Overexpression of an acidic endo-β-1,3-1,4-glucanase in transgenic maize seed for direct utilization in animal feed

Abstract

Background: Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.

Methodology/principal findings: Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0-8.0, which is in agreement with the normal environment of digestive tract.

Conclusion/significance: Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.

Figure 1. Construction of the recombinant vector and the transgenic maize seed.

A The recombinant expression vector pHP20754-bgl7Am. B The chimeric gene cassettes for expression in maize. C Ears and seeds of transgenic maize (T1 and BC3) compared with that of wild-type Zheng58.

Figure 2. PCR analysis and southern blot analysis of the transgenic maize.

A PCR detection of the gene bgl7Am in the genomic DNA of transgenic plant leaves of generation BC1. Lane 1, the plasmid PHP20754-bgl7Am as positive control; lane 2–9, the transgenic plants; lane 10, the non-transgenic Zheng58. B PCR detection of the gene actin. Lane 1, the plasmid PHP20754-bgl7Am; lane 2–9, the transgenic plants; lane 10, the non-transgenic Zheng58. C Southern blot analysis of bgl7Am in transgenic plants. The EcoRI and HindIII-digested genomic DNA was hybridized with the bgl7Am probe. Lane 1, the DIG-labeled molecular weight markers; lane 2–4, transgenic plants digested by EcoRI (arrowhead indicate the positive bands); lane 5, non-transgenic Zheng58 digested by EcoRI; lane 6, non-transgenic Zheng58 digested by HindIII; lane 7–9, transgenic plants digested by HindIII (arrowhead indicate the positive band); lane 10, PCR fragment of the bgl7Am as a positive control (arrowhead).

Figure 3. Western blot analysis of recombinant Bgl7AM from the transgenic maize.

A Western blot analysis of the Bgl7AM from transgenic maize seeds. Lane M, the protein molecular markers; Lane 1, 3, 5, the proteins isolated from transgenic maize seeds; lane 2, 4, 6, the proteins isolated from transgenic maize seeds and treated with Endo H; lane 7, the non-transgenic Zheng58 as a negative control; lane 8, the purified P. pastoris Bgl7A as a positive control. B Western blot analysis of the Bgl7AM from different tissues of the transgenic maize (leaf, stem, root and seeds). Lane M, the protein molecular markers; purified P. pastoris Bgl7A as a positive control; Z58 refers to non-transgenic Zheng58 (negative control).

Figure 4. Property comparison of recombinant endo-β-1,3-1,4-glucanase expressed in maize (BGL7AM) and P. pastoris (BGL7A).

A Effect of pH on β-glucanase activity of BGL7AM and BGL7A at 60°C. B pH stability of BGL7AM and BGL7A. After incubation at 37°C for 1 h in buffers ranging from pH 1.0 to 9.0, the β-glucanase activity was assayed in 100 mM citric acid-Na₂HPO₄ (pH 5.0) at 60°C. C Temperature-dependent activity profiles of BGL7AM and BGL7A in 100 mM citric acid-Na₂HPO₄ (pH 5.0). D Thermostability of BGL7AM and BGL7A pre-incubated at 70°C at pH 5.0. The aliquots were removed at different time points then measure residual β-glucanase activity at 60°C and pH 5.0. Error bars represent the standard deviation of triplicate measurements.