Expression of Leucaena Leucocephala de Wit Chitinase in Transgenic Koshihikari Rice

Authors: M. Kaomek, J. R. Ketudat-Cairns

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.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1061651

References:

[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.