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Characterisation of the phytase gene in trifoliate orange (Poncirus trifoliata (L.) Raf.) seedlings

Shu, Bo, Wang, Peng, Xia, Ren-Xue
Scientia horticulturae 2015 v.194 pp. 222-229
manganese, metabolism, citrus fruits, phytases, acid phosphatase, Komagataella pastoris, phytin, recombinant proteins, seedlings, iron, microorganisms, gene expression, genes, phosphates, Poncirus trifoliata, phosphorus, gene expression regulation, roots, leaves
Phosphate (P) deficiency decreases the yield and quality of citrus fruits. The P concentration, P uptake, and the expression of three predicted acid phosphatase genes in trifoliate orange seedlings were measured among inorganic phosphate (Pi), organic phosphate (Po), and no phosphate (−P) treatments to assess the ability of Po to improve trifoliate orange P deficiency. The three genes were expressed in Pichia pastoris X-33 to characterise the trifoliate orange phytase gene. Our analysis showed that trifoliate orange could utilise phytin without microorganisms in P-deficient conditions by promoting phytase and acid phosphatase activity; however, Po had a smaller effect on the improvement of trifoliate orange P deficiency than Pi fertiliser. The expression of the three genes in roots and leaves with Pi treatment did not respond to −P solutions, but nearly all three genes were up-regulated in roots or leaves with Po and −P treatment after −P solutions were supplied. Further, the PtPAP3 recombinant protein (r-PtPAP3), which was expressed in P. pastoris, showed phytase activity that was as high as that of cereal phytases. The r-PtPAP3 Km values were 46.2μM for phytate and 1631μM for ρ-nitrophenylphosphate. Incubation with Mn2+ increased the specific activity of r-PtPAP3 for phytate by approximately 1.9-fold, and Fe2+ increased it by approximately 1.2-fold. In summary, the function and expression pattern of PtPAP3 allowed a detailed understanding of the significance of phytate metabolism of a woody perennial.