Subcellular Localization Of Phytase In Transgenic Maize Kernels

In the work, four transgenic corn homozygous lines derived from four independent events obtained from phytase gene constructs ( PHP20723AO, PHP20754AO, pSPHP3303AO and pSPHP3303TAO ) respectively were choosed to study subcellular localization of phytase in tansgenic maize kernels.Firstly, histological localization of phytase in the samples was studied. The result of Western-blot shows phytase in seed from endosperm construct expressed in endosperm only; phytase in seed from embryo construct expressed in embryo not only, but in endosperm at a lower level than that found in the embryo. Determination of Pi and phytate content indicated the Pi in transgenic seeds from embryo construct containing VTS was increased about 15-fold and the phytate content was reduced by approximately 38% compared to wild-type control seeds.Immunogold labeling was used to determine the subcellular localization of phytase within maize kernels. Result reveals phytase was detected in vacuoles in 16 day-after-pollination endosperm cells derived from vector PHP20754AO and a lot of phytase accumulated in 19 day-after-pollination endosperm cells; No specific signal was detected in embryo cells; Gold particles were found in vacuoles in 19 day-after-pollination embryo cells derived from vector PHP20723AO and a few gold particles accumulated in endosperm cells; Some gold particles were detected in cytosol in 18 day-after-pollination embryo cells derived from vector pSPHP3303AO; Some gold particles were observed in intercellular space and cell wall in embryo cells 22 day-after-pollination derived from vector pSPHP3303TAO.Above results reveal proaleurain signal peptide and vacuole targeting sequence of maize lead phytase to protein body in vacuole in this work, so subcellular location in maize seed has dramatic effect on protein accumulation and its stability.In vitro simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were established in this study. Samples treated by different time to analysis tolerance of phytase to protease were carried on Western blotting analysis. Results showed that phytase was stable within 60min in SGF and was stable within 15min in SIF, indicating that phytase in transgenic corn was digested hard difficultly in SGF and digested easily in SIF.