全 文 ：Received 24 Nov. 2003 Accepted 9 Mar. 2004
Supported by the State Key Basic Research and Development Plan of China (G1999011700) and the National Natural Science Foundation of
China (30270085 and 30025004).
* Author for correspondence. Tel: +86 (0)10 62753126; Fax: +86 (0)10 62751526; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 582-587
Isolation and Characterization of Ty1/Copia-like Reverse
Transcriptase Sequences from Mung Bean
XIAO Wei-Min1, SAKAMOTO Wataru2, Sodmergen1*
(1. College of Life Sciences, Peking University, Beijing 100871, China;
2. Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, 710-0046, Japan)
Abstract : Ty1/copia-like sequences were amplified from mung bean (Vigna radiata (L.) Wilczek) genomic
DNA, by PCR with degenerate oligonucleotide primers corresponding to highly conserved domains in the
Ty1/copia-like retrotransposons. PCR fragments of roughly 270 bp were isolated and cloned, and forty
clones were sequenced. Thirty-six of the forty clones had unique nucleotide sequences, and eighteen
clones had a frameshift, a stop codon, or both. Alignment of the nucleotide sequences indicated that these
clones, denoted Tvr, fell into nine subgroups and nine ungrouped sequences. The nucleotide sequence
similarity between these elements ranged from 8% to 100%, which indicates high level of sequence
heterogeneity among these clones. A phylogenetic analysis comparing these clones with corresponding
sequences from other plant species showed that some of the Tvr clones are more closely related to Ty1/
copia-like retrotransposons from other species than to other Tvr clones. Dot blot analysis revealed that
Ty1/copia-like retrotransposons comprise about 9.3% of the mung bean genome.
Key words: mung bean; DOP-PCR; copia; retrotransposon; reverse transcriptase
Transposable elements (TEs), first discovered in maize
by McClintock (1951), are fragments of DNA that can move
from one site in a genome to another. TEs are the single
largest component of the genetic material in most eukary-
otes (SanMiguel and Bennetzen, 1998; Vicient et al., 1999;
Meyers et a l., 2001). Their transposit ion is mediated by
either RNA or DNA, a trait that divides TEs into two major
classes (Finnegan, 1989; Flavell et al., 1994). The Class-1
transposon , or retrotranspos on, are further divided into
two families, the long terminal repeat (LTR)-containing and
non-LTR families , on the bas is o f their st ructural
characteris t ics . Ty1-cop ia and Ty3-gyps y are LTR
retrotrans posons with different gene orders and different
conserved sequences (Xiong and Eickbush, 1990; Santini
et al., 2002).
The best-understood group of LTR retrotransposons is
the Ty1-copia group. These sequences are ubiquitous in
the plant kingdom, appearing in species ranging from single-
celled algae to bryophytes, gymnosperms and angiosperms
(Flavell et al., 1992; Hirochika, 1993). A partial sequence
encoding the transcriptase can be amplified easily using a
degenerated oligonucleotide primer polymerase chain re-
act ion (DOP-PCR) with p rimers based on conserved se-
quences (Hirochika et al., 1992; Matsuoka and Tsunewaki,
1996; Pearce et al., 1996; Villordon et al., 2000; Price et al.,
2002). This technique allows the specific identification of
elements of the Ty1/copia group, excluding other families
of retrotransposons (Voytas et al., 1992). The copy number
of cop ia retrotransposons is highly variable between
species, ranging from about 310 cop ies per genome in
Arabidopsis thaliana (Navarro-Quezada and Schoen, 2002)
to over 25% of the genome of Vicia faba (Flavell et al.,
1994) and 19% of the genome of maize (Meyers et al., 2001).
Des p i t e th e u b iq u i to u s n e s s o f Ty1- co p ia
retrotransposons in plants, their transcription is mostly si-
lent during plant growth and development. To date, most
activation of Ty1-copia retrotransposons in plants have
been reported to occur in plants or cells that have been
exposed to strong stress cond itions, such as cell and tis-
sue cultu res (Hirochika, 1993; Hirochika et a l., 1996),
wounding, and pathogen infections (Kimura et al., 2001).
Due to their abundance in plant genomes, their stability
during transposition and their sensitivity to stress, copia-
like retrotransposons are thought to play important roles in
plan t mutagenesis and genome evolu tion (Flavell et al.,
1994; White et al., 1994; Feschotte and Wessler, 2002).
Our group has previously reported that genetic varia-
tions appeared in the p rogeny of a mung bean scion fol-
lowing a distant g raft (Zhang et al., 2002). These graft-
induced genetic variations were fo rmerly presumed to be
XIAO Wei-Min et al.: Isolation and Characterization of Ty1/Copia-like Reverse Transcriptase Sequences from Mung Bean 583
caused by gene transmission from the s tock to the scion
(Pandey, 1976; Taller et al., 1998). However, s ubsequent
examination of the variations revealed sequence changes
in the genomic DNA instead of evidence of gene transmis-
sion (Zhang et al., 2002). We therefore suggested that the
genetic variat ions in the progeny of the mung bean scion
might be caused by stress-induced transposition. Further
studies in the area of mung bean retrotransposons neces-
sitated the isolation of such sequences, which to our knowl-
edge had not been reported previously. In the present study,
we isolated the RT (reverse transcriptase) sequences from
Ty1/copia-like retrotransposons in mung bean and investi-
gated the proportion of Ty1/copia-like retrotransposons in
the mung bean genome. The results are a necessary step in
the study of retrotranspositions in mung bean.
1 Materials and Methods
1.1 Plant DNA
Total DNA was extracted from young leaves of mung
bean (Vigna radiata (L.) Wilczek) cv. Xiaohuijiao (XHJ),
which was plan ted widely in Xinjiang with high genetic
stability, by using the CTAB method (Sant et al. 1999).
1.2 PCR amplification
The primers us ed fo r amplify ing the RT (revers e
transcriptase) sequence of the Ty1-copia retrotransposon
were 5 -GAGAATTCACNGCNTTYYTNCAYGG-3 and 5 -
GAGGATCCATRTCRTCNACRTANAR-3 . These se-
quences correspond to the highly conserved pept ide se-
quences TAFLHG and LYVDDM, which flanked internal
domain of RT. Genomic template DNA (25 ng) and 50 pmol
of each of two primers were dissolved to a final volume of
25 mL in 0.2 mmol/L of each of the four dNTPs and a unit of
Ex Taq Polymerase (Takara). The PCR cycling profile was:
35 cycles of 30 s/94 oC, 30 s/51 oC, 1 min/72 oC; and 1 cycle
of 7 min/72 oC; all performed using a GeneAmp PCR system
2400 (Perkin Elmer Norwalker CT., USA).
1.3 Cloning and sequencing
PCR products were extracted with chloroform and pre-
cipitated with ethano l. Following digestion with EcoRⅠ
and BamHⅠ, for which restriction sites had been incorpo-
rated into the primers, the amplification products were gel-
purified and inserted into pUC18 vector. Forty clones were
randomly selected for sequencing using an ABI 377 auto-
mated DNA sequencer (Applied Biosystems).
1.4 Sequence analysis
DNA sequences were translated using the Seqkit World
Wide Web (www)-bas ed Translate too l at the following
Universal Resource Locato r (URL) addres s: http://www.
pyc.pku.edu.cn/seqkit. Multiple sequence alignments were
performed using CLUSTAL W (version 1.82).
1.5 Dot blotting
Genomic DNA and a heterogeneous 0.27-kb PCR prod-
uct were serially diluted, denatured in 0.2 mol/L NaOH + 2
mol/L NaCl for 15 min, and transferred onto a nylon mem-
brane (Hybond-N+, Amersham) using a vacuum dot blotter.
For the probe, the heterogeneous 0.27-kb PCR product was
radio labeled to approximately 109 cpm/µL using random-
primed labeling (Takara, Japan). Hybridization was performed
in 6× SSC + 5× Denhardt’s solution at 65 °C for 16 h.
The filters were washed in 0.1× SSC at 65 °C and visual-
ized by autoradiography.
2 Results
2.1 RT sequences
PCR amplification with degenerate primers yielded a
single band of the expected size of approximately 270 bp.
The DNA in this band was recovered and cloned, and 40
random clones were selected for sequencing. The nucle-
otide sequence data have been deposited in the GenBank
nucleot ide databas e under the acces s ion numbers
AY233226-AY233265. Of the clones sequenced, 36 were
unique, and 18 con tained a frameshift , a s top codon, or
bo th . The divergence o f the pred icted amino acid s e-
quences among the individual clones ranged from 0 to 89%.
Based on sequence similarity, the clones were divided into
nine subgroups, Tvr 1-9, and n ine ungrouped individual
sequences , Tvr 10-18 (Table 1). For sequences to be as-
signed to a subgroup, >90% amino acid identity in pairwise
comparis ons was required. DNA sequences o f clones
within each family s howed a variat ion that ranged from
2.0% to 6.7% (Table 1). The mean variation due to replica-
tion errors during amplification was about 1.8% (Monica et
al., 1998), which means that the sequence divergence among
the clones of a given subgroup is not only due to amplifica-
tion errors, bu t also that it reflects evolu tionary changes
among these sequences. An alignment of amino acid se-
quences of one member of each of the nine subgroups and
the nine ungrouped sequences is presented in Fig.1. The
sequence similarity between the g roups varied in a large
range. Several examples, including Tvr 5 and 9 (89%), Tvr 5
and 3 (64%), and Tvr 2 and 15 (35%), are shown. For further
comparison, we identified from the GenBank database cor-
responding RT sequences from Ty1-copia-like elements of
twelve other plant species. Several Tvrs had a high level of
similarity with a corresponding sequence from another
species. For example, Tvr 11 and CAD59770 from Cicer
arietinum have a similarity of 87%, and Tvr 17 and T06281
from Lycopersicon esculentum have a similarity of 70%.


Acta Botanica Sinica 植物学报 Vol.46 No.5 2004586
transmission of genes between grafted plant parts, the ex-
act mechanism for triggering genetic variations is unknown.
The results of our examination pointed to sequence changes
in the genomic DNA as the source of variation rather than
gene transmission (Zhang et al., 2002). We therefore sug-
ges ted that the genetic variations in the progeny of the
mung bean s cion migh t be caus ed by s tress -induced
transposition.
It has been reported that copia-like retrotransposons
are activated in plants or cells that have been exposed to
strong stress conditions, such as cell and tissue cultures
(Peschke et al., 1987; Hirochika, 1993; Hirochika et al., 1996),
wounding, and pathogen infections (Kimura et al., 2001).
The results of our previous study, which showed sequence
changes in the genomic DNA, are cons istent with these
latest findings. Prior to that study, no information on mung
bean retrotransposons had been reported. In the present
study, we cloned the RT sequence of the Ty1/copia-like
retrotransposons in mung bean and investigated the pro-
portion of these trans posons in the mung bean genome.
The identified sequences have strong similarity to RT se-
quences reported previously in other species, and Ty1/
cop ia-like ret rotranspos ons compris e about 9.3% of the
mung bean genome. Furthermore, nearly half of the s e-
quences examined in this study had a frameshift mutation,
a stop codon, or both. This implies that a large majority of
the elements are transpositionally inactive. In addition,
alignment of the s equences revealed high levels of s e-
quence heterogeneity . The similarit ies in the predicted
amino acid sequences of the individual fragments vary from
11% to 100%. Such extreme heterogeneity is similar to that
seen in the RT genes of Ty1-copia group retrotransposons
from s everal other plant s pecies (Flavell et a l., 1992;
Matsuoka and Tsunewaki, 1999).
Isolation of full-length cop ia-like retrotransposon se-
quences and monitoring of transposition under s tress
conditions, such as distant grafting, is under way. The re-
sults of the present study constitute necessary background
information for further studies of the retrotransposons of
mung bean.
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