全 文 ：Received 2 Apr. 2004 Accepted 1 Aug. 2004
Supported by the State Key Basic Research and Development Plan of China (2001CB10901), the Hi-Tech Research and Development (863)
Program of China (2001AA212041, 2002AA212031) and the National Special Program for Research and Industrialization of Transgenic
Plants (JY03-B-18-03).
* Author for correspondence. Tel(Fax): +86 (0)10 64852890; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (12): 1416-1423
Cre/lox-mediated Marker Gene Excision in Elite Indica Rice Plants Trans-
formed with Genes Conferring Resistance to Lepidopteran Insects
CHEN Song-Biao1, LIU Xiang1, PENG Hai-Ying2, GONG Wang-Kui1, WANG Rui1, WANG Feng2, ZHU Zhen1*
(1. Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100101, China;
2. Fujian Provincial Key Laboratory of Agricultural Genetic Engineering, Fujian Academy of Agriculture Sciences,
Fuzhou 350003, China)
Abstract: Cre/lox -mediated gene excision in commercial rice (Oryza sativa L.) plants was studied with
a recombination-reporter gene system, in which the selectable marker hygromycin phosphotransferase
gene (hpt) flanking by two directly oriented lox sites was located between the rice actin1 promoter and a
promoterless gusA gene. This system allows visualization of GUS expression by activating promoterless
gusA after site-specific recombination. The crossing strategy was used to introduce the cre gene into the
lox plants. In 30 hybrid plants from four crosses made from T0 actin1 promoter-lox -hpt -lox -gusA plant with
T0 cre plant, 12 expressed GUS and 9 showed hygromycin-sensitive. We furthermore demonstrated the
utility of the Cre/lox in excision of hpt marker gene in an elite indica rice restorer Minghui 86 transformed
with both insecticidal modified cowpea trypsin inhibitor gene sck and Bacillus thuringiensis endotoxin gene
cryIAc. In 77 hybrid plants from nine crosses made from T2 homozygous lox -hpt -lox -sck -cryIAc plant with
T2 homozygous cre plant, 56 showed hygromycin-sensitive. Molecular analyses confirmed the excision of
hpt in all hygromycin-sensitive plants.
Key words: Cre/lox ; gene excision; transgenic rice; insect resistance; sck ; cryIAc; marker-free
Selectable marker genes are commonly used in transfor-
mation systems for the recovery of transgenic plants.
However, the maintenance of marker genes in transgenic
plants has caused environmental and consumer concerns
in recent years. Although no scientific basis has been de-
termined for these concerns, the absence of selectable
marker genes would certainly contribute to the public ac-
ceptance of transgenic crops (Zuo et al., 2001). Moreover,
the removal of marker genes is also a reasonable strategy
for gene stacking through re-transformation using the same
marker gene (Yoder and Goldsbrough, 1994). To this end,
several strategies have been proposed to generate marker-
free transgenic plants, including co-transformation,
transposon-mediated repositioning, intrachromosomal re-
combination and site-specific recombination (Ebinuma
et al., 2001).
Site-specific recombination systems, such as Cre/lox
(Austin et al., 1981), R/RS (Araki et al., 1987) and FLP/FRT
(Broach et al., 1982), consist of a recombination enzyme
and two small DNA recognition sites. These recombina-
tion enzymes can interact with its specific recognition sites
and result in excision of the intervening DNA, when two
recognition sites present in a direct repeat orientation. Use
of site-specific recombination systems has been reported
to successfully excise selectable marker genes from genome
in model plants such as Arabidopsis and tobacco (Dale
and Ow, 1991; Russell et al., 1992; Gleave et al., 1999; Sugita
et al., 1999; Sugita et al., 2000; Zuo et al., 2001) and in
economically important crops rice and maize (Endo et al.,
2002; Hoa et al., 2002; Zhang et al., 2003).
Rice (Oryza sativa) is one of the world’s most important
food crops, but is susceptible to attack by wide range of
herbivorous insects. Since there is no known rice cultivar
with natural sufficient level of resistance to the major rice
pests (Maqbool et al., 1998), transgenic breeding technol-
ogy has been considered to be a new environmentally sus-
tainable approach to control insect predation of rice. Since
1993 (Fujimoto et al., 1993), transgenic rice plants with in-
secticidal genes enhanced pest resistance have been re-
ported in many experiments (Duan et al., 1996; Xu et al.,
1996; Nayak et al., 1997; Cheng et al., 1998; Maqbool et al.,
1998; Tu et al., 2000; Khanna and Raina, 2002). In our pre-
vious research, we have also successfully developed
transgenic elite indica rice lines expressing a modified cpti
CHEN Song-Biao et al.: Cre/lox-mediated Marker Gene Excision in Elite Indica Rice Plants Transformed with Genes Conferring
Resistance to Lepidopteran Insects 1417
gene, named sck (Deng et al., 2003) or both sck and cryIAc
genes with highly resistance to lepidopteran insects
(unpublished data). In this study, we investigated the func-
tion of the Cre/lox, one of the most widely studied site-
specific recombination systems, in rice by a recombina-
tion-reporter gene system. The results showed that the Cre/
lox system could control gene excision efficiently in rice.
Based on this, we furthermore demonstrated the utility of
Cre/lox in excision of selectable marker gene in commercial
rice plants transformed with insecticidal genes sck and
cryIAc.
1 Materials and Methods
1.1 Vector construction
Three binary vectors, pCACreBar, pCALHGUS and
pCUBACloxHpt (Fig.1a, b, c) were designed and constructed
to test the function of the Cre/lox controlling gene excision
in rice. To construct plasmid pCACreBar, a cre gene di-
gested by HindⅢ from pUC19-cre (Mozo and Hooykaas,
1992) was inserted into the corresponding sites between
the rice actin1 promoter (McElroy et al., 1990) and the ter-
minator of nopaline synthase gene (Tnos) in the intermedi-
ate plasmid pAPTN. The actin1 promoter-cre-Tnos frag-
ment digested by XhoⅠ / PstⅠ from the resultant recombi-
nant plasmid pBlueACre was then inserted into the XhoⅠ/
PstⅠ site of pCMasbarm (Zhou et al., 2003) to generate
plasmid pCACrebar.
A 112-bp double strand oligonucleotide, 5 -
CTCGAGATAACTTCGTATAATGTATGCTATACGAAGT-
TATAGTTAACTACGTAAGGCCTAATAACTTCGTATAA-
TGTATGCTATACGAAGTTATCCACCATGTTGGATCGAT-
3, was synthesized (TaKaRa company, Dalian, China) and
then cloned into the XhoⅠ/ ClaⅠ site of pBlueScriptKS+
(Stratagene) to obtain plasmid pBluelox. This oligonucle-
otide contains a HpaⅠ site flanked by two direct repeat
lox sites. An expression cassette of hygromycin
phosphotransferase (hpt) gene, which driven by the pro-
moter of mannopine synthase gene (Pmas), digested by
XhoⅠ / EcoRⅤ from intermediate plasmid pMPHpt was
inserted into the HpaⅠ site of pBluelox to obtain plasmid
pBlueloxHpt. The lox-Pmas-hpt-Tnos-lox fragment was
digested by XhoⅠ/BstXⅠ from pBlueloxHpt and inserted
into the XhoⅠ/BstXⅠ site of pCAMBIA2300 (http://www.
cambia.org.au/main/r-et-camvec.htm) to form plasmid
pCloxHpt. The plasmid pCALHGUS was then derived by
inserting the rice actin1 promoter into the XhoⅠ site and
inserting a promoterless b -glucuronidase gene (gusA) fol-
lowed by the Tnos into the BstXⅠ /HindⅢ of the
pCloxHpt.
Plasmid pCUBACloxHpt was derived by inserting a frag-
ment containing both a sck gene (Deng et al., 2003) con-
trolled by the rice actin1 promoter and a cryIAc gene (Cheng
et al., 1998) controlled by the maize ubiquitin promoter
(Christensen and Quail, 1996) into the HindⅢ site of
pCloxHpt.
1.2 Transformation of rice plants and crossing of
transgenic plants
Plant expression vectors pCACreBar, pCALHGUS and
pCUBACloxHpt were transferred into Agrobacterium
tumefaciens LBA4404 by electroporation according to the
instruction of Escherichia coli pulser apparatus (BIO-
RAD). The elite indica rice restorer line, Minghui 86, was
used for transformation. Rice plants were grown under field
conditions, and immature embryos 12-15 d after pollina-
tion were isolated for tissue culture and transformation
experiments. One-month-old calli induced on N6-based
(Chu, 1978) medium were then used as explants for
Agrobacterium-mediated transformation by following es-
sentially the protocol of Hiei et al. (1994). Stably
pCACreBar-transformed plants were selected for
phosphinothricin (ppt) resistance and pCALHGUS- and
pCUBACloxHpt-transformed plants were selected for
hygromycin resistance. To excise hpt from transgenic rice
plants, the crossing strategy was used to introduce the cre
gene into the lox plants. For pCALHGUS-transformed
plants, primary T0 transformants were crossed with primary
T0 pCACreBar-transformed plants and for pCUBACloxHpt-
transformed plants, homozygous T2 generation plants were
produced to cross with homozygous T2 pCACreBar-trans-
formed plants.
1.3 Plant assays
GUS activity in leaves or in whole plants of rice seed-
lings was performed according to Jefferson et al. (1987).
Hygromycin resistance of rice seedlings was assayed by
leaf culture method as essentially described by Wang and
Waterhouse (1997). Seeds were germinated and grown in
the greenhouse for about 1 month. Leaf tip of the first ex-
panding leaf was cut and cultured on MS media containing
0.5 mg/L 6-BA and 100 mg/L hygromycin for about 6-8 d to
assay hygromycin resistance.
1.4 Molecular analysis
Total genomic DNA was extracted from leaves, as de-
scribed by Murray and Thompson (1988). PCR was carried
out under standard conditions with oligonucleotide prim-
ers for hpt, gusA, cre, sck and cryIAc genes, resulting in
fragments of 0.85 kb, 0.99 kb, 1.0 kb, 0.42 kb and 0.74 kb
r e s p e c t i v e l y . P r i m e r s e q u e n c e s w e r e 5 -
TA C A C A G C C AT C G G T C C A G A - 3 a n d 5 -
Acta Botanica Sinica 植物学报 Vol.46 No.12 20041418
TAGGAGGGCGTGGATATGTC-3 fo r hpt , 5 -
CGAACT GAACT GGCAGACTATC-3 and 5 -
GGTTCAGGCACAGCACATCAAAG-3 for gusA, 5-
AT G T C C A AT T TA C T G A C C G T- 3 a n d 5 -
CTAATCGCCATCTTCCAGCA-3 fo r cre , 5 -
A A AAT G AA G A G C A C C AT C T T C - 3 a n d 5 -
TCTAGAGTTCATCTTTCTCATC-3 for sck , 5 -
T G C AG AG AG C T T C AG AG AG T G -3 a n d 5 -
ACACCCTGACCTAGTTGAGC-3 for cryIAc. For South-
ern blotting analysis, about 20 mg of genomic DNA digested
with restriction endonuclease (NEB) was separated through
electrophoresis in 1.0% agarose gel and blotted onto
Hybond-N+ nylon membranes (Amersham Pharmacia). Blots
were hybridized with sck, cryIAc, hpt and cre, using the
PCR-generated fragments of sck, cryIAc, hpt and cre
(primers and conditions as described above) labeled with
[a-32P] dCTP as probes.
2 Results
2.1 Experimental design and transformation of rice plants
A recombination-reporter gene system allowing visual-
ization of GUS expression by activating promoterless gusA
after site-specific excision was designed and constructed
to test the function of Cre/lox system in rice. The construct,
pCALHGUS (Fig.1b), contained a selectable marker hpt gene
expression cassette flanking by two directly oriented lox
sites. This hpt cassette was located between the rice ac-
tin1 promoter and the gusA structural gene and as a result,
gusA expression from the actin1 promoter was blocked.
After the excision of the hpt cassette catalyzed by Cre
recombinase, the promoterless gusA gene will be adjacent
to the actin1 promoter (Fig.1b) and consequently, the gusA
gene will be activated and its expression could be detected.
Calli of elite indica rice restorer Minghui 86 were trans-
formed with plant expression vectors pCACreBar,
pCALHGUS and pCUBACloxHpt. For vector pCACreBar,
transformed calli were selected and regenerated on ppt-
containing medium and for vectors pCALHGUS and
pCUBACloxHpt, transformed calli were selected and regen-
erated on hygromycin-containing medium. Totally 5, 8, and
26 plants were derived from independent calli transformed
with pCACreBar, pCALHGUS and pCUBACloxHpt and
designated as cre-, Pactin-lox-hpt-lox-gusA- and lox-hpt-
lox-sck-cryIAc-plants respectively.
Fig.1. Maps of plant expression vectors. a. pCACreBar. b. pCALHGUS. c. pCUBACloxHpt. cre, recombinase gene of Cre/lox system;
bar, phosphinothricin-N-acetyltaensferase gene; gusA, b -glucuronidase gene; hpt, hygromycin phosphotransferase gene; sck, modified
cpti gene with a signal peptide and a KDEL coding sequences at 5 and 3 ends (Deng et al., 2003); cryIAc, Bacillus thuringiensis endotoxin
gene; Pactin, promoter of rice actin1 gene; Pmas, promoter of mannopine synthase gene; Pubi, promoter of maize ubiquitin gene; Tnos,
terminator of nos gene; Tocs, terminator of ocs gene; lox, specific recognition site of Cre/lox system; LB, RB, left and right borders of T-
DNA.
CHEN Song-Biao et al.: Cre/lox-mediated Marker Gene Excision in Elite Indica Rice Plants Transformed with Genes Conferring
Resistance to Lepidopteran Insects 1419
2.2 GUS expression in hybrid plants of Pactin-lox-hpt-
lox-gusA-plants ×cre-plants
PCR analyses were carried out to confirm the presence
of gusA or cre gene in selected primary Pactin-lox-hpt-lox-
gusA-plants and cre-plants. An expected 989-bp internal
fragment from gusA or an expected 1 000-bp internal frag-
ment from cre could be detected in all Pactin-lox-hpt-lox-
gusA- or cre-plants (data not shown). The eight Pactin-
lox-hpt-lox-gusA-plants and five cre-plants were then
grown in the greenhouse and the plants were crossed for
producing hybrid seeds.
Four crosses of Pactin-lox-hpt-lox-gusA-plant × cre-
plant with 30 hybrid plants (F1) in total were made. All these
four crosses yielded GUS expressing hybrid plants (12 out
of 30) (Table 1). The GUS expression in individual crosses
varied from 2/7 (28.6%) to 6/12 (50%). GUS activity in whole
plants of a few rice seedlings was performed by histochemi-
cal assay and the GUS expression could be visualized in
leaf, stem and root (Fig.2). To confirm the excision of hpt
gene mediated by the Cre recombinase, the seedlings were
assayed for hygromycin resistance. Of 12 hybrid seedlings
with GUS expression, nine were hygromycin-sensitive (Table
1; example in Fig.3). PCR analysis showed that while the
parental Pactin-lox-hpt-lox-gusA-plants yielded the ex-
pected 845-bp fragment for hpt, the GUS-positive/
hygromycin-sensitive hybrid plants yielded no PCR frag-
ment for hpt (example in Fig.4). However, the GUS-posi-
tive/hygromycin-resistant hybrid plants also yielded the
specific PCR fragment for hpt (example in Fig.4, lane 6),
suggesting the excision of hpt gene in these hybrid plants
was chimeric.
2.3 Excision of hpt in hybrid plants of lox-hpt-lox-sck-
cryIAc-plants×cre-plants
The lox-hpt-lox-sck-cryIAc-plants were also verified the
presence of sck and cryIAc genes by PCR and 22 out of 26
T0 plants yielded both expected 419-bp specific fragment
for sck and 744-bp specific fragment for cryIAc. These 22
plants and 2 T0 cre-plants, cre-1 and cre-3 (Table 1) were
planted in the greenhouse to produce selfed seeds. The
inheritance of transgenes was then tested by analyzing
segregation of hygromycin- or ppt-resistant phenotype in
selfed progeny and confirmed by PCR. Sixteen out of 22 T1
lines derived from T0 lox-hpt-lox-sck-cryIAc-plants and all
2 T1 cre-plant lines from cre-1 and cre-3 showed segrega-
tion of hygromycin-resistant : hygromycin-sensitive or ppt-
resistant : ppt-sensitive in a nearly 3 : 1 ration (data not
shown). Homozygous T2 transgenic lines from these T1
lines were obtained by selection of seedlings on
hygromycin- or ppt-containing medium and confirmed by
PCR and then used for crossing.
Nine crosses of lox-hpt-lox-sck-cryIAc-plant× cre-
plant with 77 hybrid plants in total were made. The hybrid
seedlings were assayed of hygromycin resistance by cul-
turing leaf tip on selective medium. Of 77 hybrid seedlings,
56 were hygromycin sensitive (Table 1). To corroborate the
excision of hpt gene, molecular analysis was performed on
hygromycin-sensitive hybrid plants. While expected 845-
bp fragment for hpt could be detected in parental lox-hpt-
lox-sck-cryIAc-plants, the hygromycin-sensitive hybrid
plants yielded no PCR fragment for hpt (data not shown).
Genomic DNA isolated from hybrid plants of cross lox-hpt-
lox-sck-cryIAc-8×cre-1 (Table 1) and their parental plants
Table 1 GUS expression and hygromycin sensitivity in the hybrid of lox-plant× cre-plant
Cross No. of tested F1 hybrid plants No. of GUS+ plants No. of hygS plants
Pactin-lox-hpt-lox-gusA-2× cre-1 3 1 1(1)
Pactin-lox-hpt-lox-gusA-3× cre-1 8 3 2
Pactin-lox-hpt-lox-gusA-7× cre-3 12 6 5
Pactin-lox-hpt-lox-gusA-10× cre-3 7 2 1
Total 30 12 9
lox-hpt-lox-sck-cryIAc-2× cre-3 18 18(2)
lox-hpt-lox-sck-cryIAc-8× cre-1 8 8
lox-hpt-lox-sck-cryIAc-11× cre-1 11 8
lox-hpt-lox-sck-cryIAc-11× cre-3 9 9
lox-hpt-lox-sck-cryIAc-12 × cre-1 2 2
lox-hpt-lox-sck-cryIAc-12 × cre-3 5 3
lox-hpt-lox-sck-cryIAc-20 × cre-3 12 6
lox-hpt-lox-sck-cryIAc-29 × cre-1 7 4
lox-hpt-lox-sck-cryIAc-29 × cre-3 5 4
Total 77 56
Pactin-lox-hpt-lox-gusA, plants transformed with vector pCALHGUS; cre, plants transformed with vector pCACreBar; lox-hpt-lox-sck-
cryIAc, plants transformed with vector pCUBACloxHpt; GUS+, GUS-positive; hygS, hygromycin-sensitive. (1), only GUS+ plants were tested;
(2), all F1 hybrid plants were tested.
Acta Botanica Sinica 植物学报 Vol.46 No.12 20041420
was subjected to Southern blot hybridization with sck,
cryIAc, hpt and cre probes respectively (example in Fig.5).
For hybridization with sck, cryIAc and hpt probes, genomic
DNA was digested with BamHⅠ and for hybridization
with cre probe, genomic DNA was digested with Hind Ⅲ.
The hygromycin-sensitive hybrid plants yielded sck, cryIAc
and cre but hpt hybridization signals (Fig.5, lanes 2-5),
while the parental lox-hpt-lox-sck-cryIAc-plant yielded sck,
cryIAc and hpt hybridization signals (Fig.5, lane 1) and the
parental cre-plant yielded cre hybridization signal (Fig.5,
Figs.2–3. GUS expression and hygromycin sensitivity assay of
hybrid plants of Pactin-lox-hpt-lox-gusA-plant× cre-plant. 2.
GUS expression in hybrid plant. left, non-transgenic control plant;
center, hybrid plant of Pactin-lox-hpt-lox-gusA-7×cre-3 (Table
1); right, parental Pactin-lox-hpt-lox-gusA-plant. 3. Hygromycin
sensitivity assay of rice seedling leaves. 1-2, non-transgenic con-
trol plants; 3-5, parental Pactin-lox-hpt-lox-gusA-plants; 6-9,
hybrid plants of Pactin-lox-hpt-lox-gusA-7×cre-3 (Table 1); 10-
12, parental cre-plants.
Fig.4. PCR analysis of hybrids of Pactin-lox-hpt-lox-gusA-
plant×cre-plant for the gusA, hpt and cre genes. a. Samples of
amplification fragment for gusA. b. Samples of amplification frag-
ment for hpt. c. Samples of amplification fragment for cre. M,
molecular marker; N, non-transgenic control plant; P, positive
control; 1, parental Pactin-lox-hpt-lox-gusA-plant; 2-7, hybrid
rice plants of Pactin-lox-hpt-lox-gusA-7×cre-3 (Table 1); 8, pa-
rental cre-plant.
Fig.5. Southern analysis of hybrids of lox-hpt-lox-sck-cryIAc-
plant× cre-plant. a-c. Genomic DNA was digested with the
restriction enzyme BamHⅠ and hybridizations were done with
sck, cryIAc or hpt probe respectively. d. Genomic DNA was
digested with the restriction enzyme HindⅢ and hybridizations
were done with cre probe. P, the 0.56-kp sck-Tnos fragment, 2.1-
kp cryIAc-Tnos fragment and 1.05-kp hpt fragment digested by
BamHⅠ from plasmid pCUBACloxHpt (Fig.1c) and the 1.0-kb
cre fragment digested by HindⅢ from plasmid pCACreBar (Fig.
1a) were analyzed as positive control for a-c and d respectively;
N, non-transgenic control plant; 1, parental lox-hpt-lox-sck-cryIAc-
plant; 2-5 hybrid rice plants of lox-hpt-lox-sck-cryIAc-8×cre-1
(Table 1); 6, parental cre-plant.
CHEN Song-Biao et al.: Cre/lox-mediated Marker Gene Excision in Elite Indica Rice Plants Transformed with Genes Conferring
Resistance to Lepidopteran Insects 1421
lane 6).
3 Discussion
The Cre/lox site-specific recombination system from E.
coli phage P1 has been well characterized. Cre is a 38.5-kD
protein that recognizes and interacts with two 34-bp lox
recognition sequences resulting in excision, integration or
inversion of the intervening DNA sequences depending
on the orientation of lox sites. The Cre/lox system has
been used successfully to manipulate transgenes or chro-
mosomes in the nuclear genomes of a wide variety of or-
ganisms including animals (Gorman and Bullock, 2000) and
plants (Ow, 2002). In plants, the Cre/lox-mediated manipu-
lation included chromosome recombination, translocation
or deletion (Koshinsky et al., 2000; Vergunst et al., 2000),
site-specific integration of the transgenes (Albert et al.,
1995; Vergunst and Hooykaas, 1998; Vergunst et al., 1998;
Day et al., 2000), reducing the copy number of DNA inserts
in the host genome (Srivastava et al., 1999) and deletion of
selectable marker genes from the host genomes (Dale and
Ow, 1991; Russell et al., 1992; Gleave et al., 1999; Zuo et
al., 2001; Endo et al., 2002; Zhang et al., 2003).
In the present study, we demonstrated the Cre/lox-me-
diated excision of selectable marker hpt gene in the rice
plants using a recombination-reporter gene system. The
excision of lox-hpt-lox led to the fusion of the gusA gene to
the rice actin1 promoter and consequently activated the
expression of gusA gene. The crossing strategy was used
to introduce the cre gene into the lox plants and four
crosses were made from T0 Pactin-lox-hpt-lox-gusA-plant
with T0 cre-plant. The recombination was found in all four
crosses although the efficiency of the recombination
(expressed as the percentage of GUS expression plants)
varied among individual crosses. Leaf assay on
hygromycin-containing medium and PCR amplification con-
firmed the excision of hpt from the hybrid plants genome.
Chimeric excision of transgenes in host genomes medi-
ated by site-specific recombination system was reported to
be a common phenomenon in many experiments (Dale and
Ow, 1991; Russell et al., 1992; Onouchi et al., 1995; Bar et
al., 1996; Hoa et al., 2002). In this study, we also found that
in a few cases of the tested hybrid plants, the GUS expres-
sion phenotype was inconsistent with the hygromycin-re-
sistant phenotype (Table 1) and these plants also yielded
the expected 845-bp fragment when PCR analysis was car-
ried out using the specific primers for hpt gene. These re-
sults suggested that the recombination in these plants was
induced at early germinal stage, or is random during the
somatic development stage (Hoa et al., 2002) and the
excision of hpt was incomplete. To improve the recombina-
tion efficiency, it might need to select transformants with
high expression of Cre to produce hybrid plants (Bayley et
al., 1992).
Site-specific recombination systems have been used to
successfully excise marker genes in plants, including eco-
nomically important crops rice and maize (Endo et al., 2002;
Hoa et al., 2002; Zhang et al., 2003). However, most of
these experiments were carried out based on reporter genes
such as gusA or gfp to detect the gene excision. Here we
demonstrated the utility of Cre/lox in excision of selectable
marker gene in commercial rice plants transformed with in-
secticidal genes. Instead of using reporter genes to detect
the recombination, we monitored the hpt excision by a
simple leaf assay method. In 77 hybrid plants made from T2
homozygous plants carrying sck and cryIAc with T2 ho-
mozygous plants carrying cre, 56 were found with
hygromycin-sensitive phenotype and confirmed of the ex-
cision of hpt by molecular analysis. Therefore, after removal
of the cre and bar gene constructs in hybrid plants by
genetic segregation, plants might be obtained that con-
tained only the sck and cryIAc genes and used to develop
commercial insect-resistant lines. The absence of select-
able marker genes would contribute to the public accep-
tance of transgenic rice.
Acknowledgements: The authors wish to thank Dr. P J J
Hooykaas and Dr. A C Vergunst, Institute of Molecular
Plant Sciences, Leiden University, for kindly providing plas-
mid pUC19-cre.
References:
Albert H, Dale E C, Lee E, Ow D W. 1995. Site-specific integra-
tion of DNA into wild-type and mutant lox sites in the plant
genome. Plant J, 7: 649-659.
Araki H, Jearnpipatkul A, Tatsumi H, Sakurai T, Ushino K, Muta
T, Oshima Y. 1987. Molecular and functional organization of
yeast plasmid pSR1. J Mol Biol, 182: 191-203.
Austin S, Ziese M, Sternberg N. 1981. A novel role of site-spe-
cific recombination in maintenance of bacterial replicons. Cell,
25: 729-736.
Bar M, Leshem B, Gilboa N, Gidoni D. 1996. Visual characteriza-
tion of recombination at FRT-gusA loci in transgenic tobacco
mediated by constitutive expression of the native FLP
recombinase. Theor Appl Genet, 93: 407-413.
Bayley C, Morgan M, Dale E C, Ow D W. 1992. Exchange of
gene activity in transgenic plants catalyzed by the Cre-lox
site-specific recombination system. Plant Mol Biol, 18: 353-
281.
Broach J R, Guarascio V R, Jayaram M. 1982. Recombination
Acta Botanica Sinica 植物学报 Vol.46 No.12 20041422
within the yeast plasmid 2 micron circle is site specific. Cell,
29: 227-234.
Cheng X, Sardana R, Kaplan H, Altosaar I. 1998. Agrobacterium-
transformed rice plants expressing synthetic cryIA(b) and cryIA
(c) genes are highly toxic to striped stem borer and yellow
stem borer. Proc Natl Acad Sci USA, 95: 2767-2772.
Chu C-C . 1978. The N6 medium and its applications to anther
culture of cereal crops. Proceedings of Symposium in Plant
Tissue Culture. Peking: Science Press. 43-50.
Christensen A H, Quail P H. 1996. Ubiquitin promoter-based
vectors for high-level expression of selectable and/or screenable
marker genes in monocotyledonous plants. Transgenic Res, 5:
213-218.
Dale E C, Ow D W. 1991. Gene transfer with subsequent removal
of the selection gene from the host genome. Proc Natl Acad Sci
USA, 88: 10558-10562.
Day C D, Lee E, Kobayashi J, Holappa L D, Albert H, Ow D W.
2000. Transgene integration into the same chromosome loca-
tion can produce alleles that express at a predictable level, or
alleles that are differentially silenced. Genes Dev, 14: 2869-
2880.
Deng C-Y, Song G-S, Xu J-W , Zhu Z. 2003. Increasing accumu-
lation level of foreign protein in transgenic plant through protein
targeting. Acta Bot Sin , 45: 1084-1089.
Duan X, Li X, Xue Q, Abo-EI-Saad M, Xu D, Wu R. 1996.
Transgenic rice plants harboring an introduced potato pro-
teinase inhibitor Ⅱ gene are insect resistant. Nat Biotechnol,
14: 494-498.
Ebinuma H, Sugita K, Matsunaga E, Endo S, Yamada K, Komamine
A. 2001. Systems for the removal of a selection marker and
their combination with a positive marker. Plant Cell Rep, 20:
383-392.
Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H. 2002. Single-
step transformation for generating marker-free transgenic rice
using the ipt-type MAT vector system. Plant J, 30: 115-122.
Fujimoto H, Itoh K, Yamamoto M, Kyozuk J, Shimamoto K.
1993. Insect resistant rice generated by introduction of a modi-
fied d-endotoxin gene by Bacillus thuringiensis. Bio/Technology,
11: 1151-1155.
Gleave A P, Mitra D S, Mudge S R, Morris B A M. 1999. Select-
able marker-free transgenic plants without sexual crossing:
transient expression of cre recombinase and use of a condi-
tional lethal dominant gene. Plant Mol Biol, 40: 223-235.
Gorman C, Bullock C. 2000. Site-specific gene targeting for gene
expression in eukaryotes. Curr Opin Biotech, 11: 455-460.
Hiei Y, Shozo O, Komari T, Takashi K. 1994. Efficient transfor-
mation of rice (Oryza sativa L): mediated by Agrobacterium
and sequence analysis of the boundaries of the T-DNA. Plant
J, 6: 271-282.
Hoa T T C, Bong B B, Huq E, Hodge T K. 2002. Cre/lox site-
specific recombination controls the excision of a transgene
from the rice genome. Theor Appl Genet, 104: 518-525.
Jefferson R A, Kavanagh T A, Bevan M W. 1987. GUS fusions:
b-glucuronidase as a sensitive and versatile gene fusion marker
in higher plants. EMBO J, 6: 3901-3907.
Khanna H, Raina S. 2002. Elite indica transgenic rice plants ex-
pressing modified CryIAc endotoxin of Bacillus thuringiensis
show enhanced resistance to yellow stem borer (Scirpophaga
incertulas). Transgenic Res, 11: 411-423.
Koshinsky H A, Lee E, Ow D W. 2000. Cre/lox site-specific
recombination between Arabidopsis and tobacco chromosomes.
Plant J, 23: 715-722.
Maqbool S B, Husnain T, Riazuddin S, Masson L, Christou P.
1998. Effective control of yellow stem borer and rice leaf
folder in transgenic rice indica varieties Basmati 370 and m 7
using the novel d-endotoxin cry2A Bacillus thuringiensis gene.
Mol Breeding, 4: 501-507.
McElroy D, Zhang W, Cao J, Wu R. 1990. Isolation of an effi-
cient actin promoter for use in rice transformation. Plant Cell,
2: 163-171.
Mozo T, Hooykaas P J J. 1992. Design of a novel system for the
construction of vectors for Agrobacterium-mediated plant
transformation. Mol Gen Genet, 236: 1-7.
Murray H G, Thompson W P. 1988. Rapid isolation of high
molecular weight DNA. Nucleic Acids Res, 8: 4321-4325.
Nayak P, Basu D, Das S, Basu A, Ghosh D, Ramakrishnan N A,
Ghosh M, Sen S K. 1997. Transgenic elite indica rice plants
expressing CryIAc ¶-endotoxin of Bacillus thuringiensis are
resistant against yellow stem borer (Scirpophaga incertulas).
Proc Natl Acad Sci USA, 94: 2111-2116.
Onouchi H, Nishihama R, Kudo M, Machida Y, Machida C.
1995. Visualization of site-specific recombination catalyzed
by a recombinase from Zygosaccharomyces rouxii in
Arabidopsis thaliana. Mol Gen Genet, 247: 653-660.
Ow D W. 2002. Recombinase-directed plant transformation for
the post-genomic era. Plant Mol Biol, 48: 183-200.
Russell S H, Hoopes J L, Odell J T. 1992. Directed excision of a
transgene from the plant genome. Mol Gen Genet, 234: 49-
59.
Srivastava V, Anderson O D, Ow D W. 1999. Single-copy
transgenic wheat generated through the resolution of comolex
integration patterns. Proc Natl Acad Sci USA, 96: 11117-
11121.
Sugita K, Matsunaga E, Ebinuma H. 1999. Effective selection
system for generating marker-free transgenic plants indepen-
dent of sexual crossing. Plant Cell Rep, 18: 941-947.
Sugita K, Kasahara T, Matsunaga E, Ebinuma H. 2000. A trans-
formation vector for the production of marker-free transgenic
CHEN Song-Biao et al.: Cre/lox-mediated Marker Gene Excision in Elite Indica Rice Plants Transformed with Genes Conferring
Resistance to Lepidopteran Insects 1423
plants containing a single copy transgene at high frequency.
Plant J, 22: 461-469.
Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush G S, Datta
S K. 2000. Field performance of transgenic elite commercial
hybrid rice expressing Bacillus thuringiensis d-endotoxin. Nat
Biotechnol, 18: 1101-1104.
Vergunst A C, Hooykaas P J J. 1998. Cre/lox-mediated site-spe-
cific integration of Agrobacterium T-DNA in Arabidopsis
thaliana by transient expression of Cre. Plant Mol Biol, 38:
393-406.
Vergunst A C, Jansen L E T, Fransz P F, Hans de Jong J, Hooykaas
P J J. 2000. Cre/lox-mediated recombination in Arabidopsis:
evidence for transmission of a translocation of a translocation
and a deletion event. Chromosoma, 109: 287-297.
Vergunst A C, Jansen L E T, Hooykaas P J J. 1998. Site-specific
integration of Agrobacterium T-DNA in Arabidopsis thaliana
mediated by Cre recombinase. Nucleic Acids Res, 26: 2729-
2734.
Wang M B, Waterhouse P M. 1997. A rapid and simple method of
assaying plants transformated with hygromycin or PPT re-
sistance genes. Plant Mol Biol Rep, 15: 209-215. (Managing editor: ZHAO Li-Hui)
Xu D, Xue Q, Mcelroy D, Mawal Y, Hilder V A, Wu R. 1996.
Constitutive expression of a cowpea trypsin inhibitor gene,
CpTi, in transgenic rice plants confers resistance to two major
rice insect pests. Mol Breeding, 2: 167-173.
Yoder J I, Goldsbrough A P. 1994. Transformation system for
generating marker-free transgenic plants. Bio/Technology, 12:
263-267.
Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V,
Gilbertson L. 2003. Cre/lox-mediated marker gene excision in
transgenic maize (Zea mays L.) plants. Theor Appl Genet,
107: 1157-1168.
Zhou H-Y , Chen S-B, Li X-G , Xiao G-F , Wei X-L , Zhu Z (朱祯).
2003. Generating marker-free transgenic tobacco plants by
Agrobacterium-mediated transformation with double T-DNA
binary vector. Acta Bot Sin , 45: 1103-1108.
Zuo J R, Nui Q W, Geir M S, Chua N H. 2001. Chemical regulated,
site-specific DNA excision in transgenic plants. Nat Biotechnol,
19: 157-161.