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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.

Show MeSH
PCR analysis of the genomic DNA from leaves of generation BC1 of transgenic and non-transgenic plants.A) PCR detection of the gene man5As. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the vector pHP20754-man5As; lane 12, the non-transgenic Zheng58. B) PCR detection of the gene actin. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the non-transgenic Zheng58.
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pone-0056146-g002: PCR analysis of the genomic DNA from leaves of generation BC1 of transgenic and non-transgenic plants.A) PCR detection of the gene man5As. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the vector pHP20754-man5As; lane 12, the non-transgenic Zheng58. B) PCR detection of the gene actin. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the non-transgenic Zheng58.

Mentions: PCR assay with primers specific for man5As was used to evaluate the inheritance of transgenic maizes from generation T1 to BC3. PCR results of actin gene (∼300 bp) indicated the high quality of genomic DNA (Figure 2B). Gene fragments of about 450 bp were detected in the transformation events 29 and 22 (Figure 2A). The positive rates of all generations based on PCR results (Table 3) showed a rising trend, suggesting the stability for future generations.


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)

PCR analysis of the genomic DNA from leaves of generation BC1 of transgenic and non-transgenic plants.A) PCR detection of the gene man5As. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the vector pHP20754-man5As; lane 12, the non-transgenic Zheng58. B) PCR detection of the gene actin. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the non-transgenic Zheng58.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056146-g002: PCR analysis of the genomic DNA from leaves of generation BC1 of transgenic and non-transgenic plants.A) PCR detection of the gene man5As. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the vector pHP20754-man5As; lane 12, the non-transgenic Zheng58. B) PCR detection of the gene actin. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the non-transgenic Zheng58.
Mentions: PCR assay with primers specific for man5As was used to evaluate the inheritance of transgenic maizes from generation T1 to BC3. PCR results of actin gene (∼300 bp) indicated the high quality of genomic DNA (Figure 2B). Gene fragments of about 450 bp were detected in the transformation events 29 and 22 (Figure 2A). The positive rates of all generations based on PCR results (Table 3) showed a rising trend, suggesting the stability for future generations.

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