Botanical Studies (2007) 48: 1-11.
*
Corresponding author: E-mail: bocharng@ccms.ntu.edu.
tw; Tel: +886-2-33664774; Fax: +886-2-23620879.
INTRODUCTION
Rice has become a model for the study of monocot
plants because of the accumulating molecular information
for this species (Harushima et al., 1998; Sakata et al.,
2000; Temnykh et al., 2000; Yuan et al., 2000; for
review see Jeon and An, 2001). Rice also has a high
transformation efficiency (Tyagi and Mohanty, 2000),
a small (430 Mb) genome (Arumuganathan and Earle,
1991), and is economically important (David, 1991).
The complete DNA sequence for the rice genome is now
known. Questions are now being asked about the function
of the genes within it, and techniques are in development
to address these questions on a genome-wide, cross-
species scale. Because of the high degree of conservation
among the gene sequences and orders among cereals,
the structural and functional analyses of rice should have
broad practical implications for developing products and
technologies in both rice and other economically important
cereals. Mutants represent one of the most effective ways
to acquire information on a gene¡¦s function. Various
mutants, such as gene knockouts or null mutations, are
invaluable for understanding biological variability when
assigning functions to such a large quantity of sequence
information. To make full use of the information mutants
provide, developing methods that efficiently utilize vast
quantities of information regarding function are critical.
Transposon tagging has become a powerful tool
to create mutants for isolating new genes. Several
experimental approaches have been undertaken to
develop rice lines in which genes are randomly tagged
by insertion elements (Greco et al., 2001b; Hirochika,
2001, 2004; Izawa et al., 1997; Jeon and An, 2001).
Since the first introduction of maize Ac/D s into rice
(Izawa et al., 1991; Shimamoto et al., 1993), a variety of
modified constructs have been introduced into rice (Chin
et al., 1999; Greco et al., 2001a; Nakagawa et al., 2000).
Some important features of the Ac element have been
characterized in transgenic rice plants: for example (1)
The Ac element transposes in 18.9% of transformed rice;
(2) The transposed Ac element continues to transpose
and is transmitted to subsequent generations; (3) Ac
transactivates transposition of the non-autonomous Ds
element; (4) The germinal excision frequency of Ac could
be as high as 40%; and (5) Ac transposes preferentially
into protein-coding regions (Enoki et al., 1999). These
observations indicate that Ac/Ds gene tagging systems are
valuable for rice functional genomics. Although another
transposon system, En/Spm, has been introduced into rice
plants, its transposition efficiency seems quite low (Greco
et al., 2004). Later, Kumar et al. (2005) showed that the
En/Spm system works very well in rice. For the Ac/Ds
system, several successful transposon tagging experiments
The inducible transposon system for rice functional
genomics
Yuh-Chyang CHARNG
1,
*, Gideon WU
1
, Chia-Shan HSIEH
1
, Han-Ning CHUANG
1
, Ji-Ying
HUANG
1
, Ling-Chun YEH
1
, Ya-Hsiung SHIEH
1
, and Jenn TU
2
1
Department of Agronomy, National Taiwan University, No.1 Sec. 4 Roosevelt Rd., Taipei, Taiwan, Republic of China
2
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China
(Received January 17, 2006; Accepted May 11, 2006)
ABSTRACT.
The one-component inducible transposon system for rice functional genomic studies was
assessed. In contrast to the native Ac transposon, INAc contains the transposase gene under the control of the
inducible promoter (PR-1a) from tobacco. To examine whether INAc can be used in cereals, the behavior of
INAc was analyzed with transgenic rice plants containing a single copy of the INAc element. Treatment of
rice calli with salicylic acid induced transposition of INAc in somatic tissue, and the transposition efficiency
of INAc was dose dependent. Furthermore, a high throughput method for detection of new transposed INAc
was developed. Analyzing the flanking sequences of the transposed INAc indicated the independent insertions.
Given the fact that a number of different types of Ac/Ds vectors have been already examined in rice, the
importance of a "controlled" transposon system to yield knockout mutants or new transgenic plants was
discussed.
Keywords: Ac transposase; Inducible promoter; Salicylic acid; Transposon tagging.
MOLECULAR BIOLOGY
pg_0002
2
Botanical Studies, Vol. 48, 2007
in rice plants indicate that it is a valuable tool for rice
functional genomic studies (Zhu et al., 2003; Komatsu
et al., 2003; Zhu et al., 2004). However, one major
concern when using a native Ac element is that it could
undergo frequent excision from the target gene, resulting
in variegation. This disadvantage can be eliminated with
the use of a two-component system or a self-stabilizing
Ac derivative (Schmitz et al., 1994). However, the
integration of a stable non-autonomous element in the
target gene requires segregation of the two components,
which is very inconvenient. With this mind, we previously
designed an inducible one-component transposon, INAc,
and demonstrated that it could be induced to transpose in
dicotyledon plants (Charng et al., 2000 and 2004). Then,
in a review of strategies for producing rice gene tags,
Jeon and An (2001) suggested that the development of an
inducible transposable element system would be valuable.
This encouraged us in the present study to introduce the
INAc element into rice plants. We observed that the INAc
transposition events increase after applying the inducer,
salicylic acid (SA), and that this higher transposition
efficiency of INAc is dose dependent. In this paper,
we conclude with a discussion of the promotion of the
inducible transposon to perform rice functional genomic
studies.
MATERIALS AND METHODS
Plants and constructs
Construction of the plasmid pINAc and its introduction
into the Agrobacterium tumefaciens strain LBA4404 has
been reported previously (Charng et al., 2000). The rice
variety used in this study was Oryza sativa L. Japonica cv.
TNG67. Calli induced from immature rice seeds were co-
cultured with Agrobacterium using the methods described
by Hiei et al. (1994) and Toki (1997). Putative transformed
calli were selected with hygromycin B.
For induction experiments, the T1 rice seeds of each
transformed line were incubated on callus induction
medium (CIM) containing hygromycin for 4 weeks in
order to yield enough calli for induction experiments. The
effect of SA on INAc transposition was determined by
incubating Hyg
R
calli for various times on CIM containing
0 mM, 5 mM, or 10 mM SA. The calli from the same
seed were evenly divided (about 25 calli per plate) and
incubated on CIM containing 0 mM, 5 mM, or 10 mM SA
with respect to the induction time of 7, 5, and 3 days. The
calli were then transferred to CIM without SA for about 2
weeks before sampling for PCR analysis.
Isolation of genomic DNA and Southern
hybridization analysis
Genomic DNA was isolated from transformed plants
with the use of a kit (BIO 101, Vista, CA). In brief, fresh
leaves (2 g) or callus tissue (0.1 g) was frozen in liquid
nitrogen in a mortar and ground with a pestle. Nuclei were
isolated and lysed by protease treatment, and genomic
DNA was precipitated with ethanol and dissolved in TE
buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8.0). About
10 £gg of each DNA was digested with the appropriate
restriction enzyme under the conditions specified by
the suppliers and fractionated on 0.8% agarose gels (in
1¡ÑTAE) overnight at 1 V/cm. Southern analysis was
performed as described by Charng and Pfitzner (1994).
PCR analysis of
INAc
excision events
Transposition of INAc from the INAc::LUC construct
in transgenic plants was analyzed by polymerase chain
reaction (PCR) with three oligonucleotide primers: primer
1P (identical to the T-DNA promoter sequence from
position 285 to 303 as numbered by Velten et al. (1984),
5¡¦-GGTTGCCATGTCCTACACG-3¡¦); primer LUC2
(complementary to the luciferase coding sequence from
position 367 to 347 as numbered by De Wet et al. (1987),
5¡¦-GCGGGCGCAACTGCAACTCCG-3¡¦); and primer
AC1 (complementary to the Ac sequence from position
155 to 137 as numbered by Muller-Neumann et al. (1984),
5¡¦-ACCCGACCGGATCGTATCG-3¡¦). Each reaction
mixture contained ~0.1 £gg of template DNA, 0.25 £gg of
each primer, 0.2 mM deoxynucleoside triphosphates, 1 U
of Taq DNA polymerase, 10 mM Tris-HCl (pH 8.3), 50
mM KCl, 1.5 mM MgCl
2
, and 0.01% (w/v) gelatin. The
amplification protocol comprised 30 cycles of 1 min at 94
¢XC, 2 min at 55¢XC, and 2 min at 72¢XC and was performed
in a T-gradient Thermocycler (Biometra, Gottingen,
Germany).
The re-intergrated INAc after excision in transgenic
rice was analyzed by single-primer PCR with AC1 as the
primer, and the amplification protocol comprised 45 cycles
of 1 min at 94¢XC, 1 min at 32¢XC, and 3 min at 72¢XC.
The flanking sequences of the INAc element in
transgenic plants were amplified by TAIL-PCR analysis
with the following oligonucleotide primers: primer
AC8 (CCCGTTTCCGTTCCGTTTTC), primer AC2
(CTCGGGTTCGAAATCGATC), and primer AC10 (CG
GTTATACGATAACGGTCGGTAC), which are identical
to the Ac sequences from position 4446 to 4465, 4475 to
4493, and 4513 to 4536 as numbered by Muller-Neumann
et al. (1984), respectively. Three arbitrary degenerate (AD)
primers and the TAIL-PCR procedure were according to
a previous report (Liu et al., 1995), except in the present
study the primary TAIL-PCR contained about 150 ng of
rice genomic DNA.
RESULTS
INAc construct and transgenic rice plants
The construction of the plasmid pINAc has been
reported previously (Charng et al., 2000). This construct
was transformed into rice varieties Oryza sativa L.
Japonica cv. TNG67. 45 independent transformed lines
were collected for primary analysis. The results of induced
transposition of INAc were obtained from transformed rice
plants containing a single copy of INAc.
pg_0003
CHARNG et al. ¡X The inducible transposon in rice
3
Spontaneous transposition of INAc in rice
INAc transposes spontaneously in primary transformed
shoots of tomato harboring the INAc element. The first
step to assess the facilitation of INAc is to examine the
spontaneous transposition events in transgenic rice. We
analyzed genomic DNA from primary rice calli by PCR
analysis (Figure 1) and Southern blot analysis (Figure 2).
Two sets of primers were used to verify transposed vs.
un-transposed INAc elements. With the primers AC1 and
LUC2, a 580-bp PCR product was obtained with DNA
from Hyg
R
rice plants, which harbor the un-transposed
INAc element (Figure 1A).
With the primers 1P and
LUC2, a 670-bp PCR product was obtained with DNA
from transformed rice plants, indicating the excision of the
INAc element. As shown in Figure 1, the transformed lines
R7, R51 and R20 yielded a 580-bp fragment with primers
AC1 and LUC2 (Figure 1B) but yielded no product with
primers 1P and LUC2. These findings indicate that these
Hyg
R
rice plants represented successfully transformed
lines and that the INAc elements were stable without
induction treatment. In contrast, transformed line R44
yielded both a 580-bp fragment and a 670-bp fragment
with these two sets of primers, indicating that this rice
plant contained cells in which INAc had undergone
spontaneous transposition and cells in which it had not.
By using these two sets of primers, 8 out of 45 transgenic
rice showed spontaneous transposition (data not shown).
For the transformed lines yielding no 670-bp fragment,
the spontaneous transposition event was monitored during
the development of the T0 generation. PCR analysis
indicated that the un-induced INAc element remained
stable and transmitted to subsequent generations (data
not shown). Also, genomic DNA from these transformed
lines was subjected to Southern blot analyses. As probes,
the 1.4-kb Bam HI/Eco RV fragment comprising the
LUC reporter gene, and the 2.56-kb Bam HI fragment
comprising the transposase gene were used. The DNA
samples were digested with Eco RV and hybridized with
LUC probe. Figure 2 shows that the transformed lines
R7, R20, R44 and R51 yielded hybridizing fragments
of 23-kb, 10-kb, 13-kb and 13-kb, respectively. These
results demonstrate each transformed line harboring a
single copy of T-DNA. After removal of the LUC probe,
the same filter was hybridized with the TPase probe. In
Figure 1. Construction of the INAc element and PCR analysis of
its excision. (A) Structure of the INAc element and the location
of primers (shown as solid triangles) used for PCR analysis. The
INAc element contains a PR-1a::TPase fusion and a hygromycin
resistant gene (HPT). The sizes of expected PCR products (580-
and 670-bp before and after excision of INAc, respectively)
are indicated. The sequential primers for TAIL-PCR, Eco RV
site and probes for DNA blot analysis were also indicated. (B)
Ethidium bromide-stained agarose gel on which PCR products
were separated. PCR was performed with genomic DNA from
the indicated transformants and with the indicated primers .
The hatched boxes indicate the DNA fragments used as probes
for S outhern blot anal ysi s. La ne M, 100-bp DNA l adder.
Abbreviations: LB and RB, left and right borders of T-DNA; p,
1¡¦ promoter; HPT, hygromycin phosphotransferase gene; LUC,
luciferase gene.
Figu re 2. Southern blot hybridization of Eco RV digeste d
genomic DNAs isolated from different transgenic rice lines with
the LUC probe or with the TPase probe (for construction see
Figure 1). The LUC probe (left) revealed the T-DNA copy and
together with the TPase probe (right) revealed the un-transposed
(indicated by the arrow) or transposed INAc (star).
pg_0004
4
Botanical Studies, Vol. 48, 2007
agreement with the PCR experiment, the transformed lines
R7, R20 and R51 yielded the same hybridizing patterns,
indicating the primary donor sites of the un-transposed
INAc element. For the transformed line R44, in addition to
the hybridizing fragment of 13-kb (un-transposed INAc),
bands of 14-kb and 7-kb indicated the transposition of
INAc (Figure 2).
Induction of INAc transposition
The fact that INAc transposition occurred spontaneously
in transformed line R44 indicates INAc is active in rice
plants. To determine whether the INAc element could be
induced in rice plants, we studied the induction of INAc
transposition using salicylic acid (SA) as the inducer in
transgenic rice calli and plants. The behavior of INAc
in rice plants was studied with T1 seed-derived calli
from four transformed rice lines, R7, R20, R44 and R51.
Lines R7, R20 and R51 contain a single copy of the
INAc element. The T1 rice seeds of each transformed
line were incubated on CIM containing hygromycin to
ensure the presence of the INAc element (transposed or
un-transposed). Each progeny line was established by
incubating the seed on CIM without SA, in order to yield
enough calli for induction experiments. The effect of SA
on INAc transposition was determined by incubating calli
for various times on CIM containing 0, 5 mM or 10 mM
SA. Since abundant calli could be regenerated from the
rice seeds on the CIM, the calli regenerated from each
seed (determined as an independent progeny line) were
then divided into three portions and incubated on CIM
with or without SA. Five Hyg
R
seeds each from lines R7,
R20, and R51 and 30 Hyg
R
seeds from line R44 were used
for induction experiments.
After the induction process, the regeneration calli
of each line were collected for DNA extraction. The
transposition events were determined by multiplex PCR
analysis using three primers: AC1, LUC2 and 1P. The
induced transposition efficiency of each transformed line
was determined by the presence of the 580-bp fragment
and/or the 670-bp fragment as shown in Figure 3 for
progeny lines from R7, R20 and R51. For progeny lines
of R44, the presence of the PCR products were expressed
as "X" or "O" as described in Table 1. It is notable that
exposure to 10 mM SA resulted in a marked decrease in
the efficiency of callus regeneration. For some progeny
lines treated with 10 mM SA, new calli formation was
not observed, even after four weeks of propagation. For
these progeny lines, DNAs could not be extracted for PCR
analysis and are expressed as "¢w" (Table 1).
For transformed lines R7, R20 and R51, in which no
spontaneous transposition event was detected, all un-
induced progeny lines yielded a 580-bp fragment and one
(line R50-1) out of 15 progeny lines also yielded a 670-bp
fragment. These findings suggest that a spontaneous
transposition event occurred in these transformed lines but
with low frequency. However, when these progeny lines
were treated with 5 mM SA, 8 out of 15 calli exhibited
transposition events; when these same line were treated
with 10 mM, 7 out of 11 calli exhibited transposition
events. In addition to the spontaneous transposition events
observed in un-induced progeny line R51-1, a 670-bp
product was also detected in SA-induced progeny lines
R7-2 (at both 5 mM SA and 10 mM SA), R7-4 (at both 5
mM SA and 10 mM SA), R20-1 (at 10 mM SA), R20-2
(at both 5 mM and 10 mM SA), R20-3 (at 5 mM SA),
R20-4 (at 5 mM SA), R20-5 (at both 5 mM and 10 mM
SA), R51-2 (at 5 mM SA), and R51-4 (at 10 mM SA). In
summary, these findings demonstrate that transposition
events were triggered under un-induced conditions with
0.6% efficiency while 5 mM- and 10 mM-SA induction
produced a 53% and 64% efficiency, respectively.
Taken together, these results show that the transposition
efficiency of INAc was induced by SA in a dose-dependent
manner. On the other hand, the transposition efficiency
in line R7, R20, and R51 was 40%, 80% and 20%,
respectively. These findings suggest that the location of
the INAc element in the plant genome also contributes to
the efficiency of transposition.
F igu re 3. PCR a na lysis for detec tion t rans position events
for progeny lines of R7, R20 and R51. E ach PCR reaction
was performed with 1P, AC1 and LUC primers. The reaction
yielded only 580-bp indicated un-transposed INAc. The reaction
yielded only 670-bp, indicating completely-trans pos ed INAc.
The reaction yielded both 670 and 580-bp, indicating partially-
transposed INAc. Lane M, 100-bp DNA ladder.
pg_0005
CHARNG et al. ¡X The inducible transposon in rice
5
Interestingly, among the progeny lines of the
transformed line R-44 that demonstrated spontaneous
transposition, 15 out of 30 un-induced progeny lines
yielded a 670-bp fragment when using primers 1P and
LUC2 (Table 1). These findings indicated that new
spontaneous transposition events occurred in the un-
induced progeny of transformed line R-44. When the same
30 progeny lines were treated with 5 mM SA, 29 lines
yielded a 580-bp fragment, and 24 lines yielded a 670-bp
fragment. Exposure to 10 mM SA resulted in a marked
decrease in the efficiency of callus regeneration, and hence
21 progeny lines were obtained for analysis. Among these
21 lines, 18 lines yielded a 580-bp fragment when using
primers AC1 and LUC2, and 19 lines yielded a 670-bp
fragment when using primers 1P and LUC2. Thus, the
transposition efficiencies of 0 mM SA- (spontaneous),
5 mM SA-, and 10 mM SA-induced progeny lines were
50%, 80% and 90%, respectively. These results suggest
that SA increases the frequency of production of the
670-bp product in a dose-dependent manner although,
as mentioned above, treatment of plants with 10 mM SA
resulted in a marked decrease in the efficiency of callus
regeneration.
Table 1. PCR analysis for detecting transposition events for progeny lines of R-44.
Induction
no SA
5 mm SA (5 days)
10 mm SA (3 days)
PCR products
Progeny lines 580-bp 670-bp Single primer 580-bp 670-bp Single primer 580-bp 670-bp Single primer
1
O
X
O
X
O
X
2
O
O
O
X
O
O
3
O
O
O
O
¡X
¡X
¡X
4
O
X
O
O
¡X
¡X
¡X
5
O
O
O
O
¡X
¡X
¡X
6
O
O
O
O
¡X
¡X
¡X
7
O
X
O
O
¡X
¡X
¡X
8
O
O
O
O
O
O
9
O
X
X
O
O
O
O
10
O
X
O
X
¡X
¡X
¡X
11
O
O
O
O
O
O
O
12
O
X
O
O
O
O
O
13
O
O
O
O
O
O
14
O
O
O
O
O
O
O
15
O
X
O
O
O
O
O
16
O
X
O
O
¡X
¡X
¡X
17
O
O
O
O
O
O
18
O
X
O
O
O
O
19
O
O
O
O
O
O
O
20
O
O
O
O
O
O
21
O
X
O
X
X
O
22
O
O
O
O
O
O
23
O
X
O
O
X
O
24
O
X
O
X
¡X
¡X
¡X
25
O
O
O
O
¡X
¡X
¡X
26
O
X
O
O
X
O
27
O
O
O
O
O
X
28
O
O
O
O
O
O
O
O
29
O
X
O
X
O
O
30
O
X
O
O
O
O
"O": symbol denotes the existence of PCR products (580-bp products for primers AC1 and LUC2; 670-bp products for primers 1P and
LUC2; different size patterns for single primer (AC1) PCR; "X": no PCR product; "¡X": DNAs were not extracted for PCR analysis.
pg_0006
6
Botanical Studies, Vol. 48, 2007
It is important to understand why some SA-induced
progeny lines yielded both a 580 bp and a 670 bp product
while others yielded only one of these products. Typical
examples of this phenomenon were in progeny lines
R20-2 (Figure 3, 10 mM SA induction), and R44-21,
-23, and -26 (Table 1, 10 mM SA induction). When these
progeny lines were incubated on CIM without SA, only
a 580-bp fragment could be detected. When these same
progeny lines were induced with 10 mM SA, only a
670-bp fragment could be detected. When progeny line
R44-23 and R44-26 were induced with 5 mM SA, both a
580-bp fragment and a 670-bp product could be detected.
It is likely that in these SA-treated progeny lines, INAc
was transposed in most cells after 10 mM SA treatment.
As a result, no 580-bp fragment was yielded when using
AC1 and LUC2 primers for PCR analysis. We found that
this phenomenon could also serve as an additional method
to determine the effect of SA concentration. In Table 1,
the transposition events of each progeny line with various
treatments were interpreted as an un-transposed line,
a partially-transposed line, or a completely-transposed
line. The un-transposed lines were determined as those
lines which yielded only a 580-bp fragment but no
670-bp fragment, e.g., progeny line R44-1 and R44-24.
The partially-transposed lines were determined as those
lines which yielded both a 580-bp fragment and a 670-bp
fragment, e.g., progeny lines R44-3 to R44-8 (Table 1,
5 mM SA induction) and R7-2 (Figure 3, 5 and 10 mM
SA induction). The completely-transposed lines were
determined as those lines which yielded only a 670-bp
fragment but no 580-bp fragment, e.g., progeny lines
R20-2 (Figure 3, 5 mM and 10 mM SA induction), and
R44-21, -23 and -26 (Table 1, 10 mM SA induction). The
SA-induced transposition efficiency was then measured
by the percentage of un-transposed, partially-transposed,
and completly-transposed progeny lines of an individual
transformed line. Using this method, we observed that
partially-transposed efficiency reached 50%, 77% and
76% in un-treated, 5 mM SA- and 10 mM SA-treated
progeny lines, respectively. Furthermore, completedly-
transposed efficiency reached 0%, 3% and 14% in un-
treated, 5 mM SA- and 10 mM SA-treated progeny lines,
respectively. Once again, these results demonstrated
that SA can induce the expression of PR-1a::TPase and
transposition events in transgenic rice plants in a dose-
dependent manner.
Detection of new transposition rice lines and
the flanking genomic DNA
An important feature of an inducible transposable
element for functional genomic studies is its tendency
to create new transposition lines after induction. Thus, a
high throughput method for screening the independent
transposants (transposition lines) markedly increases
the probability of success of transposon tagging when
attempting to isolate important plant genes. Toward this
end, in the present study we developed an identification
system for new transposants in rice plants based on the
single-primer PCR concept described by Karlyshev et
al. (2000). The procedure involves just one transposon-
specific primer and a single PCR reaction involving
a low annealing temperature (32¢XC) for amplification
of the region adjacent to the transposon insertion site.
Amplification of this region is possible because at low
annealing temperatures, the primer will bind to sites
of limited sequence complementarity that are on the
opposite strand from the specific primer binding site and
close enough to the transposon insertion site for PCR to
work. Subsequently, when the single primer PCR results
indicate the existence of new independent transposants,
the flanking sequence of the re-integrated INAc can be
obtained by the TAIL-PCR technique.
As indicated in Table 1, the DNA extracts prepared
from the progeny lines of R44 were analyzed by single-
primer PCR. Principally, a single-primer PCR reaction
produces several bands composed of specific and non-
specific products. In this experiment, one end of the
specific products was primed by AC1 containing a portion
of DNA (155-bp) identical to the end of INAc while the
non-specific products were amplified from the unknown
sequence dispersed on the chromosome. All un-transposed
progeny lines of R44 principally yielded the same DNA
pattern, composed of non-specific products and/or a
specific product. However, when the INAc element was
transposed from the original site and integrated into
another site on the chromosome, observing different
DNA patterns by a single-primer PCR analysis became
possible. Figure 4 presents a typical example of this
analysis. For most of the progeny lines, single-primer PCR
produced three major bands of 600-bp, 800-bp and 1.3-kb
in size. However, for progeny line R44-14 (with 5 mM
SA treatment), a 450-bp band was detected instead of a
800-bp band. These results indicate that for progeny lines
harboring the un-transposed INAc, the 800-bp band was
the specific product primed by the AC1 primer. Additional
bands of various sizes but larger than 155-bp indicate new
and different transposition sites of INAc. For example, the
appearance of the 450-bp band in progeny line R44-14
strongly suggested a new transposition site of INAc.
According to this, we detected 2 out of 30 progeny lines
(no SA), 3 out of 30 progeny lines (5 mM SA-treated) and
3 out of 21 progeny lines (10 mM SA-treated) of R44 that
yielded new PCR product patterns (Table 1).
The experiments were expanded to amplify the
flanking sequences of the T-DNA and the transposed
INAc elements. Genomic sequences flanking the INAc
transposons were isolated from transgenic lines of
R-44 and R-20 with induced somatic transposition, due
to observation of the new single-primer PCR product
patterns. The flanking sequences were isolated using
TAIL PCR (Liu et al., 1995, see Materials and Methods).
Three specific primers AC8, AC2, and AC10 were used
for the primary, secondary, and tertiary PCR reactions,
respectively (Figure 1). A summary of the significant
homologies obtained after comparison of the flanking
sequences obtained in public database is shown in Table
pg_0007
CHARNG et al. ¡X The inducible transposon in rice
7
2. For the transformed line R-20, only 1 out of 3 calli
showed an un-linked transposition event of INAc, yet for
the transformed line R-44, 4 out of 6 calli harbored the
transposed INAc un-linked to the T-DNA. These results
indicate that the independent transposition events of INAc
could be induced efficiently by SA.
DISCUSSION
Rice, with its small genome size and well-characterized
molecular information, is an ideal model plant for cereal
genomics research. The entire sequence of the rice (Oryza
sativa) genome has been determined by the International
Rice Genome Sequencing Project (IRGSP). Still, based on
current technologies, a large population of mutant plants
will likely be required to adequately assign function to the
abundance of sequence information.
Jeon and An have summarized various strategies
for producing gene tags that may be invaluable for
understanding the functional genomics of rice (2001).
Both T-DNA and Ac element have been used as the
insertional mutagen for rice gene tagging. Previously,
we reported an inducible transposable element for higher
plants (Charng et al., 2000). In the present study, we
studied the activity of INAc in order to further develop rice
gene tagging systems.
For the INAc construct, the native PR-1a promoter was
fused with the transposase gene of the Ac element, and
this PR-1a::TPase construct was inserted together with
the HPT gene directly into a small Ds element (Figure
1A). The resulting INAc element was placed between
the 1¡¦ promoter and the LUC gene. In principal, LUC
expression should be triggered after excision of the
transposable element. However, when the INAc construct
Figure 4. S i n g l e - pr i m e r
P C R a n a l y s i s o f t he n e w
t r a n s p o s i t i o n e v e n t s o f
INAc. G eno m ic DNA from
transformed rice progeny lines
were s et for P CR rea ctions ,
whi ch co nta i ned onl y A C1
p ri m er, a nd t he an ne a l in g
temperature was set as 32¢XC.
Table 2. Genomic sequences flanking INAc insertions in transgenic rice plants. The T-DNA integration site of each line is indicated
after its designation.
Line
Chromosome
BACs/PACs Insertion position (bp) GenBank accession no. Identities
R-44
(T-DNA) 3
OJ1124_H03
135923
AC087852
102/102 (100%)
0-11
10
OSJNBa0041L14
76009
AC099042
340/352 (96%)
0-28
1
P0443D08
120609
AP003250
467/478 (97%)
5-9
nd
nd
nd
nd
nd
5-14
3
OSJNBb0081B07
85729
AC093018
67/67 (100%)
5-28
9
P0528B09
88111
AP004703
89/89 (100%)
10-12
10
*Pseudo10p0.0-10p4.4
768203
AC145127
(100%)
10-15
nd
nd
nd
nd
nd
10-19
3
OSJNBa0002I03
72340
AC091246
40/40 (100%)
R-20
(T-DNA) 12
5-2
9
OSJNBa0047P18
154242
AP005864
47/47 (100%)
12
OSJNBa0014C10
45261
AL731787
27/27 (100%)
10-5
12
OSJNBa0052H10
29041
BX000494
104/104 (100%)
"nd": indicates no detectable product is obtained after TAIL PCR amplification.
pg_0008
8
Botanical Studies, Vol. 48, 2007
was introduced into rice plants, our preliminary analyses
indicated that although the transposition events could be
detected by PCR analysis, no luciferase activities were
detected (data not shown). This result is likely because the
1¡¦ promoter is less functional in rice plants. Consequently,
we set out to determine the transposition events using
PCR analysis. 8 out of 45 primary transformed rice lines
yielded a 670-bp band with primers 1P and LUC2 in the
absence of the inducer SA. This result is likely attributable
to activation of the PR-1a promoter by nearby enhancer
elements in the plant genome (Beilmann et al., 1992).
In the transformed lines that exhibited no spontaneous
transposition events, the INAc element remained stable
during the development of the plant, and it was transmitted
to the next generation (data not shown). The induction
experiments were performed with the T1 progeny lines,
which harbored the INAc element. We used PCR to
determine transposition events by observing the 670-bp
fragment with primers 1P and LUC2. The results shown in
Figure 3 and Table 1 demonstrate that transposition events
could be induced by SA treatment (5 mM or/and 10 mM)
calli with various efficiencies. Generally, we incubated
rice seeds on CIM for 4 weeks to produce enough calli for
induction experiments. Spontaneous transposition events
may have occurred during this calli regeneration process.
Since the calli produced for induction experiments were
divided into three equal portions prior to any artificial
stimulation for transposition, it would seem that a
spontaneous transposition event would happen equally in
all three portions of the calli (control, 5 mM or 10 mM
SA-treated). However, the fact that only one transposition
event (1 out of 15) was detected in un-treated calli of
progeny lines from R20, R7 and R50 strongly suggests
that the transposition events detected in SA-treated calli
mainly resulted in response to the induction treatment.
For those calli regenerated from the transformed line
R44, in which spontaneous transposition events were
detected in the T0 generation, it was necessary to consider
that the 670-bp products detected in the SA-treated calli
may have been spontaneous transposition events before
or during SA treatment. To rule out this possibility,
we determined the induced transposition events and
efficiencies by comparing the presence of both a 580-bp
(un-transposed) and a 670-bp (transposed) product.
Any 670-bp product with primers 1P and LUC2 was
recorded as a transposition event (spontaneous or non-
spontaneous). The transposition efficiencies were based
upon the presence of a 670-bp product in the progeny lines
of transformed line R44. We found that, the transposition
efficiency of un-induced, 5 mM SA- and 10 mM SA-
treated R44 progeny lines were 50%, 80% and 90%,
respectively. Alternatively, for progeny lines R44-24,
R44-34 and R44-55, 10 mM SA-treated calli yielded only
a 670-bp fragment but no 580-bp product. This was likely
because transposition events were triggered in most cells
of these calli, and hence the un-transposed INAc could
not be detected. We considered these calli as completely-
transposed after treatment. The presence of completely-
transposed calli from each progeny line served as a means
to determine the effect of SA concentration. On this basis,
completedly-transposed efficiency reached 0%, 3% and
14% by the un-treated, 5 mM SA- and 10 mM SA-treated
progeny lines, respectively. Taken together, these results
strongly suggest that the transposition efficiency of INAc
can be induced by SA in a dose-dependent manner.
An important feature of an inducible transposable
element is its ability to induce transposition in germinal
tissue. Induction of the germinal transposition in rice
is supposedly more complicated than in tobacco, since
the rice floral tissues embed themselves in the flag
leaves when (or before) meiosis occurs. For our primary
induction experiments, the highest germinal transposition
efficiency was detected by flooding the transgenic rice
with 5 mM SA solution; two out of 40 R20 progeny
lines showed transposition events under those condition,
indicating a 5% germinal transposition efficiency (data
not shown). Compared with the germinal transposition of
the native Ac element in rice (up to 40%; Nakagawa et al.,
2000), optimization of induction conditions for germinal
transposition of INAc requires further experimentation.
Still, since rice is an ideal model plant due to the
availability of its complete DNA sequence and its high
regeneration efficiency from calli, even heterozygous
mutants created by INAc are also valuable for reverse
genetics. We believe that the induced INAc-mutants
regenerated from rice calli are also valuable.
Our experiences in the present study lead us to suggest
a procedure for inducible transposon tagging in rice.
First, the INAc was induced to create abundant mutant
lines. Then, as a high throughput tool, the single-primer
PCR method was used to identify the new transposants.
Finally, these transposants were collected for TAIL-PCR
to identify the flanking sequence of the INAc element.
By aligning the DNA sequences obtained from the TAIL-
PCR technique to ensure the existence of the region from
the specific primer for tertiary TAIL-PCR (primer AC10
in this paper) to the end of INAc (5-end in this paper), the
new transposants could be recorded for insertion-tagged
sequence libraries for rice.
All the features described above indicate that the
inducible transposon system would be a valuable tool
for Agro-biotechnology, e.g. either to create knockout
mutants or to generate selectable marker-free transgenic
plants. Nevertheless, several improvements must be
considered in order to design a new inducible transposon
for plant gene tagging systems. First, the fact that INAc
can transpose spontaneously in tomato, tobacco, and
rice indicates that the PR-1a inducible promoter can
be activated by endogenous stimuli in these plants.
Although the expression level in this condition might
be low, the resulting abundance of the transposase is
likely sufficient to drive transposition events. Previous
studies indicated that Ac/ Ds transposons are easily
triggered by fewer transposases (Fusswinkel et al., 1991).
Thus, it seems that spontaneous transposition events of
pg_0009
CHARNG et al. ¡X The inducible transposon in rice
9
INAc may occur generally in heterologous plants, with
various efficiencies. This fact encourages us to consider
the use of an animal inducible system for constructing
new inducible transposons. Recently, several inducible
systems from animals were used in plants (Padidam
et al., 2003; Ouwerkerk et al., 2001; Zuo et al., 2000;
Bohner et al., 1999). All of these systems should be
assessed for the creation of new inducible transposon.
On the other hand, since a self-stabilizing Ac concept
has been reported (Schmitz and Theres, 1994), a one-
step inducible transposon that undergoes inducible
transposition but is stable after integration should also be
valuable. Alternatively, many activation-tagging systems
have been developed in Arabidopsis for cloning genes
(Weigel et al., 2000; Marsch-Martinez et al., 2002). An
entrapment tagging system allows for monitoring gene
activity by creating fusions between tagged genes and
a reporter gene (Meissner et al., 2000). According to
these, several new inducible transposons based on INAc
have been demonstrated to be functional and as efficient
as INAc in rice (Charng et al., unpublished data). All of
these strategies, combined with the one-step inducible
transposon concept, will allow us to develop more efficient
transposon systems for future rice functional genomic
studies.
Acknowledgments. This project was supported by
the National Science Council (Grant No. NSC93-
2317-B-002-007-) of Taiwan.
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pg_0012