Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing

PLoS One. 2015 Jun 8;10(6):e0129294. doi: 10.1371/journal.pone.0129294. eCollection 2015.

Abstract

Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50 °C) and lower stability over alkaline pH range, but better thermal stability at 60 °C to 70 °C and resistance to feed pelleting inactivation (80 °C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animal Feed*
  • Blotting, Southern
  • Blotting, Western
  • Codon / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Gene Expression Regulation, Plant
  • Genetic Vectors / metabolism
  • Gibberella / enzymology*
  • Molecular Sequence Data
  • Peptide Hydrolases / metabolism*
  • Peptides / chemistry
  • Phenotype
  • Pichia / genetics
  • Plants, Genetically Modified
  • Polymerase Chain Reaction
  • Regeneration
  • Seeds / genetics*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Temperature
  • Transformation, Genetic
  • Zea mays / genetics*
  • alpha-Galactosidase / biosynthesis*

Substances

  • Codon
  • Peptides
  • alpha-Galactosidase
  • Peptide Hydrolases

Grants and funding

This work was supported by the Key Program of Transgenic Plant Breeding of China (2014ZX08003-002), and the National High-Tech Research and Development Program of China (2012AA022208), and the China Modern Agriculture Research System(CARS-42).