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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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Southern blot analysis of MAN5AS in three transgenic plants of event 22 after digestion with HindIII and BamHI.Lane 1, the DIG-labeled molecular weight markers; lane 2–4, MAN5AS with BamHI digestion; lane 5 and 6, non-transgenic Zheng58 digested by BamHI and HindIII, respectively; lane 7–9, MAN5AS with HindIII digestion; lane 10, the digested expression cassettes as a positive control.
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pone-0056146-g003: Southern blot analysis of MAN5AS in three transgenic plants of event 22 after digestion with HindIII and BamHI.Lane 1, the DIG-labeled molecular weight markers; lane 2–4, MAN5AS with BamHI digestion; lane 5 and 6, non-transgenic Zheng58 digested by BamHI and HindIII, respectively; lane 7–9, MAN5AS with HindIII digestion; lane 10, the digested expression cassettes as a positive control.

Mentions: To confirm the gene integration and the copy number of man5As in transgenic plants, the genomic DNAs of three positive transgenic plants of event 22 were analyzed by Southern blot after restriction digest with HindIII and BamHI. A band of ∼1.4 kb was detected in the positive lane, but not in non-transgenic Zheng58. HindIII cut the chimeric man5As twice that relieved an internal fragment of 2.4 kb from the gene expression cassettes (Figure 3). BamHI and XmaI were the ligation sites of transgene man5As and vector pHP20754. After BamHI digest, only one band migrated (Figure 3), indicating that there is only one copy of man5As in event 22.


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)

Southern blot analysis of MAN5AS in three transgenic plants of event 22 after digestion with HindIII and BamHI.Lane 1, the DIG-labeled molecular weight markers; lane 2–4, MAN5AS with BamHI digestion; lane 5 and 6, non-transgenic Zheng58 digested by BamHI and HindIII, respectively; lane 7–9, MAN5AS with HindIII digestion; lane 10, the digested expression cassettes as a positive control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056146-g003: Southern blot analysis of MAN5AS in three transgenic plants of event 22 after digestion with HindIII and BamHI.Lane 1, the DIG-labeled molecular weight markers; lane 2–4, MAN5AS with BamHI digestion; lane 5 and 6, non-transgenic Zheng58 digested by BamHI and HindIII, respectively; lane 7–9, MAN5AS with HindIII digestion; lane 10, the digested expression cassettes as a positive control.
Mentions: To confirm the gene integration and the copy number of man5As in transgenic plants, the genomic DNAs of three positive transgenic plants of event 22 were analyzed by Southern blot after restriction digest with HindIII and BamHI. A band of ∼1.4 kb was detected in the positive lane, but not in non-transgenic Zheng58. HindIII cut the chimeric man5As twice that relieved an internal fragment of 2.4 kb from the gene expression cassettes (Figure 3). BamHI and XmaI were the ligation sites of transgene man5As and vector pHP20754. After BamHI digest, only one band migrated (Figure 3), indicating that there is only one copy of man5As in event 22.

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