A Study of the Expression of Beta-Propeller Phytase in Transgenic Plants

This dissertation, "A Study of the Expression of Beta-propeller Phytase in Transgenic Plants" by Wing-lee, Chan, 陳永利, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled A STUDY OF THE EXPRESSION OF BETA-PROPELLER PHYTASE IN TRANSGENIC PLANTS Submitted by CHAN WING LEE for the degree of Master of Philosophy at The University of Hong Kong in August 2004 Phytases are enzymes that liberate inorganic phosphate from phytate. Beta-propeller phytases (BPPs) belong to a family of phytases exclusively found in Bacillus Species. In the first part of this study, a recombinant BPP, 168phyA from Bacillus subtilis, expressed in transgenic tobacco (t168phyA) was purified by a three-step purification scheme. The biochemical properties of t168phyA were compared with its bacterial counterpart. t168phyA was glycosylated and showed a 4kDa increase in molecular size in SDS-PAGE (44kDa vs. 40kDa). Although its thermostability remained unchanged, the temperature and pH optima of t168phyA shifted from 60℃ to 45-50℃ and from 5.5 to 6.0, respectively. Despite these changes, t168phyA remained catalytically active and had a specific activity of 20U/mg phytase. These results verified the activity of recombinant Bacillus phytase expressed in plant. In the second part of this study, 168phyA was introduced into Arabidopsis thaliana (ecotype Landsberg) by different expression cassettes for intracellular and extracellular expression. Two transgenic lines, 41 and A2, which expressed 168phyA intracellularly, were generated. When the plants were grown on MSO agar, both lines displayed improved phosphorus-nutrition when compared to the wild type. Line A2 even attained higher dry weight than the wild type. However, when agar was supplemented with phytate as the sole phosphorus source, the growth performances of both lines were similar to the wild type. When the plants were grown in phosphate-rich soil, line A2 was taller and bore more peduncles than the wild type or line 41. The seeds from both transgenic lines were similar in morphology and fertility to the wild type seeds. For extracellular expression, three transgenic lines that secreted 168phyA from roots were generated. Lines A32 and A33 had an Arabidopsis extensin 3 signal peptide, while line C2 utilized the leader sequence of the carrot extensin for secretion. The secretion level of 168phyA was in the order of A33>C2>A32. When phytate was supplied as the sole phosphorus source in agar, the wild type and the null lines showed retarded growth (27-60% reductions in dry weight). In contrast, the transgenic lines A32 and C2 did not show any growth retardation, while line A33 showed enhanced growth and phosphorus accumulation over the controls. The seed morphology of lines A32 and C2 were examined and compared with the wild type seeds. A large number of vacuoles were found in these two lines. Their seeds also contained fewer protein bodies with irregular arrangement, which are the major storage form of phytate in seeds. Nonetheless, the seeds remained fertile and had a germination frequency of about 90%. These results suggested that intracellular expression of Bacillus phytase in Arabidopsis may shift the equilibrium of the inositol phosphate biosynthesis pathway, thereby providing more phosphate for metabolism. A similar observation was reported for transgenic tobacco. Secretion of Bacillus phytase from roots enabled plants to assimilate soluble phytate in agar. Phytase expression may lower seed phytate content but has no significant effect on seed fe