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Current IssueEditorial BoardOnline First ArticlesArchive

volume 38 issue 2 (april 2015) : 178-181,   Doi: 10.5958/0976-0571.2015.00079.X

Cloning and characterization of protease inhibitor genes from some legumes

M
Manoj Saini
P
P . Nandeesha
M
Mayank Kaashyap
P
Prasoonpal Gupta
M
Mukesh Mohan
S
Subhojit Datta*
1Biotechnology Unit, Indian Institute of Pulses Research, Kanpur 208 024, India.
Cite article:- Saini Manoj, Nandeesha . P, Kaashyap Mayank, Gupta Prasoonpal, Mohan Mukesh, Datta* Subhojit (2025). Cloning and characterization of protease inhibitor genes from some legumes. Legume Research. 38(2): 178-181. doi: 10.5958/0976-0571.2015.00079.X.

ABSTRACT

Protease inhibitors are natural defense proteins that inhibit the proteolytic activity of proteases. Legume protease inhibitors are diverse in their molecular mechanism and many of them have successfully been deployed in crop plants to confer resistance against insect pests. Nine combinations of degenerated primers,designed, based on “multiple aligned” gene sequences from NCBI Gene bank database were used to amplify the protease inhibitor genes in of ten different legume crops (two varieties each)viz.fieldpea,chickpea, lentil, lathyrus, pigeonpea , frenchbean,mungbean ,clusterbean , horsegramand cowpea .The primer combination BBF2/BBR2 amplified the expectedamplicon of 800 bp invarities ofmungbean,horsegramand cowpea. These amplicons obtained from each genotype were further ligated to the pJET1.2/blunt vector and transformed into XL1 blue strain of E.coli. Out of 41 clones sequenced, two clone sequences obtained from cowpea (DCS 6) showed homology with substilisin-like serine protease inhibitor of Desulfovibria vulgaris and serine/threonine protein kinase of Epichlocfastucae.These genes can be transformed into Agrobacterium based binary vectors and used for transformation of crops. Therefore these findings offer new genes for transgenic development against lepidopteron pests.

REFERENCES

  1. Abdelnoor R.V., Barros E.G. and Moreira M.A. (1995). Determination of genetic diversity within Brazilian soybean germplasm using random amplified polymorphic DNA techniques and comparative analysis with pedigree data. Braz J Genet 18: 265-273.
  2. Barrett A. J, Rawlings N. D and Woessner J.F. (1998). Hand book of Proteolytic Enzymes, Academic Press New York:.
  3. Christeller J.T. (2005). Evolutionary mechanisms acting on proteinase inhibitor variability.FEBS J 272: 5710-5722.
  4. Datta S. and Tiwari K.K. (2006).Cloning of Bowman Birk Type Protease inhibitor gene from pulse crops.Indian JAgriBiochem 19:33-35.
  5. Habib H. and Fazili K.M. (2007). Plant protease inhibitors: a defense strategy in plants. Biotech MolBiol Rev 2: 68-85.
  6. Hilder V.A., Gatehouse A.M.R., Sherman S.E., Barker R.F. and Boulter D. (1987). A novel mechanism of insect resistance engineered into tobacco. Nature 300: 160-163.
  7. Holton T.A, Christopher J.T., Clure L.M, Harker N and Henry R.J.( 2002). Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat. Mol Breed 9: 63-71.
  8. Johnson R., Narvaez J., An G. and Ryan C. (1989). Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manducasexta larvae. Proc Natl Acad Sci USA 86: 9871-9875.
  9. Kapur R., Tan-Wilson A.L., and Wilson K. (1989).Isolation and partial characterization of a subtilisin inhibitor from the mungbean (Vignaradiata). Pl Physiol 91: 106-112.
  10. Koiwa H., Bressan R.A. and Hasegawa P.M. (1997).Regulation of protease inhibitors and plant defense. Trends Pl Sci 2: 379-384.
  11. Krattiger A.F. (1997). Insect resistance in crops: a case study of Bacillus thuringiensis (Bt) and its transfer to developing countries. ISAAA Briefs 2: 42
  12. Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., Thompson J.D., Gibson T.J. and Higgins D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947-8
  13. NCBI (http:// www.ncbi.nim.nih.gov).
  14. Ribeiro A., Akkermans A.D.L., van Dammen A., Bisseling T. and Pawlowski K.(1995). A nodule-specific gene encoding a subtilisinlike protease is expressed in early stages of actinorhizal nodule development. Plant Cell 7: 785–794.
  15. Richardson M. (1977).The protease inhibitors of plants and micro-organism. Phytochem 16: 159-169.
  16. Sambrook J., Fritsch E.F. and Maniatis T. (1989). A Molecular Cloning: A laboratory Manual (Second Edition) Cold Spring Lab press. Cold Spring Harbor. New York.
  17. Valueva T.A. and Mosolov V.V. (2004).Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms. Biochem 69: 1305-1309.
  18. Vartak H.G., Rele M.V. and Jagannathan V. (1980).Proteinase inhibitors from Vignaunguiculata subsp. Cuylindrica. III. Properties and kinetics of inhibitors of papain, subtilisin and trypsin. Arch Biochem Biophy 204: 134-140.
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    volume 38 issue 2 (april 2015) : 178-181,   Doi: 10.5958/0976-0571.2015.00079.X

    Cloning and characterization of protease inhibitor genes from some legumes

    M
    Manoj Saini
    P
    P . Nandeesha
    M
    Mayank Kaashyap
    P
    Prasoonpal Gupta
    M
    Mukesh Mohan
    S
    Subhojit Datta*
    1Biotechnology Unit, Indian Institute of Pulses Research, Kanpur 208 024, India.
    Cite article:- Saini Manoj, Nandeesha . P, Kaashyap Mayank, Gupta Prasoonpal, Mohan Mukesh, Datta* Subhojit (2025). Cloning and characterization of protease inhibitor genes from some legumes. Legume Research. 38(2): 178-181. doi: 10.5958/0976-0571.2015.00079.X.

    ABSTRACT

    Protease inhibitors are natural defense proteins that inhibit the proteolytic activity of proteases. Legume protease inhibitors are diverse in their molecular mechanism and many of them have successfully been deployed in crop plants to confer resistance against insect pests. Nine combinations of degenerated primers,designed, based on “multiple aligned” gene sequences from NCBI Gene bank database were used to amplify the protease inhibitor genes in of ten different legume crops (two varieties each)viz.fieldpea,chickpea, lentil, lathyrus, pigeonpea , frenchbean,mungbean ,clusterbean , horsegramand cowpea .The primer combination BBF2/BBR2 amplified the expectedamplicon of 800 bp invarities ofmungbean,horsegramand cowpea. These amplicons obtained from each genotype were further ligated to the pJET1.2/blunt vector and transformed into XL1 blue strain of E.coli. Out of 41 clones sequenced, two clone sequences obtained from cowpea (DCS 6) showed homology with substilisin-like serine protease inhibitor of Desulfovibria vulgaris and serine/threonine protein kinase of Epichlocfastucae.These genes can be transformed into Agrobacterium based binary vectors and used for transformation of crops. Therefore these findings offer new genes for transgenic development against lepidopteron pests.

    REFERENCES

    1. Abdelnoor R.V., Barros E.G. and Moreira M.A. (1995). Determination of genetic diversity within Brazilian soybean germplasm using random amplified polymorphic DNA techniques and comparative analysis with pedigree data. Braz J Genet 18: 265-273.
    2. Barrett A. J, Rawlings N. D and Woessner J.F. (1998). Hand book of Proteolytic Enzymes, Academic Press New York:.
    3. Christeller J.T. (2005). Evolutionary mechanisms acting on proteinase inhibitor variability.FEBS J 272: 5710-5722.
    4. Datta S. and Tiwari K.K. (2006).Cloning of Bowman Birk Type Protease inhibitor gene from pulse crops.Indian JAgriBiochem 19:33-35.
    5. Habib H. and Fazili K.M. (2007). Plant protease inhibitors: a defense strategy in plants. Biotech MolBiol Rev 2: 68-85.
    6. Hilder V.A., Gatehouse A.M.R., Sherman S.E., Barker R.F. and Boulter D. (1987). A novel mechanism of insect resistance engineered into tobacco. Nature 300: 160-163.
    7. Holton T.A, Christopher J.T., Clure L.M, Harker N and Henry R.J.( 2002). Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat. Mol Breed 9: 63-71.
    8. Johnson R., Narvaez J., An G. and Ryan C. (1989). Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manducasexta larvae. Proc Natl Acad Sci USA 86: 9871-9875.
    9. Kapur R., Tan-Wilson A.L., and Wilson K. (1989).Isolation and partial characterization of a subtilisin inhibitor from the mungbean (Vignaradiata). Pl Physiol 91: 106-112.
    10. Koiwa H., Bressan R.A. and Hasegawa P.M. (1997).Regulation of protease inhibitors and plant defense. Trends Pl Sci 2: 379-384.
    11. Krattiger A.F. (1997). Insect resistance in crops: a case study of Bacillus thuringiensis (Bt) and its transfer to developing countries. ISAAA Briefs 2: 42
    12. Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., Thompson J.D., Gibson T.J. and Higgins D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947-8
    13. NCBI (http:// www.ncbi.nim.nih.gov).
    14. Ribeiro A., Akkermans A.D.L., van Dammen A., Bisseling T. and Pawlowski K.(1995). A nodule-specific gene encoding a subtilisinlike protease is expressed in early stages of actinorhizal nodule development. Plant Cell 7: 785–794.
    15. Richardson M. (1977).The protease inhibitors of plants and micro-organism. Phytochem 16: 159-169.
    16. Sambrook J., Fritsch E.F. and Maniatis T. (1989). A Molecular Cloning: A laboratory Manual (Second Edition) Cold Spring Lab press. Cold Spring Harbor. New York.
    17. Valueva T.A. and Mosolov V.V. (2004).Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms. Biochem 69: 1305-1309.
    18. Vartak H.G., Rele M.V. and Jagannathan V. (1980).Proteinase inhibitors from Vignaunguiculata subsp. Cuylindrica. III. Properties and kinetics of inhibitors of papain, subtilisin and trypsin. Arch Biochem Biophy 204: 134-140.
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