Secretion of active recombinant phytase from stably transformed soybean cells
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The objective of this research was to express a fungal phytase gene in transgenic soybean cells to to study the potential for improving phosphorus utilization in soybean meaL A simple and inexpensive particle inflow gene gun was constructed and bombardment was optimized as assayed by Î²-glucuronidase reporter gene expression. A somatic embryogenesis approach was used for soybean regeneration from culture. The efficiencies of embryo induction and embryo conversion to form roots and shoots were compared in commercial soybean cultivars to identify optimal cultivars for recovery of transgenic plants.
To study the expression of a recombinant fungal phytase gene (PhyA from Aspergillus niger), four expression vectors were constructed in soybean transformation vectors. PhyA was placed under the control of either a constitutive cauliflower mosaic virus 35S promoter or a soybean seed specific Î²-conglycinin promoter, each with or without a patatin endoplasmic reticulum (ER) signal sequence. All four vectors were sequenced and introduced into 'Williams 82' suspension culture cells by particle bombardment. Stably transformed cell lines were selected and tested for stable integration by Southern analysis. The presence of the phytase protein product was detected by immunoblotting. Activity of recombinant phytase was characterized by enzyme assay. Cell lines containing the phyA gene under control of the CaMV 35S promoter and ER signal sequence secreted active phytase into the culture medium. The pH and temperature optima were determined for recombinant phytase.
- Doctoral Dissertations