Introduction and constitutive expression of a tobacco hornworm (Manduca sexta) chitinase gene in soybean
Embryogenic soybean [Glycine max (L.) Merrill] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration inheritance, and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes. Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of 48kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode.
Key wordsplant transformation Glycine max bombardment soybean cyst nematode sCN msc
Unable to display preview. Download preview PDF.
- Bernard, R. L.; Noel, G. R.; Anand, S. C.; Shannon, J. G. Registration of Fayette soybean. Crop Sci. 28:1028–1029; 1988.Google Scholar
- Dellaporta, S. L.; Wood, J.; Hicks, J. B. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1:19–21; 1983.Google Scholar
- Finer, J. J.; McMullen, M. D. Transformation of soybean via particle bombardment of embryogenic suspension culture tissue. In Vitro Cell. Dev. Biol. Plant 27:175–182; 1991.Google Scholar
- Jefferson, R. A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5:387–405; 1987.Google Scholar
- Koga, D.; Funakoshi, T.; Mizuki, K.; Kramer, K. J.; Zen, K. C.; Choi, H. K.; Muthukrishnan, S. Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of silkworm, Bombyx mori, and tobacco hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 22:305–311; 1992.CrossRefGoogle Scholar
- Niblack, T. L.; Heinz, R. D.; Smith, G. S.; Donald, P. A. Distribution, density, and diversity of Heterodea glycines in Missouri. Suppl. J. Nematol. 25:880–886; 1993.Google Scholar
- Perry, R. N.; Trett, M. W. Ultrastructure of the eggshell of Heterodera schachtii and H. glycines (Nematoda: Tylenchida). Revue Nématol. 9:399–403; 1986.Google Scholar
- Qiu, J.; Hallmann, J.; Kokalis-Burelle, N.; Weaver, D. B.; Rodriguez-Kabana, R.; Tuzun, S. Activity and differential induction of chitinase isozymes in soybean cultivars resistant or susceptible to root-knot nematodes. J. Nematol. 29:523–530; 1997.Google Scholar
- Stewart, C. N. Jr.; Adang M. G.; All, J. N.; Boerma, H. R.; Cardineau, G.; Tucker, D.; Parrot, W. A. Genetic transformation, recovery, and characterization of soybean (Glycine max [L.] Merrill) transgenic for a synthetic Bacillus thuringiensis CRY IA(c) gene. Plant Physiol. 112:121–129; 1996.PubMedCrossRefGoogle Scholar
- Trick, H. N.; Dinkins, R. D.; Santarém, E. R.; Di, R.; Samoylov, V.; Meurer, C. A.; Walker, D. R.; Parrott, W. R.; Finer, J. J.; Collins, G. B. Recent advances in soybean transformation. Plant Tiss. Cult. Biotechnol. 3:9–26; 1997.Google Scholar
- Wrather, J. A.; Anderson, T. R.; Arsyad, D. M.; Gai, J.; Ploper, L. D.; Porta-Puglia, A.; Ram, H.; Yorinori, J. T. Soybean disease loss estimates for the top 10 soybean producing countries in 1994. Plant Dis. 81:107–110; 1997.Google Scholar
- Wright, M. S. Method of regenerating soybeans form cultured soybean cotyledonary nodes. US patent no. 4,992,375; 1991.Google Scholar