The Protein Journal

, Volume 24, Issue 2, pp 113–123

Molecular Cloning and Expression of an α-Amylase Inhibitor from Rye with Potential for Controlling Insect Pests

  • Simoni C. Dias
  • Octávio L. Franco
  • Cláudio P. Magalhães
  • Osmundo B. de Oliveira-Neto
  • Raú A. Laumann
  • Edson L. Z. Figueira
  • Francislete R. Melo
  • Maria F. Grossi-de-Sá
Article

Abstract

Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new α-amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of α-amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic α-amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.

Keywords

α-amylase inhibitor CM protein insect pest molecular cloning plant defense. 

Abbreviations

AgA

Anthonomus grandis α-amylase

AoA

Acanthoscelides obtectus α-amylase

BIII

α-amylase inhibitor from rye seeds

Bt

Bacillus thuringiensis

CM

proteins chloroform–methanol proteins

HSA

human salivar a-amylase

pBIII

partial nucleotide sequence of BIII

PPA

porcine pancreatic α-amylase

RBI

Ragi bifunctional inhibitor

RIPs

ribosome inactivating proteins

TMA

Tenebrio molitor α-amylase

ZSA

Zabrotes subfasciatus α-amylase

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Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Simoni C. Dias
    • 1
    • 2
    • 3
  • Octávio L. Franco
    • 1
    • 3
  • Cláudio P. Magalhães
    • 1
    • 4
  • Osmundo B. de Oliveira-Neto
    • 1
  • Raú A. Laumann
    • 1
    • 3
  • Edson L. Z. Figueira
    • 1
  • Francislete R. Melo
    • 1
    • 5
  • Maria F. Grossi-de-Sá
    • 1
    • 3
    • 6
  1. 1.EMBRAPA-Recursor Genéticos e BiotechnologiaBrasília-DFBrazil
  2. 2.Universidade de BrasíliaBrasilia-DFBrazil
  3. 3.Universidade Católica de BrasíliaBrasília-DFBrazil
  4. 4.Faculdade JKBrasília-DFBrazil
  5. 5.União Pioneira de Integraçao SocialBrasília-DFBrazil
  6. 6.Cenargen/ partial nucleotide sequence of BIII; PPA, porcine pancreatic EmbrapaS.A.I.N. Parque Estaçã o BiológicaBrasíliaBrazil

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