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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Secretion of active recombinant phytase from stably transformed soybean cells

Li, Jia 04 October 2006 (has links)
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 β-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 (<i>Phy</i>A from <i>Aspergillus niger</i>), four expression vectors were constructed in soybean transformation vectors. <i>Phy</i>A was placed under the control of either a constitutive cauliflower mosaic virus 35S promoter or a soybean seed specific β-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 <i>phy</i>A 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. / Ph. D.

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