Abstract
BackgroundMannans 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 FindingsThe 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/SignificanceThis 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.
Highlights
Mannan is the second most abundant hemicellulosic polysaccharide after xylan in nature [1]
Man5As was inserted into the expression vector pHP20754 between the embryo-specific ZM-leg1A promoter and ZM-leg1 terminator (Figure 1B), which is a transcriptionally active spacer region that allows highly efficient transgene expression
In 2011, the plantation area of transgenic plants reached about 160 million hectares and was distributed in 29 countries
Summary
Mannan is the second most abundant hemicellulosic polysaccharide after xylan in nature [1]. Mannans have been defined as one of the intense anti-nutritional factors [7]; they often combine with water, increase the viscosity of chyme, block the intestinal surface partially, and reduce the feed conversion and limit the efficiency of carbohydrate utilization [8,9]. These anti-nutritional factors can cause flatulence, and sometimes are responsible for digestive disorders and metabolic diseases [10,11]. The objective of this study was to overexpress a b-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production
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