Cultivation Of Transgenic Maize Expressing α-Galactosidase From Gibberella Sp. F75

Soybean meal is the main raw material of protein in the feedstuff. However, in most leguminous plants, there are galactooligosaccharides linked by α-galactosidic bonds, which degradation in the intestine of monogastric animals can cause flatulence and diarrhea. To alleviate the anti-nutritional effect, α-galactosidase is generally supplemented in the feed. Although α-galactosidase production by microbial fermentation is flexible and convenient, this process accompanies with a high cost in equipment and energy consumption. In contrast, producing feed enzyme directly in feed grains can reduce environmental pollution, save energy, and simplify the production and supplementation of feed enzymes. Maize is another feed ingredient of supplier and thus represents an ideal bioreactor to produce feed enzymes. The objective of this study is to develop a genetically stable transgenic maize line with favorable α-galactosidase activity, which can be directly used for feed production.The α-galactosidase from Gibberella sp. F75 was selected due to its excellent enzyme characteristics, protease-resistance, and high application values. For efficient expression in maize, the gene aga-F75 was modified to aga-F75 m with codon optimization. The nucleotide sequence of aga-F75 m shared 77.3% identity with that of aga-F75, and its CAI value and GC content were increased from 0.71 to 0.82 and 51.29% to 55.82%, respectively. aga-F75 m was cloned into plant expression vector pHP20754 harboring a seed-specific expression promoter to construct the expression cassette, which was then transformed into Hi-II maize cells with high-velocity microprojectiles. A total of 33 independent transgenic events were achieved, and T0 transgenic maize plants were obtained by screening, differentiation and rooting. The excellent maize inbred line Zheng58 was selected as the male parent to produce seeds by being pollinated with transgenic maize. Comparison of the agronomic traits of transgenic lines and Zheng58 indicated that the introduction of exogenous aga-F75 m had no significant effect on the phenotype of transgenic maize.Analysis of the α-galactosidase activity in maize seeds showed that the maximum activity of Aga-F75 M was 10 000 U/kg. The average activity was 3 500 U/kg. Southern blot analysis indicated that the copy number of aga-F75 m in transgenic plant of event 8-1 was at least two. According to western blot result, Aga-F75 M had a molecular weight of 95 kDa, higher than its predicted value(82 kDa), and was specifically expressed in seeds other than root, stem, and leaf tissue of transgenic maize. N-glycosylation occurred in Aga-F75 M since the reduction in molecular weight was detected after treatment with Peptide-N-Glycosidase F. In comparison to Aga-F75 produced in Pichia pastoris, Aga-F75 M produced in maize showed similar properties on optimal pH, pH stability, and thermostability, but had a decreased optimal temperature of 50 °C(10 °C lower). After feed pelleting, Aga-F75 M lost less activity(80%) than Aga-F75(100%), indicating that maize drived Aga-F75 M had better thermostability during feed pelleting.The study for the first time expressed exogenous α-galactosidase gene in transgenic maize, and developed an α-galactosidase transgenic maize with excellent plant trait, high-level expression and genetic stability. The results verified the possibility to produce feed enzymes of large molecular weights in transgenic crop. The direct utilization of α-galactosidase transgenic maize in feed industry will not only avoid microbial fermentation, purification and supplementation, but also reduce energy consumption and environment pollution. Thus, α-galactosidase transgenic maize has a significant application prospect and benefits both the ecology and environment.