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Aleurone protoplasts were transfected to express synthetic genes encoding cytosolic and secreted forms of the alkalophilic Bacillus alpha -amylase, alkBA. The alpha -amylase activity in the cytosol of transfected protoplasts was increased 4 fold compared to the controls. Secreted alkBA was probably glycosylated, consistent with its higher apparent molecular mass, whereas the molecular mass of cytosolic alkBA was comparable to that of unglycosylated alkBA of bacterial origin. During micromashing, activity was better retained for the secreted enzyme than for the cytosolic form. Subsequently, barley was transformed with the gene encoding the secreted form of alkBA. Out of eight selected transgenic callus lines, three were propagated to fertile T0 plants. PCR analyses verified the presence of transgene segments encoding alkBA. The alpha -amylase activity in extracts of germinated transgenic grains was increased by 30–100% compared with the control, suggesting that the amylolytic activity of barley can be manipulated through transgenic expression. Immunoblots of these extracts confirmed the presence of both alkBA and endogenous alpha -amylases. The average content of alkBA was calculated to ∼1·5–5·0 μg per germinated grain, corresponding to 0·4–0·8% of the water-soluble protein. |
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