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Overexpression of a fungal β-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization.

Xu X, Zhang Y, Meng Q, Meng K, Zhang W, Zhou X, Luo H, Chen R, Yang P, Yao B - PLoS ONE (2013)

Bottom Line: The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds.Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.

ABSTRACT

Background: Mannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.

Methodology/principal findings: The man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.

Conclusion/significance: This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

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Related in: MedlinePlus

Construction of the recombinant vector and regeneration of transgenic maize.A) The recombinant expression vector pHP20754-man5As. B) The chimeric gene cassettes for expression in maize. C) PCR analysis of five putative calli. Lane 1, the DNA molecular weight markers; lane 2, the expression vector pHP20754-man5As (positive control); lane 3–7, the calli of transgenic maize Hi-II; lane 8, the calli of non-transgenic maize Hi-II (negative control). D) Embryogenic calli in selective medium. E) Plantlets in rooting medium. F) Regenerated maize plants in the greenhouse. G) Transgenic maize in fields. H) Ears of generation T1 of transgenic plant and non-transgenic maize Zheng58. I) Seeds of generation T1 of transgenic plant and non-transgenic maize Zheng58.
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pone-0056146-g001: Construction of the recombinant vector and regeneration of transgenic maize.A) The recombinant expression vector pHP20754-man5As. B) The chimeric gene cassettes for expression in maize. C) PCR analysis of five putative calli. Lane 1, the DNA molecular weight markers; lane 2, the expression vector pHP20754-man5As (positive control); lane 3–7, the calli of transgenic maize Hi-II; lane 8, the calli of non-transgenic maize Hi-II (negative control). D) Embryogenic calli in selective medium. E) Plantlets in rooting medium. F) Regenerated maize plants in the greenhouse. G) Transgenic maize in fields. H) Ears of generation T1 of transgenic plant and non-transgenic maize Zheng58. I) Seeds of generation T1 of transgenic plant and non-transgenic maize Zheng58.

Mentions: The vector pHP20754 consists of the ZM-leg1A promoter, the ZM-leg1 terminator, the maize proaleurain signal peptide (SP) and the vaculoe targeting sequence (VTS) (Figure 1A). The ZM-leg1A promoter is endosperm specific. A pair of specific primer (1417man-F and 1417man-R contaning the BamHI and XmaI sites, respectively; Table 1) was used to amplify the mutant gene man5As from pUC57MCS. The PCR conditions were as follows: 5 min at 95°C, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s. The PCR products were purified with a DNA purfication kit (TaKaRa, Osaka, Japan) and were ligated to the vector pEASY-T3 (TransGen, Beijing, China) for sequencing. Both the vector pHP20754 and man5As were digested with BamHI and XmaI, and ligated together with T4 DNA ligase to construct the chimeric gene cassettes for expression (Figure 1A). The recombinant vector pHP20754-man5As was then digested with PvuII for transformation. All the restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA).


Overexpression of a fungal β-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization.

Xu X, Zhang Y, Meng Q, Meng K, Zhang W, Zhou X, Luo H, Chen R, Yang P, Yao B - PLoS ONE (2013)

Construction of the recombinant vector and regeneration of transgenic maize.A) The recombinant expression vector pHP20754-man5As. B) The chimeric gene cassettes for expression in maize. C) PCR analysis of five putative calli. Lane 1, the DNA molecular weight markers; lane 2, the expression vector pHP20754-man5As (positive control); lane 3–7, the calli of transgenic maize Hi-II; lane 8, the calli of non-transgenic maize Hi-II (negative control). D) Embryogenic calli in selective medium. E) Plantlets in rooting medium. F) Regenerated maize plants in the greenhouse. G) Transgenic maize in fields. H) Ears of generation T1 of transgenic plant and non-transgenic maize Zheng58. I) Seeds of generation T1 of transgenic plant and non-transgenic maize Zheng58.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569411&req=5

pone-0056146-g001: Construction of the recombinant vector and regeneration of transgenic maize.A) The recombinant expression vector pHP20754-man5As. B) The chimeric gene cassettes for expression in maize. C) PCR analysis of five putative calli. Lane 1, the DNA molecular weight markers; lane 2, the expression vector pHP20754-man5As (positive control); lane 3–7, the calli of transgenic maize Hi-II; lane 8, the calli of non-transgenic maize Hi-II (negative control). D) Embryogenic calli in selective medium. E) Plantlets in rooting medium. F) Regenerated maize plants in the greenhouse. G) Transgenic maize in fields. H) Ears of generation T1 of transgenic plant and non-transgenic maize Zheng58. I) Seeds of generation T1 of transgenic plant and non-transgenic maize Zheng58.
Mentions: The vector pHP20754 consists of the ZM-leg1A promoter, the ZM-leg1 terminator, the maize proaleurain signal peptide (SP) and the vaculoe targeting sequence (VTS) (Figure 1A). The ZM-leg1A promoter is endosperm specific. A pair of specific primer (1417man-F and 1417man-R contaning the BamHI and XmaI sites, respectively; Table 1) was used to amplify the mutant gene man5As from pUC57MCS. The PCR conditions were as follows: 5 min at 95°C, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s. The PCR products were purified with a DNA purfication kit (TaKaRa, Osaka, Japan) and were ligated to the vector pEASY-T3 (TransGen, Beijing, China) for sequencing. Both the vector pHP20754 and man5As were digested with BamHI and XmaI, and ligated together with T4 DNA ligase to construct the chimeric gene cassettes for expression (Figure 1A). The recombinant vector pHP20754-man5As was then digested with PvuII for transformation. All the restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA).

Bottom Line: The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds.Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.

ABSTRACT

Background: Mannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.

Methodology/principal findings: The man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.

Conclusion/significance: This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

Show MeSH
Related in: MedlinePlus