Biosci. Biotechnol. Biochem., 70 (7), 1780–1783, 2006
Note
Simple Identification of Transgenic Arabidopsis Plants Carrying
a Single Copy of the Integrated Gene
Tomonori K IHARA,1 Cheng-Ri Z HAO,2 Yuriko K OBAYASHI,2 Eiji T AKITA,3
Tetsu K AWAZU,1 and Hiroyuki K OYAMA2; y
1
Forestry Research Institute, Oji Paper, 24-9 Nobono, Kameyama, Mie 519-0212, Japan
Laboratory of Plant Cell Technology, Faculty of Applied Biological Sciences, Gifu University,
1-1 Yanagido, Gifu 501-1193, Japan
3
Graduate School of Biological Sciences, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0192, Japan
2
Received December 20, 2005; Accepted March 22, 2006; Online Publication, July 23, 2006
[doi:10.1271/bbb.50687]
Transgenic Arabidopsis thaliana plants carrying a
single copy of integrated DNA can be identified by
single-step genomic polymerase chain reaction. The
reaction employs two sets of primer pairs with the same
melting temperature that amplify the amplicons derived
from the integrated T-DNA together with those from an
endogenous single-copy gene as a reference. When the
band intensity ratio is one, this means that the transgenic plants are carrying a single copy of the integrated
gene per haploid.
Key words:
Arabidopsis thaliana; integrated gene copy
number; genomic polymerase chain reaction
(PCR); transgenic plant
Transgenic plants derived from Agrobacterium-mediated transformation often carry multiple copies of
integrated T-DNA.1) Consequently, the integrated gene
can become unstable due to enhanced gene silencing as
a result of the multiple copies of the ectopic gene.2)
Isolation of transgenic plants harboring a single copy of
the integrated gene is therefore important in transgenic
studies.
The segregation test is useful for identifying transgenic plants with single-locus DNA integration, but it
does not allow for determination of the copy number at
the locus. Southern blotting analysis is a standard
procedure to determine the copy number of an integrated
gene in transgenic plants, but it possesses several
problems (e.g., it is labor-intensive and time consuming)
when applied to large-scale screening. Quantitative
polymerase chain reaction (PCR)-based methods are
an alternative, real-time and quantitative competitive
PCR (QC-PCR)-based procedures having become popular for estimations of integrated gene copy number.3–5)
Previous studies have indicated, however, that the
reliability of these procedures is affected by several
factors influencing the efficiency of the PCR reaction.
For example, the amplification efficiency is highly
sensitive to the amount and quality (e.g., intactness
and contamination) of the template DNA. Moreover, in
QC-PCR, the quality of competitor DNA (i.e., purity and
intactness) can also have a considerable effect on the
accuracy of amplification.5) To minimize errors, both
procedures usually ensure highly accurate quantification
of DNA and normalization of the PCR reaction using
a known single-copy gene as an internal standard. With
this process, real-time PCR is deemed more advantageous than QC-PCR, because the latter requires a set of
PCR reactions with a dilution series of competitor DNA.
But, real-time PCR requires a specific detector to
monitor the PCR reactions, which potentially limits
the applicability of this procedure as a standard
laboratory protocol. In the present study, we developed
a new PCR-based procedure, quantitative dual target
PCR (QD-PCR), which can select transgenic plants with
a single integrated gene using standard laboratory
equipment.
Genomic DNA was extracted from leaves (approximately 100 mg fresh weight) of adult plants using a
DNeasy plant mini kit (Qiagen, Hilden, Germany), and
quantified using a PicoGreen dsDNA Quantification Kit
(Invitrogen, Carlsbad, CA). Genomic DNA samples
(20 ng) were then used as templates in a total volume of
10 ml of PCR mixture in individual PCR tubes. The PCR
mixture consisted of standard grade Taq DNA polymer-
y
To whom correspondence should be addressed. Fax: +81-58-293-2911; E-mail: koyama@cc.gifu-u.ac.jp
Abbreviations: GUS, -glucuronidase; NPTII, neomycine phosphotransferase II; 4HPPD, 4-hydroxyphenylpyruvate dioxygenase; HPT,
hygromycin phosphotransferase; PCR, polymerase chain reaction; PetC, photosynthetic electron transfer c; Q-PCR, quantitative PCR; QD-PCR,
quantitative dual target PCR; Tm , melting temperature
Identification of a Single Copy Transgenic Plant
1781
Table 1. Sequences and Tm Values of Primers Used in Quantitative Dual Target PCR for Determination of the Copy Number of T-DNA Integrated
into the Arabidopsis thaliana Genome
4HPPD
F1
R1
PetC
F1
R1
HPT
F1
R1
F2
R2
T-DNA
F1
R1
A. thaliana endogenous (At1g06570)
50 -GCGCTTCCATCACATCGAGTTC
50 -AATCCAATGGGAACGACGACGC
A. thaliana endogenous (At4g03280)
50 -TAAGACTCATGGTCCCGGTGAC
50 -ACCATGGAGCATCACCAGTCCT
Transgene
50 -GAGGGCGAAGAATCTCGTGC
50 -GATGTTGGCGACCTCGTATTGG
50 -CGAGAAGTTTCTGATCGAAA
50 -CAGTCAATGACCGCTGTTAT
Transgene
50 -CCTTGGTTTGTGAAGCAAGCCTTGAA
50 -TGGCTCTAATTCCCAAATGGCTCAAG
Tm
( C)
Amplicon size
(bp)
60.4
60.4
520
60.4
60.4
507
60.4
60.4
52.2
54.2
609
60.5
60.5
618
624
The Tm value was calculated according to the following equation: Tm ð CÞ ¼ 60:8 þ 0:41 ð%GCÞ ð500=nÞ, where %GC is the GC content in the oligonucleotide
(%) and n is the length of the oligonucleotide (bp).
4HPPD, 4-hydroxyphenylpyruvate dioxygenase; PetC, photosynthetic electron transfer c; HPT, hygromycin phosphotransferase
ase (0.35 units per tube; Invitrogen), 100 mM dNTPs and
0.5 mM each of two sets of primer pairs targeting the
integrated T-DNA (HPT gene; AY818364, or the TDNA region itself in SALK T-DNA lines; http://signal.
salk.edu/pBIN-pROK2.txt-new), and a known single
copy gene (4-hydroxyphenylpyruvate dioxygenase
[4HPPD; At1g06570] and photosynthetic electron transfer c [PetC; At4g03280] respectively) (Table 1).6,7) The
PCR reaction was carried out with up to 22 cycles of
94 C for 30 s, 58 C for 30 s, and 72 C for 1 min. After
PCR, samples were loaded onto regular agarose gel
(1.5%, w/v) and visualized by ethidium bromide
staining. The gel image was captured by FAS III
(Toyobo, Osaka, Japan), and the band intensity was
quantified using NIH image software (http://rsb.info.
nih.gov/nih-image). As a reference for determination of
the integrated T-DNA copy number, real-time PCR was
performed using LightCycler (Roche Diagnostics, Basel,
Switzerland) with LightCycler FastStart DNA Master
SYBR Green I (Roche Diagnostics) according to the
manufacturer’s protocol. Copy numbers of the target and
internal standard gene in 20 ng genomic DNA were
quantified relative to the known amount (0.032–4 atto
mol) of plasmid DNA containing each gene. The copy
number of the integrated gene was then normalized by
that of the internal standard gene.
As described above, with PCR, normalization of
template DNA is necessary to quantify the copy number
of an integrated gene, the ‘‘target gene,’’ because
amplification efficiency is sensitive to the purity and
intactness of the template DNA. This step is usually
carried out by quantification of an internal single-copy
gene as a reference. Both QC-PCR and real-time PCR
carry out this step separately from quantification of the
target DNA, but the proposed QD-PCR technique can
run both steps simultaneously in the same PCR reaction.
This reduces the necessary labor and cost of isolation of
transgenic plants carrying a single copy of the integrated
DNA, and minimizes errors that can occur as a result of
slight differences between PCR reactions (e.g., differences in the amount of template genomic DNA in the
various PCR reactions).
In QD-PCR, the target gene is amplified together with
the internal standard gene using two sets of PCR primer
pairs in a single PCR reaction. As described above, this
is advantageous as compared to other PCR-based
methods. But, if the amplification efficiency were to
vary between the target and the internal standard gene,
errors would occur during identification of transgenic
plants carrying a single copy of the integrated gene.
Hence we determined the amplification efficiency of
HPT in the integrated T-DNA and authentic single-copy
genes (4HPPD and PetC) in transgenic plants carrying
a single copy of the integrated gene as a result of
separated real-time PCR reactions (Fig. 1A). When we
used primer pairs with exactly the same Tm values (see
Table 1), all amplicons showed very similar amplifications. Hence we inferred that both the target (HPT) and
internal control genes (4HPPD and PetC) are amplified
at a very similar rate in the QD-PCR reaction. To
determine whether this does in fact take place, we
performed a series of reactions with genomic DNA
extracted from transgenic Arabidopsis carrying single
and double copies of the integrated T-DNA, which was
identified by southern blotting analysis and quantification of the copy number of the integrated T-DNA
containing the HPT gene by competitive PCR in our
previous studies.5,8)
First we examined the effect of PCR cycles on the
band intensity ratio of the target (HPT) and the internal
control gene (4HPPD) (Fig. 1B, C). We were able to
detect both bands after 18 cycles, after which the band
intensity sharply increased (Fig. 1B). The ratio of HPT
to 4HPPD amplicon in the single-copy plant was close to
1782
T. KIHARA et al.
A
A
Fluorescence
100
1
2
3
4
5
HPT
4HPPD
1.1 1.2 0.5 0.4 Ratio
± 0.1 ± 0.0 ± 0.0 ± 0.0 (HPT/4HPPD)
10
1
Amplification efficiency
HPT ( )
1.84±0.01
4HPPD( ) 1.85±0.02
PetC ( ) 1.81±0.01
0.1
15 17 19 21 23 25 27 29 31
Number of cycles
0*
97* 88* 70* 64*
Segregation test
(Survival/100 seeds)
B
Homozygous Heterozygous
B Single copy
18
19
C Double copy
20 21 22
1.2 0.9 1.0 1.2 1.1
± 0.1 ± 0.0 ± 0.0 ± 0.1 ± 0.0
D
I
18 19 20 21 22 cycles
HPT
4HPPD
Ratio
1.9 1.7 1.8 1.4 1.3
± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 (HPT/4HPPD)
1
HPT
4HPPD
3
1
2
3
T-DNA
PetC
5.5 1.1 0.9 8.8 0.5 0.6 Ratio
± 0.7 ± 0.1 ± 0.0 ± 0.5 ± 0.1 ± 0.0 (T-DNA/PetC)
6
II
2
1
1
9
0.5 0.5
Copy number
estimated by real-time PCR
Fig. 1. Amplification of HPT, 4HPPD, and PetC Genes from the
Genome of Transgenic Arabidopsis Plants Using Real-Time PCR
and QD-PCR.
Real-time PCR and QD-PCR reactions were carried out with
20 ng of each genomic DNA derived from single- (A, B, and D) or
double- (C) copy transgenic plants. The increase in SYBR green
fluorescence was caused by amplification of either the internal
standard gene (4HPPD and PetC) or the integrated gene (HPT) in the
real-time PCR reaction (A). The QD-PCR reaction was carried out
for 18–22 cycles in B and C and 20 cycles in D. In the QD-PCR
results, the lower band represents the internal single-copy gene
(4HPPD). The Tm values of all primers were identical in the
reactions shown in A, B, C, and DI, but that of HPT in DII was
lower than the others. Values shown in the bottom of B and C
represent the band intensity ratio determined by the NIH image
(means SD, n ¼ 3).
Fig. 2. Identification of Transgenic Arabidopsis Plants Carrying a
Single Copy of Integrated T-DNA by QD-PCR.
The ratios of target (A, HPT; B, T-DNA region) to control (A,
4HPPD; B, PetC) genes were obtained using gel images (n ¼ 3,
means SD). A, The QD-PCR reaction was carried out with
homozygous (Lane 2, 3) and heterozygous (Lane 4, 5) transgenic
lines carrying a single copy of the integrated HPT gene. The upper
band represents the HPT amplicons. Lane 1 is a null line. The
number of Hyg-resistant plants among 100 seed progenies is shown
below the gel image. Asterisks were attached if the value could be
adapted to Mendelian segregation at P ¼ 0:01 using the 2 test. B,
The QD-PCR reaction was carried out with one homozygous and
heterozygous T-DNA insertion line selected from three different
SALK seed lots according to the method of Siebert et al.9)
Homozygous and heterozygous plants derived from the same seed
lot were given the same number. Lower bands indicate the PetC
amplicons. The copy number estimated using real-time PCR is
shown below the gel image (n ¼ 3).
one, while that in the double-copy transgenic plant was
close to two after 18–20 cycles (Fig. 1C). However, the
ratio of the latter sharply decreased after 21–22 cycles.
These results indicate that estimation of copy number
using QD-PCR is reliable only when the PCR reaction is
not saturated (< 20 cycles).
Secondly, we examined the effect of the melting
temperature (Tm ) of the PCR primers on the accuracy of
the QD-PCR reaction. In the above experiments, we
designed PCR primers with exactly the same Tm value
(60.4 C; Table 1). This gave both the target gene (HPT)
and the control gene (4HPPD) the same amplification
efficiency (Fig. 1B, D, lane I), but the intensity of the
HPT amplicon was weaker than that of the control gene
when the Tm values of the former primers (52.2 and
54.2 C; Table 1) were lower than those of the control
gene (Fig. 1D, lane II). From these results, we concluded that the Tm values of the primers must be equal
for an accurate QD-PCR reaction.
Finally, we tested the capability of the QD-PCR
method in identification of homozygous transgenic
plants carrying a single copy of the integrated DNA.
Using HPT and 4HPPD as target and control genes
respectively, we applied the QD-PCR method to the
same T2 population from which we obtained single copy
homozygous plants, confirmed using quantitative competitive PCR.5) In this case, homozygous transgenic
plants should give the same intensity for both target and
control amplicons, while the intensity of the target
amplicon in heterozygous (i.e., 0.5 copies per haploid)
plants should be almost half that of the control gene. As
shown in Fig. 2A, two transgenic plants (lanes 2 and 3)
were judged homozygous because of similar band
intensities. On the other hand, two transgenic plants
(lanes 4 and 5) appeared to be heterozygous because the
intensity of the target (HPT) amplicon was half that of
the control. These estimations were in accordance with
the genotypes determined using the segregation test with
seed progenies of each plant (Fig. 2A).
Because amplicons of HPT and 4HPPD, which have
a different GC content, were amplified with the same
efficiency, the method should be reliable when a
different combination of target and control genes is
used. SALK T-DNA lines carrying the NPTII gene in
the integrated DNA are usually supplied as a mixture of
heterozygous, homozygous, and null segregants in terms
Identification of a Single Copy Transgenic Plant
of the genotype at the identified locus of the T-DNA
insertion in the SALK catalog (http://signal.salk.edu).
Using the method of Siebert et al.,9) we examined the
genotype of the T-DNA insertion at the identified locus
in the SALK catalog, isolating one homozygous and
heterozygous plant from three different lines. When we
applied the QD-PCR method with two primer sets for
the T-DNA region as a target and PetC as an internal
control gene, we were able to identify lines 2 and 3
as carrying a single copy of the integrated T-DNA
(Fig. 2B). This was identical to the copy number of the
NPTII gene, as quantified by real-time PCR. On the
other hand, line 1 contained multiple copies as a result
of T-DNA insertion at multiple loci, as also mentioned
by SALK. In this case, plants heterozygous at the locus
identified by SALK appeared to include a larger amount
of T-DNA in the genome than the plant identified as
being homozygous at the same locus. Copy numbers
judged by QD-PCR were almost identical to those
confirmed by real-time PCR, but a relatively large
standard deviation was observed (Fig. 2B). Hence we
concluded that QD-PCR can be performed with various
combinations of target and internal standard genes.
In conclusion, QD-PCR was shown to be highly
reliable for identification of transgenic lines with a
single copy of the integrated gene (Fig. 2). The
sensitivity of QD-PCR quantification appeared to be
sufficient for direct identification of two-fold differences
in the copy number of the integrated DNA using gel
images (i.e., single and double [Fig. 1B, C] or heterozygous and homozygous single-copy transgenic plants
[Fig. 2]). This suggests that single-copy transgenic
plants can easily be identified in the T1 generation,
where single-copy plants are expected to show about
half the intensity of signals for the target amplicons
compared to the control genes. Moreover, because of the
simple handling of the reaction, this method is useful for
the selection of single-copy transgenic plants in transgenic studies and crop breeding.
Acknowledgments
This work was supported by the Research Institute of
Innovative Technology for the Earth (RITE) and the
Ministry of Economy, Trade and Industry of Japan. We
1783
are grateful to Miss Hiromi Inagaki of Gifu University
for technical assistance.
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