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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

Analysis of recombinant MAN5AS from two transgenic maizes.A) SDS–PAGE. B) Western blot. Lane 1 and 3, the transgenic maize; lane 2, the protein molecular markers; lane 4, the non-transgenic Zheng58; lane 5, the purified MAN5A-SST produced in P. pastoris. C) Specific promoter analysis. Lane 1, the protein molecular markers; lane 2 and 4, the protein isolated from seeds of T042-5 and T041-20; lane 3 and 5, the two transgenic plant proteins pretreated with Endo H; lane 6, the protein isolated from seeds of non-transgenic Zheng58; lane 7, the purified MAN5A-SST produced in P. pastoris; lane 8–10, the proteins collected from root, stem and leaf tissue of a transgenic plant.
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pone-0056146-g004: Analysis of recombinant MAN5AS from two transgenic maizes.A) SDS–PAGE. B) Western blot. Lane 1 and 3, the transgenic maize; lane 2, the protein molecular markers; lane 4, the non-transgenic Zheng58; lane 5, the purified MAN5A-SST produced in P. pastoris. C) Specific promoter analysis. Lane 1, the protein molecular markers; lane 2 and 4, the protein isolated from seeds of T042-5 and T041-20; lane 3 and 5, the two transgenic plant proteins pretreated with Endo H; lane 6, the protein isolated from seeds of non-transgenic Zheng58; lane 7, the purified MAN5A-SST produced in P. pastoris; lane 8–10, the proteins collected from root, stem and leaf tissue of a transgenic plant.

Mentions: To determine the expression efficiency of exogenous MAN5AS, proteins were extracted from two BC2 plants (T042-5 and T041-20) of event 29 that had high β-mannanase activities (33,468 U/kg, 32,592 U/kg). Compared with the image on SDS–PAGE (Figure 4A), three main bands were identified on the PVDF membrane after hybridization with the untreated primary antibody (Figure 4B). Only two bands of approximately 40 kDa and 50 kDa were developed when the primary antibody was pre-hybridizated with the proteins extracted from P. pastoris harboring the empty vector or Zheng58 (Figure 3C). Both bands were verified to be MAN5AS through MALDI-TOF analysis (the protein scores C.I. % are 99.84614% and 92.39621% for MAN5AS and MAN5A-SST, respectively). With Endo H treatment, the ∼50 kDa band showed some reduction in molecular weight while the ∼40 kDa that was similar to the predicted molecular weight kept intact. No band was detected on negative control. The positive control, MAN5A-SST expressed by P. pastoris, showed a band of about 90 kDa, the same as that reported in [29]. Proteins extracted from the root, stem and leaf of the positive lines had no objective band (Figure 4C), indicating the tissue specificity of MAN5AS by using the endosperm specific ZM-leg1A promoter. This promoter made exogenous MAN5AS specifically expressed in the seeds of transgenic maize and could lessen the potential impairment to the plants. Moreover, MAN5AS present in seeds are more convenient for storage and transportation.


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)

Analysis of recombinant MAN5AS from two transgenic maizes.A) SDS–PAGE. B) Western blot. Lane 1 and 3, the transgenic maize; lane 2, the protein molecular markers; lane 4, the non-transgenic Zheng58; lane 5, the purified MAN5A-SST produced in P. pastoris. C) Specific promoter analysis. Lane 1, the protein molecular markers; lane 2 and 4, the protein isolated from seeds of T042-5 and T041-20; lane 3 and 5, the two transgenic plant proteins pretreated with Endo H; lane 6, the protein isolated from seeds of non-transgenic Zheng58; lane 7, the purified MAN5A-SST produced in P. pastoris; lane 8–10, the proteins collected from root, stem and leaf tissue of a transgenic plant.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056146-g004: Analysis of recombinant MAN5AS from two transgenic maizes.A) SDS–PAGE. B) Western blot. Lane 1 and 3, the transgenic maize; lane 2, the protein molecular markers; lane 4, the non-transgenic Zheng58; lane 5, the purified MAN5A-SST produced in P. pastoris. C) Specific promoter analysis. Lane 1, the protein molecular markers; lane 2 and 4, the protein isolated from seeds of T042-5 and T041-20; lane 3 and 5, the two transgenic plant proteins pretreated with Endo H; lane 6, the protein isolated from seeds of non-transgenic Zheng58; lane 7, the purified MAN5A-SST produced in P. pastoris; lane 8–10, the proteins collected from root, stem and leaf tissue of a transgenic plant.
Mentions: To determine the expression efficiency of exogenous MAN5AS, proteins were extracted from two BC2 plants (T042-5 and T041-20) of event 29 that had high β-mannanase activities (33,468 U/kg, 32,592 U/kg). Compared with the image on SDS–PAGE (Figure 4A), three main bands were identified on the PVDF membrane after hybridization with the untreated primary antibody (Figure 4B). Only two bands of approximately 40 kDa and 50 kDa were developed when the primary antibody was pre-hybridizated with the proteins extracted from P. pastoris harboring the empty vector or Zheng58 (Figure 3C). Both bands were verified to be MAN5AS through MALDI-TOF analysis (the protein scores C.I. % are 99.84614% and 92.39621% for MAN5AS and MAN5A-SST, respectively). With Endo H treatment, the ∼50 kDa band showed some reduction in molecular weight while the ∼40 kDa that was similar to the predicted molecular weight kept intact. No band was detected on negative control. The positive control, MAN5A-SST expressed by P. pastoris, showed a band of about 90 kDa, the same as that reported in [29]. Proteins extracted from the root, stem and leaf of the positive lines had no objective band (Figure 4C), indicating the tissue specificity of MAN5AS by using the endosperm specific ZM-leg1A promoter. This promoter made exogenous MAN5AS specifically expressed in the seeds of transgenic maize and could lessen the potential impairment to the plants. Moreover, MAN5AS present in seeds are more convenient for storage and transportation.

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