetic analyses of
transgenic plants: considerations and approaches. Plant Sci
163(4):673681
Blair MW, Munoz C, Garza R, Cardona C (2006) Molecular mapping
of genes for resistance to the bean pod weevil (Apion godmani
Wagner) in common bean. Theor Appl Genet 112:913923

123

Broadway RM, Duffey SS (1986) Plant proteinase inhibitors:

mechanism of action and effect on the growth and digestive
physiology of larval Heliothis zea and Spodoptera exiqua.
J Insect Physiol 32:827833
Cassab GI (1998) Plant cell wall proteins. Annu Rev Plant Biol
49:281309
Casu RE, Selivanova A, Perroux JM (2012) High-throughput
assessment of transgene copy number in sugarcane using realtime quantitative PCR. Plant Cell Rep 31(1):167177
Chen H, Jones AD, Howe GA (2006) Constitutive activation of the
jasmonate signaling pathway enhances the production of
secondary metabolites in tomato. FEBS Lett 580:25402546
Chen H, Seguin P, Archambault A, Constan L, Jabaji S (2009) Gene
expression and isoflavone concentrations in soybean sprouts
treated with chitosan. Crop Sci 49:224236
Cheong YH, Chang HS, Gupta R, Wang X, Zhu T, Luan S (2002)
Transcriptional profiling reveals novel interactions between
wounding, pathogen, abiotic stress, and hormonal responses in
Arabidopsis. Plant Physiol 129:661677
Chung HS, Koo AJ, Gao X, Jayanty S, Thines B, Jones AD, Howe
GA (2008) Regulation and function of Arabidopsis Jasmonate
ZIM-domain genes in response to wounding and herbivory. Plant
Physiol 146:952964
Conesa A, Gotz S, Garca-Gomez JM, Terol J, Talon M, Robles M
(2005) Blast2GO: a universal tool for annotation, visualization
and analysis in functional genomics research. Bioinformatics
21:36743676
Constabel CP, Bergey DR, Ryan CA (1996) Polyphenol oxidase as
a component of the inducible defense response in tomato
against herbivores. In: Phytochemical diversity and redundancy in ecological interactions. Springer, New York,
pp 231252
Cui ZL, Gai JY (1997) A study of leaf-feeding insect species on
soybeans in Nanjing area. Soybean Sci 16:1220
DeBoer KD, Lye JC, Aitken CD, Su AKK, Hamill JD (2009) The
A622 gene in Nicotiana glauca (tree tobacco): evidence for a
functional role in pyridine alkaloid synthesis. Plant Mol Biol
69:299312
Despres L, David JP, Gallet C (2007) The evolutionary ecology of
insect resistance to plant chemicals. Trends Ecol Evol
22:298307
DeWald DB, Mason HS, Mullet JE (1992) The soybean vegetative
storage proteins VSP alpha and VSP beta are acid phosphatases active on polyphosphates. J Biol Chem
267:1595815964
Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MS, Wang L (2002)
The phenylpropanoid pathway and plant defence: a genomics
perspective. Mol Plant Pathol 3:371390
Du B, Zhang W, Liu B, Hu J, Wei Z, Shi Z, He R, Zhu L, Chen R,
Han B, He G (2009) Identification and characterization of
Bph14, a gene conferring resistance to brown planthopper in
rice. Proc Natl Acad Sci USA 106:2216322168
Edens RM, Anand SC, Bolla RI (1995) Enzymes of the phenylpropanoid pathway in soybean infected with Meloidogyne
incognita or Heterodera glycines. J Nematol 27:292303
Ehlting J, Chowrira SG, Mattheus N, Aeschliman DS, Arimura G-I,
Bohlmann J (2008) Comparative transcriptome analysis of
Arabidopsis thaliana infested by diamond back moth (Plutella
xylostella) larvae reveals signatures of stress response, secondary
metabolism, and signaling. BMC Genomics 9:154
Felton GW, Donato KK, Broadway RM, Duffey SS (1992) Impact of
oxidized plant phenolics on the nutritional quality of dietar
protein to a noctuid herbivore, Spodoptera exigua. J Insect
Physiol 38:277285

Author's personal copy

Plant Cell Tiss Organ Cult
Gatehouse AM, Gatehouse JA (1998) Identifying proteins with
insecticidal activity: use of encoding genes to produce insectresistant transgenic crops. Pestic Sci 52:165175
Gatehouse AM, Hilder VA, Gatehouse JA (1992) Control of insect
pests by plant genetic engineering. Proc R Soc Edinb Sect B Biol
Sci 99:5160
Gordon AJ, Minchin FR, Skot L, James CL (1997) Stress-induced
declines in soybean N2 fixation are related to nodule sucrose
synthase activity. Plant Physiol 114:937946
Govenor HL, Schultz JC, Appel HM (1997) Impact of dietary
allelochemicals on gypsy moth (Lymantria dispar) caterpillars:
importance of midgut alkalinity. J Insect Physiol 43:11691175
Green ES, Zangerl AR, Berenbaum MR (2001) Effects of phytic acid
and xanthotoxin on growth and detoxification in caterpillars.
J Chem Ecol 27:17631773
Gulati J, Kim SG, Baldwin IT, Gaquerel E (2013) Deciphering
herbivory-induced gene-to-metabolite dynamics in Nicotiana
attenuata tissues using a multifactorial approach. Plant Physiol
162:10421059
Guo AY, Zhu QH, Chen X, Luo JC (2007) GSDS: a gene structure
display server. Yichuan 29:10231026 [in Chinese]
Halitschke R, Baldwin IT (2003) Antisense LOX expression increases
herbivore performance by decreasing defense responses and
inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J 36(6):794807
Hart SV, Kogan M, Paxton JD (1983) Effect of soybean phytoalexins
on the herbivorous insects Mexican bean beetle and soybean
looper. J Chem Ecol 9:657672
Harvey JA, van Dam NM, Witjes L, Soler R, Gols R (2007) Effects of
dietary nicotine on the development of an insect herbivore, its
parasitoid and secondary hyperparasitoid over four trophic
levels. Ecol Entomol 32:1523
Heim U, Weber H, Baumlein H, Wobus U (1993) A sucrose-synthase
gene of Vicia faba L.: expression pattern in developing seeds in
relation to starch synthesis and metabolic regulation. Planta
191:394401
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SA, Fraley
RT (1985) A simple and general method for transferring genes
into plants. Science 227:12291231
Howe GA (2008) New weapons and a rapid response against insect
attack. Plant Physiol 146:832838
Iseli C, Jongeneel CV, Bucher P (1999) ESTScan: a program for
detecting, evaluating, and reconstructing potential coding
regions in EST sequences. ISMB 99:138148
Jouanin L, Bonade-Bottino M, Girard C, Morrot G, Giband M (1998)
Transgenic plants for insect resistance. Plant Sci 131:111
Kang YJ, Kim KH, Shim S, Yoon MY, Sun S, Kim MY, Van K, Lee
SH (2012) Genome-wide mapping of NBS-LRR genes and their
association with disease resistance in soybean. BMC Plant Biol
12:139
Kant MR, Baldwin IT (2007) The ecogenetics and ecogenomics of
plantherbivore interactions: rapid progress on a slippery road.
Curr Opin Genet Dev 17:519524
Kessler A, Halitschke R, Baldwin IT (2004) Silencing the jasmonate
cascade: induced plant defenses and insect populations. Science
305:665668
Kidd SK, Melillo AA, Lu RH, Reed DG, Kuno N, Uchida K, Furuya
M, Jelesko JG (2006) The A and B loci in tobacco regulate a
network of stress response genes, few of which are associated
with nicotine biosynthesis. Plant Mol Biol 60:699716
Kogan M (1972) Feeding and nutrition of insects associated with
soybeans 2 Soybean resistance and host preferences of the
Mexican bean beetle, Epilachna varivest. Ann Entomol Soc Am
65:675683
Krugel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002)
Agrobacterium-mediated transformation of Nicotiana attenuata,

a model ecological expression system. Chemoecology

12:177183
Li Y, Hu Y, Bolund L, Wang J (2010) State of the art de novo
assembly of human genomes from massively parallel sequencing
data. Hum Genomics 4:271277
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
data using real-time quantitative PCR and the 2-DDCT method.
Methods 25:402408
Mahanil S, Attajarusit J, Stout MJ, Thipyapong P (2008) Overexpression of tomato polyphenol oxidase increases resistance to
common cutworm. Plant Sci 174(4):456466
Marchetti S, Delledonne M, Fogher C, Chiaba C, Chiesa F, Savazzini
F, Giordano A (2000) Soybean Kunitz, C-II and PI-IV inhibitor
genes confer different levels of insect resistance to tobacco and
potato transgenic plants. Theor Appl Genet 101:519526
Mason HS, Mullet JE (1990) Expression of two soybean vegetative
storage protein genes during development and in response to
water deficit, wounding, and jasmonic acid. Plant Cell
2:569579
Mishra AK, Agarwal S, Jain CK, Rani V (2009) High GC content:
critical parameter for predicting stress regulated miRNAs in
Arabidopsis thaliana. Bioinformation 4:151
Miyazaki K, Inoue S, Yamada K, Watanabe M, Liu Q, Watanabe T,
Adachi MT, Tanaka Y, Kitajima S (2009) Differential usage of
alternate promoters of the human stress response gene ATF3 in
stress response and cancer cells. Nucleic Acids Res
37:14381451
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008)
Mapping and quantifying mammalian transcriptomes by RNASeq. Nat Methods 5:621628
Orozco-Cardenas M, McGurl B, Ryan CA (1993) Expression of an
antisense prosystemin gene in tomato plants reduces resistance
toward Manduca sexta larvae. Proc Natl Acad Sci USA
90:82738276
Paiva NL, Edwards R, Sun Y, Hrazdina G, Dixon RA (1991) Stress
responses in alfalfa (Medicago sativa L.) 11: molecular cloning
and expression of alfalfa isoflavone reductase, a key enzyme of
isoflavonoid phytoalexin biosynthesis. Plant Mol Biol
17:653667
Parr JC, Thurston R (1972) Toxicity of nicotine in synthetic diets to
larvae of the tobacco hornworm. Ann Entomol Soc Am
65:11851188
Risso D, Schwartz K, Sherlock G, Dudoit S (2011) GC-content
normalization for RNA-Seq data. BMC Bioinform 12:480
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users
and for biologist programmers. Methods Mol Biol 132:365386
Russell GB, Sutherland ORW, Hutchins RFN, Christmas PE (1978)
Vestitol: a phytoalexin with insect feeding-deterrent activity.
J Chem Ecol 4:571579
Sasaki-Sekimoto Y, Taki N, Obayashi T, Aono M, Matsumoto F,
Sakurai N, Suzuki H, Hirai MY, Noji M, Saito K, Masuda T,
Takamiya K, Shibata D, Ohta H (2005) Coordinated activation
of metabolic pathways for antioxidants and defence compounds
by jasmonates and their roles in stress tolerance in Arabidopsis.
Plant J 44:653668
Schmidt S, Baldwin IT (2009) Down-regulation of systemin after
herbivory is associated with increased root allocation and
competitive ability in Solanum nigrum. Oecologia 159:473482
Schmidt GW, Delaney SK (2010) Stable internal reference genes for
normalization of real-time RT-PCR in tobacco (Nicotiana
tabacum) during development and abiotic stress. Mol Genet
Genomics 283(3):233241
Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten
DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu
Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D,
Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein

123

Author's personal copy

Plant Cell Tiss Organ Cult
D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L,
Gill N, Joshi T, Libault M, Sethuraman A, Zhang XC, Shinozaki
K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J,
Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010)
Genome sequence of the palaeopolyploid soybean. Nature
463:178183
Schuler TH, Poppy GM, Kerry BR, Denholm I (1998) Insect-resistant
transgenic plants. Trends Biotechnol 16:168175
Self LS, Guthrie FE, Hodgson E (1964) Adaptation of tobacco
hornworms to the ingestion of nicotine. J Insect Physiol
10:907914
Shewry PR, Lucas JA (1997) Plant proteins that confer resistance to
pests and pathogens. Adv Bot Res 26:135192
Shoji T, Ogawa T, Hashimoto T (2008) Jasmonate-induced nicotine
formation in tobacco is mediated by tobacco COI1 and JAZ
genes. Plant Cell Physiol 49:10031012
Sinha R, Kumar D, Datta R, Hazra S, Bhattacharyya D, Mazumdar
AB, Mukhopadhyay R, Sultana A, Chattopadhyay S (2015)
Integrated transcriptomic and proteomic analysis of Arabidopsis
thaliana exposed to glutathione unravels its role in plant defense.
Plant Cell Tissue Organ Cult 120:975988
Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J,
Osborne E, Paredez A, Persson S, Raab T, Vorwerk S, Youngs H
(2004) Toward a systems approach to understanding plant cell
walls. Science 306:22062211
Staswick PE, Zhang Z, Clemente TE, Specht JE (2001) Efficient
down-regulation of the major vegetative storage protein genes in
transgenic soybean does not compromise plant productivity.
Plant Physiol 127:18191826
Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin IT (2004)
Nicotines defensive function in nature. PLoS Biol 2:e217
Sutherland OR, Russell GB, Biggs DR, Lane GA (1980) Insect
feeding deterrent activity of phytoalexin isoflavonoids. Biochem
Syst Ecol 8:7375
Tiemann K, Inze D, Montagu M, Barz W (1991) Pterocarpan
phytoalexin biosynthesis in elicitor-challenged chickpea (Cicer
arietinum L.) cell cultures European. J Biochem 200:751757
Truman W, Bennett MH, Kubigsteltig I, Turnbull C, Grant M (2007)
Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates. Proc Natl Acad Sci
USA 104:10751080
van Dam NM, Hadwich K, Baldwin IT (2000) Induced responses in
Nicotiana attenuata affect behavior and growth of the specialist
herbivore Manduca sexta. Oecologia 122:371379
Varshney RK, Nayak SN, May GD, Jackson SA (2009) Nextgeneration sequencing technologies and their implications for
crop genetics and breeding. Trends Biotechnol 27:522530

123

Vidal RO, Nascimento LCD, Maurcio Costa Mondego J, Amarante

Guimaraes Pereira G, Falsarella Carazzolle M (2012) Identification of SNPs in RNA-seq data of two cultivars of Glycine max
(soybean) differing in drought resistance. Genet Mol Biol
35:331334
Walling LL (2000) The myriad plant responses to herbivores. J Plant
Growth Regul 19:195216
Wang H, Gao ZJ, Fan R, Zhang YS, Wu Q, Yu DY (2011) Evaluation
of resistance of soybean germplasm to CCW based on three
resistance mechanisms. Soybean Sci 30:814
Wang Y, Wang H, Fan R, Yang Q, Yu D (2014) Transcriptome
analysis of soybean lines reveals transcript diversity and genes
involved in the response to CCW (Spodoptera litura Fabricius)
feeding. Plant Cell Environ 37:20862101
Wilson RF (2008) Chapter 1 Soybean: market driven research needs.
In: Stacey G (ed) Genetics and genomics of soybean. Springer,
New York, pp 315
Wu JJ, Wu Q, Wu QJ, Gai JY, Yu DY (2008) Constitutive
overexpression of AOS-like gene from soybean enhanced
tolerance to insect attack in transgenic tobacco. Biotechnol Lett
30:16931698
Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D (2005)
Estimating the copy number of transgenes in transformed rice by
real-time quantitative PCR. Plant Cell Rep 23(1011):759763
Yauk C, Berndt M (2007) Review of the literature examining the
correlation among DNA microarray technologies. Environ Mol
Mutagen 48:80394
Yuan JS, Burris J, Stewart NR, Mentewab A, Stewart CN (2007)
Statistical tools for transgene copy number estimation based on
real-time PCR. BMC Bioinform 8(S7):14712105
Zabala G, Zou J, Tuteja J, Gonzalez DO, Clough SJ, Vodkin LO
(2006) Transcriptome changes in the phenylpropanoid pathway
of Glycine max in response to Pseudomonas syringae infection.
BMC Plant Biol 6:26
Zhang PJ, Shu JP, Fu CX, Zhou Y, Hu Y, Zalucki MP, Liu SS (2008)
Trade-offs between constitutive and induced resistance in wild
crucifers shown by a natural but not an artificial elicitor.
Oecologia 157:8392
Zhang J, Lu L, Ji L, Yang G, Zheng C (2009) Functional
characterization of a tobacco matrix attachment region-mediated
enhancement of transgene expression. Transgenic Res
18(3):377385
Zhu-Salzman K, Ahn JE, Salzman RA, Koiwa H, Shade RE, Balfe S
(2003) Fusion of a soybean cysteine protease inhibitor and a
legume lectin enhances anti-insect activity synergistically. Agric
Forest Entomol 5:317323