Introduction and constitutive expression of a tobacco hornworm (Manduca sexta) chitinase gene in soybean

  • W. Ornatowski
  • J. Jayaraj
  • T. C. Todd
  • W. T. Schapauch
  • S. Muthukrishnan
  • H. N. Trick


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 words

plant transformation Glycine max bombardment soybean cyst nematode sCN msc 


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  1. Arakane, Y.; Zhu, Q.; Matsumia, M.; Muthukrishnan, S.; Kramer, K. J. Properties of catalytic, linker and chitin binding domains of insect chitinase. Insect Biochem. Mol. Biol., 33:631–648; 2003.PubMedCrossRefGoogle Scholar
  2. Bernard, R. L.; Noel, G. R.; Anand, S. C.; Shannon, J. G. Registration of Fayette soybean. Crop Sci. 28:1028–1029; 1988.Google Scholar
  3. Brants, A.; Brown, C. R.; Earle, E. D. Trichoderma harzianum endochitinase does not provide resistance to Meloidogyne hapla in transgenic tobacco. J. Nematol. 32:289–296; 2000.PubMedGoogle Scholar
  4. Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159; 1987.PubMedCrossRefGoogle Scholar
  5. Church, G. M.; Gilbert, W. Genomic sequencing. Proc. Natl Acad. Sci. USA 81:1991–1995; 1984.PubMedCrossRefGoogle Scholar
  6. Dellaporta, S. L.; Wood, J.; Hicks, J. B. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1:19–21; 1983.Google Scholar
  7. Ding, X.; Gopalakrishnan, B.; Johnson, L. B.; White, F. F.; Wang, X.; Morgan, T. D.; Kramer, K. J.; Muthukrishnan, S. Insect resistance of transgenic tobacco expressing an insect chitinase gene. Transgen. Res. 7:77–84; 1998.CrossRefGoogle Scholar
  8. Edwards, K.; Johnstone, C.; Thompson, C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19:1349; 1991.PubMedCrossRefGoogle Scholar
  9. Finer, J. J. Apical proliferation of embryogenic tissue of soybean [Glycine max (L.) Merrill] Plant Cell Rep. 7:238–241; 1988.CrossRefGoogle Scholar
  10. Finer, J. J.; McMullen, M. D. Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep. 8:586–589; 1990.CrossRefGoogle Scholar
  11. 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
  12. Finer, J. J.; Vain, P.; Jones, M. W. McMullen, M. D. Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep. 11:323–328; 1992.CrossRefGoogle Scholar
  13. Jackson, S. A.; Zhang, P.; Chen, W. P.; Phillips, R. L.; Friebe, B.; Muthukrishnan, S.; Gill, B. S. High-resolution structural analysis of biolistic transgene integration into the genome of wheat. Theor. Appl. Genet. 103:56–62; 2001.CrossRefGoogle Scholar
  14. Jefferson, R. A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5:387–405; 1987.Google Scholar
  15. 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
  16. Kramer, K. J.; Corpuz, L. M.; Choi, H.; Muthukrishnan, S. Sequence of a CDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta. Insect Biochem. Mol. Biol. 23:691–701; 1993.PubMedCrossRefGoogle Scholar
  17. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–684; 1970.CrossRefGoogle Scholar
  18. Mercer, C. F.; Greenwood, D. R.; Grant, J. L. Effect of plant and microbial chitinases on the eggs and juveniles of Meloidogyne hapla Chitwood (Nematoda: Tylenchida). Nematologica 38:227–236; 1992.CrossRefGoogle Scholar
  19. 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
  20. Nickell, C. D.; Noel, G. R.; Thomas, D. J.; Waller, R. Registration of Jack soybean. Crop Sci. 30:1365; 1990.CrossRefGoogle Scholar
  21. Pawlowski, W. P.; Somers, D. A. Transgene inheritance in plants genetically engineered by microprojectile bombardment. Mol. Biotechnol. 6:17–30; 1996.PubMedGoogle Scholar
  22. Pawlowski, W. P.; Somers, D. A. Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc. Natl Acad. Sci. USA 95:12106–12110; 1998.PubMedCrossRefGoogle Scholar
  23. 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
  24. 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
  25. Rahimi, S.; Perry, R. N.; Wright, D. J. Identification of pathogenesis-related proteins induced in leaves of potato plants infected with potato cyst nematodes, Globodera species. Physiol. Mol. Plant Pathol. 49:49–59; 1996.CrossRefGoogle Scholar
  26. Roberts, C. A.; Marek, S. H.; Niblack, T. L.; Karr, A. L. Parasite Meloidogyne and mutualistic Acremonium increase chitinase in tall fescue. J. Chem. Ecol. 18:1107–1116; 1992.CrossRefGoogle Scholar
  27. Rohan, R. M.; King, D.; Frels, W. I. Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes. Nucleic Acids Res. 18:6089–6095; 1990.PubMedCrossRefGoogle Scholar
  28. Spencer, T. M.; Gordon-Kamm, W. J.; Daines, R. J.; Start, W. G.; Lemaux, P. G. Bialaphos selection of stable transformants from maize cell culture. Theor. Appl. Genet. 79:625–631; 1990.CrossRefGoogle Scholar
  29. 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
  30. Tian, H.; Riggs, R. D.; Crippen, D. L. control of soybean cyst nematode by chitinolytic bacteria with chitin substrate. J. Nematol. 32:370–376; 2000.PubMedGoogle Scholar
  31. 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
  32. Wang, X.; Ding, X.; Gopalakrishnan, B.; Morgan, T. D.; Johnson, L. B.; White, F. F.; Kramer, K. J. Characterization of a 46kDa insect chitinase from transgenic tobacco. Insect Biochem. Mol. Biol. 26:1055–1064; 1996.CrossRefGoogle Scholar
  33. 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
  34. Wright, M. S. Method of regenerating soybeans form cultured soybean cotyledonary nodes. US patent no. 4,992,375; 1991.Google Scholar

Copyright information

© Society for In Vitro Biology 2004

Authors and Affiliations

  • W. Ornatowski
    • 1
  • J. Jayaraj
    • 2
  • T. C. Todd
    • 3
  • W. T. Schapauch
    • 1
  • S. Muthukrishnan
    • 2
  • H. N. Trick
    • 3
  1. 1.Department of AgronomyKansas State UniversityManhattan
  2. 2.Department of BiochemistryKansas State UniversityManhattan
  3. 3.Department of Plant PathologyKansas State UniversityManhattan

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