Isolation and characterization of a rice cysteine protease gene, OsCP1,
using T-DNA gene-trap system
, Ki-Hong Jung
, Gynheung An
and Yong-Yoon Chung
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea (*author for corre-
spondence; e-mail: email@example.com);
Division of Molecular and Life Sciences, Pohang University of
Science and Technology (POSTECH), Pohang 790-784, Korea
Received 10 October 2003; accepted in revised form 20 April 2004
Key words: anther, cell death, cysteine protease, rice, tapetum, T-DNA insertional mutagenesis
The T-DNA gene-trap system has been eﬃciently used to elucidate gene functions in plants. We report here
a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice.
GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice
anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to
those of papain family cyteine proteases containing the highly conserved interspersed amino acid motif,
ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identiﬁed a
suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a signiﬁcant defect in
pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that
might play an important role in pollen development.
Abbreviations: GUS, b-glucuronidase; ORF, open reading frame; PCR, polymerase chain reaction
T-DNA insertional mutagenesis has been success-
fully used as a powerful tool to identify a number
of genes in diﬀerent plant species (Azpiroz-Leehan
and Feldman 1997; Krysan et al., 1999; Nesi et al.,
2000; Takechi et al., 2000; Weigel et al., 2000;
Huang et al., 2001; Ishiguro et al., 2001; Jung
et al., 2003). In this system, vector used for the
mutagenesis carries a promoter-less reporter gene
such as the b-glucuronidase (GUS) gene located
immediately next to the right border of T-DNA.
When the T-DNA is integrated into the plant
genome in a random fashion, the inserted gene at a
proper orientation under a certain promoter is
stably expressed through multiple generations
reporting temporal and spatial activity of the
promoter during plant growth and development
(Jeon et al., 2000; Gothandam et al., 2003). This
integration often allows identiﬁcation of knockout
mutants from the T-DNA insertion pools (Azp-
iroz-Leehan and Feldman 1997, Jeon et al., 2000).
In this study, we report identiﬁcation of a
mutant which displays defects in plant growth and
development from a T-DNA insertion pool of rice.
Isolation and analysis of the T-DNA ﬂanking
sequence from the mutant revealed that the inter-
rupted gene could encode a cysteine protease.
Cysteine proteases belong to a family of enzymes
that play important role in intracellular protein
degradation in widely distributed systems; ani-
mals, plants, and microorganisms. Amino acids
from the degradation of proteins can be recycled
for new protein synthesis. Cysteine proteases (CPs)
Plant Molecular Biology 54: 755–765, 2004.
Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.