Expression of Leucaena Leucocephala de Wit Chitinase in Transgenic Koshihikari Rice
The cDNA encoding the 326 amino acids of a Class I
basic chitinase gene from Leucaena leucocephala de Wit (KB3,
Genbank accession: AAM49597) was cloned under the control of
CaMV35S promoter in pCAMBIA 1300 and transferred to
Koshihikari. Calli of Koshihikari rice was transformed with
agrobacterium with this construct expressing the chitinase and β-
glucouronidase (GUS). The frequencies of calli 90 % has been
obtained from rice seedlings cultured on NB medium. The high
regeneration frequencies, 74% was obtained from calli cultured on
regeneration medium containing 4 mg/l BAP, and 7 g/l phytagel at
25°C. Various factors were studied in order to establish a procedure
for the transformation of Koshihikari Agrobacterium tumefaciens.
Supplementation of 50 mM acetosyringone to the medium during
coculivation was important to enhance the frequency to transient
transformation. The 4 week-old scutellum-derived calli were
excellent starting materials. Selection medium based on NB medium
supplement with 40 mg/l hygromycin and 400 mg/l cefotaxime were
an optimized medium for selection of transformed rice calli. The
percentage of transformation 70 was obtained. Recombinant calli and
regenerated rice plants were checked the expression of chitinase and
gus by PCR, northern blot gel, southern blot gel, and gus assay.
Chitinase and gus were expressed in all parts of recombinant rice.
The rice line expressing the KB3 chiitnase was more resistant to the
blast fungus Fusarium monoliforme than control line.
[1] T.Boller, "Ethylene and the regulation of antifungal hydrolases in
plants," In Surveys of plant Molecular and Cell Biology, vol. 5 edited by
Miflin, B.J., Oxford University Press, Oxford, UK, 1998, pp. 145-174.
[2] K. Broglie, I. Chet, M. Hollyday, R. Cressman, P. Biddle, S. Knowlton,
C. J. Mauvais, and R. Broglie, "Transgenic plants with enhanced
resistance to the fungal pathogen Rhizoctonia solani," Science, vol. 254,
pp. 1194-1197, 1991.
[3] Y. Nishizawa, Z. Nishio, K. Nakazono, M. Soma, E. Nakajima, M.
Ugaki, and T. Hibi, "Enhanced resistance to blast (Magnaporthe grisea)
in transgenic Japonica rice by constitutive expression of rice chitinase,"
Theor. Appl. Genet. Vol. 99, pp. 383-390, 1999.
[4] D. Bowles, "Defense-related proteins in higher plants," Ann. Rev.
Biochem., vol. 59, pp. 873-907, 1990.
[5] H. J. M. Linthorst, "Pathogenesis-related proteins of plants," Crit. Rev.
Plant Sci., vol. 10, pp. 123-150, 1991.
[6] K. E. Broglie, J. J. Gaynor, and R. M. Broglie, "Ethylene-regulated gene
expression: molecular cloning of the genes encoding an endochitinase
from Phaseolus vulgaris," Proc. Natl. Acad. Sci. USA., vol. 83, pp.
6820-6824, 1986.
[7] A. Watanabe, V. H. Nong, D. Zhang, M. Arahira, N. A. Yeboah, K.
Udaka, and C. Fukazawa, "Molecular cloning and ethylene-inducible
expression of Chib1 chitinase form Soybean (Glycine max (L.) Merr.),"
Biosci. Biotechnol. Biochem., vol. 63, pp. 251-256, 1999.
[8] C. Staehelin, M. Schultze, E. Kondorosi, R. B. Mellor, T. Boller, and A.
Kondorosi, "Structural modifications in Rhizobium meliloti Nod factors
influence their stability against hydrolysis by root chitinases," Plant J.,
vol. 5, pp. 319-330, 1994.
[9] R. Zhong, S. J. Kays, B. P. Schroeder, and Z-H. Ye, "Mutation of a
chitinase like gene causes ectopic deposition of lignin, aberrant cell
shapes, and over production of ethylene," Plant Cell, vol. 14, pp. 165-
179, 2002.
[10] A. J. De Jong, R. Heidstra, H. P. Spaink, M. V. Hartog, E. A. Meijer, T.
Hendriks, F. Lo Schiavo, M. Terzi, T. Bisseling, A. Van Kammen, and
S. C. De Vries, "Rhizobium lipooligosaccharides rescue a carrot somatic
embryo mutant," Plant Cell, vol. 5, pp. 615-620, 1993.
[11] B. Henrissat, P. M. Coutinho, G. J. Davies, "A census of carbohydrateactive
enzymes in the genome of Arabidopsis thaliana," Plant Mol. Biol.,
vol. 47, pp. 55-72, 2001.
[12] M. Kaomek, K. Mizuno, T. Fujimura, P. Sriyotha, J. R. Ketudat-Cairns,
"Cloning, Expression and Characterization of an Anti-Fungal Chitinase
from Leucaena leucocephala de Wit," Biosci. Biotech. Biochem. vol. 67
no. 4, pp. 667-676, 2003.
[13] J. G. Verburg, and Q. K. Huynh, "Purification and Charaterization of an
Antifungal Chitinase from Arabidopsis thaliana," Plant Physiol., vol. 95,
pp. 450-455, 1991.
[14] A. Watanabe, V. H. Nong, D. Zhang, M. Arahira, N. A. Yeboah, K.
Udaka, and C. Fukazawa, "Molecular clongin and ethylene-inducible
expression of Chib1 chitinase form Soybean (Glycine max (L.) Merr.),"
Biosci. Biotechnol. Biochem., vol. 63, 251-256, 1999.
[15] D. B. Collinge, K. M. Kragh, I. D. Mikkelsen, K. K. Nieler, U.
Rasmussen, and K. Vad, "Plant chitinases," Plant J., vol. 3, 31-40, 1993.
[16] Y. Hiei, T. Komari, and T. Kumashiro, "Efficient transformation of rice
(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of
the boundaries od the T-DNA," Plant J., vol. 6, 271-282, 1994.
[17] K. A. Budelier, A. G. Smith, and C. S. Gasser, "Regulation of astylar
transmitting tissue-specific gene in wild-type and transgenic tomato and
tobacco," Mol. Gen. Genet., Vol. 224, 183-192, 1990.
[1] T.Boller, "Ethylene and the regulation of antifungal hydrolases in
plants," In Surveys of plant Molecular and Cell Biology, vol. 5 edited by
Miflin, B.J., Oxford University Press, Oxford, UK, 1998, pp. 145-174.
[2] K. Broglie, I. Chet, M. Hollyday, R. Cressman, P. Biddle, S. Knowlton,
C. J. Mauvais, and R. Broglie, "Transgenic plants with enhanced
resistance to the fungal pathogen Rhizoctonia solani," Science, vol. 254,
pp. 1194-1197, 1991.
[3] Y. Nishizawa, Z. Nishio, K. Nakazono, M. Soma, E. Nakajima, M.
Ugaki, and T. Hibi, "Enhanced resistance to blast (Magnaporthe grisea)
in transgenic Japonica rice by constitutive expression of rice chitinase,"
Theor. Appl. Genet. Vol. 99, pp. 383-390, 1999.
[4] D. Bowles, "Defense-related proteins in higher plants," Ann. Rev.
Biochem., vol. 59, pp. 873-907, 1990.
[5] H. J. M. Linthorst, "Pathogenesis-related proteins of plants," Crit. Rev.
Plant Sci., vol. 10, pp. 123-150, 1991.
[6] K. E. Broglie, J. J. Gaynor, and R. M. Broglie, "Ethylene-regulated gene
expression: molecular cloning of the genes encoding an endochitinase
from Phaseolus vulgaris," Proc. Natl. Acad. Sci. USA., vol. 83, pp.
6820-6824, 1986.
[7] A. Watanabe, V. H. Nong, D. Zhang, M. Arahira, N. A. Yeboah, K.
Udaka, and C. Fukazawa, "Molecular cloning and ethylene-inducible
expression of Chib1 chitinase form Soybean (Glycine max (L.) Merr.),"
Biosci. Biotechnol. Biochem., vol. 63, pp. 251-256, 1999.
[8] C. Staehelin, M. Schultze, E. Kondorosi, R. B. Mellor, T. Boller, and A.
Kondorosi, "Structural modifications in Rhizobium meliloti Nod factors
influence their stability against hydrolysis by root chitinases," Plant J.,
vol. 5, pp. 319-330, 1994.
[9] R. Zhong, S. J. Kays, B. P. Schroeder, and Z-H. Ye, "Mutation of a
chitinase like gene causes ectopic deposition of lignin, aberrant cell
shapes, and over production of ethylene," Plant Cell, vol. 14, pp. 165-
179, 2002.
[10] A. J. De Jong, R. Heidstra, H. P. Spaink, M. V. Hartog, E. A. Meijer, T.
Hendriks, F. Lo Schiavo, M. Terzi, T. Bisseling, A. Van Kammen, and
S. C. De Vries, "Rhizobium lipooligosaccharides rescue a carrot somatic
embryo mutant," Plant Cell, vol. 5, pp. 615-620, 1993.
[11] B. Henrissat, P. M. Coutinho, G. J. Davies, "A census of carbohydrateactive
enzymes in the genome of Arabidopsis thaliana," Plant Mol. Biol.,
vol. 47, pp. 55-72, 2001.
[12] M. Kaomek, K. Mizuno, T. Fujimura, P. Sriyotha, J. R. Ketudat-Cairns,
"Cloning, Expression and Characterization of an Anti-Fungal Chitinase
from Leucaena leucocephala de Wit," Biosci. Biotech. Biochem. vol. 67
no. 4, pp. 667-676, 2003.
[13] J. G. Verburg, and Q. K. Huynh, "Purification and Charaterization of an
Antifungal Chitinase from Arabidopsis thaliana," Plant Physiol., vol. 95,
pp. 450-455, 1991.
[14] A. Watanabe, V. H. Nong, D. Zhang, M. Arahira, N. A. Yeboah, K.
Udaka, and C. Fukazawa, "Molecular clongin and ethylene-inducible
expression of Chib1 chitinase form Soybean (Glycine max (L.) Merr.),"
Biosci. Biotechnol. Biochem., vol. 63, 251-256, 1999.
[15] D. B. Collinge, K. M. Kragh, I. D. Mikkelsen, K. K. Nieler, U.
Rasmussen, and K. Vad, "Plant chitinases," Plant J., vol. 3, 31-40, 1993.
[16] Y. Hiei, T. Komari, and T. Kumashiro, "Efficient transformation of rice
(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of
the boundaries od the T-DNA," Plant J., vol. 6, 271-282, 1994.
[17] K. A. Budelier, A. G. Smith, and C. S. Gasser, "Regulation of astylar
transmitting tissue-specific gene in wild-type and transgenic tomato and
tobacco," Mol. Gen. Genet., Vol. 224, 183-192, 1990.
@article{"International Journal of Biological, Life and Agricultural Sciences:54856", author = "M. Kaomek and J. R. Ketudat-Cairns", title = "Expression of Leucaena Leucocephala de Wit Chitinase in Transgenic Koshihikari Rice", abstract = "The cDNA encoding the 326 amino acids of a Class I
basic chitinase gene from Leucaena leucocephala de Wit (KB3,
Genbank accession: AAM49597) was cloned under the control of
CaMV35S promoter in pCAMBIA 1300 and transferred to
Koshihikari. Calli of Koshihikari rice was transformed with
agrobacterium with this construct expressing the chitinase and β-
glucouronidase (GUS). The frequencies of calli 90 % has been
obtained from rice seedlings cultured on NB medium. The high
regeneration frequencies, 74% was obtained from calli cultured on
regeneration medium containing 4 mg/l BAP, and 7 g/l phytagel at
25°C. Various factors were studied in order to establish a procedure
for the transformation of Koshihikari Agrobacterium tumefaciens.
Supplementation of 50 mM acetosyringone to the medium during
coculivation was important to enhance the frequency to transient
transformation. The 4 week-old scutellum-derived calli were
excellent starting materials. Selection medium based on NB medium
supplement with 40 mg/l hygromycin and 400 mg/l cefotaxime were
an optimized medium for selection of transformed rice calli. The
percentage of transformation 70 was obtained. Recombinant calli and
regenerated rice plants were checked the expression of chitinase and
gus by PCR, northern blot gel, southern blot gel, and gus assay.
Chitinase and gus were expressed in all parts of recombinant rice.
The rice line expressing the KB3 chiitnase was more resistant to the
blast fungus Fusarium monoliforme than control line.", keywords = "chitinase, Leucaena leucocephala de Wit,Koshihikari, transgenic rice.", volume = "3", number = "11", pages = "546-6", }