Cloning, Expression Of Two Novel Phytase Genes OsPHY1 And OsPHY2 In Rice And Gene Genetic Transformation

Phytases are enzymes involved in degradation of phytic acid and its derivatives, with inositol and Pi being the final products. In cereal crop rice, the phosphorus is existed as the organic forms in seed, such as phytins. The Pi provided for seed germination and young seedling growth is derived from the degardation of the phytins stroed in the seeds. In this study, two novel rice phytase genes, OsPHY1 and OsPHY2, were cloned, and further subjected to expression analysis, biochemical identification, and functional characterization. The main results are as follows:1. Using the cDNA from 6-d germinated seeds as the template, the open reading frame (ORF) of rice phytase gene OsPHY1 and OsPHY2 was specifically amplified. The ORF of OsPHY1 is 1620 bp in length, encoding a polypeptide of 539-aa with a molecular weight of 59.71 and an isoelctric point (pI) of 5.85. OsPHY1 contains a conserved domain referred to MPP_PAPs, a motif generally identified in purple acid phosphatases, and three other conserved domians including MPP_ACP5, MPP_CSTP1 and MPP. The ORF of OsPHY2 is 1560 bp in length, encoding a polypeptide of 519-aa with a molecular weight of 57.99 and an isoelctric point (pI) of 7.59. OsPHY2 contains a conserved domain generally identified in histidine acid phosphatases (HAPs).2. The phylogenetic trees that OsPHY1 and OsPHY2 and their corresponding homologous in plant species have been generated at the nucleic acid level. OsPHY1 was found to share high similarities to the purple acid phosphatases (PAPs) showing phytase acitivities that derived from Arabidopsis, rice, and wheat. OsPHY2 displayed high similarities with the wheat phytase genes such as T. aestivum PhyIIa1, T. aestivum PhyIIa2, T. aestivum PhyIIb and T. aestivum PhyIIac, and barley phytase genes such as H. vulgare PhyIIa1, H. vulgare PhyIIa2 and H. vulgare PhyIIc.3. Using semi-quantitative RT-PCR approach, the expression patterns of OsPHY1 and OsPHY2 were analysed. During a 10-d germination process, OsPHY1 was detected to be expressed at high levels in the novel growing embro with a pattern of single-peak curve, approximately peaking at the 4-d. Similar to OsPHY1, much more abundant OsPHY2 transcripts were detected in novel growing embros than in the leaves and roots. Taken together, the much higher expression of OsPHY1 and OsPHY2 have implicated their critical roles on degradation of phytate in the seeds during the germination process.4. Based on the recombinant plasmids harboring the ORFs of OsPHY1 and OsPHY2, the prokaryotic expression plasmids integrated the ORFs of OsPHY1 and OsPHY2 that referred to pET28a(+)-OsPHY1 and pET28a(+)-OsPHY2 were constructed. After introduction of the plasmids into E.Coli host BL21 and induced by IPTG, the predicted proteins were expressed.5. Biochemical analysis was performed on the prokaryotic expressed OsPHY1 and OsPHY2. The optimal values of temperature and pH for OsPHY1 were 57℃and pH3.5, repectively. Under the conditions of high temperature and pre-treatment of high temperature, OsPHY1 still sustained a relative high catalytic capacity. Similar to OsPHY1, OsPHY2 also possessed strong functions on degradation of phytate, with optimal values of temperature and pH for OsPHY1 were 47℃and pH3.5, repectively. However, OsPHY2 had relative low enduence to high temperature compared to OsPHY1.6. Using DNA recombinant technology, the expression plasmids of pCAMBIA3301-OsPHY1 and pCAMBIA3301-OsPHY2 were constructed by fusing the ORFs of OsPHY1 and OsPHY2 into empty expression vector pCAMBIA3301. The tobacco variety Wiscosin was transformed by the expression plasmids based on Agrobacterium-tumafeciens mediated approach. Most transgenic tobacco plant lines were PCR positive, with high target gene expression levels detected. Compared with the control (transformed with the empty expression vector pCAMBIA3301), the transgenic lines with higher expressions of OsPHY1 and OsPHY2 behaved higher phytase activities.7. Under the condition using phytate as the sole phosphorus source, the transgenic plants showed much better growth features, and much more accumulative phsphorus amount, and dry weight than those of the control plants. Therefore, the plants with overexpression of OsPHY1 and OsPHY2 were endowed a strong capacities on degradation of the phytate in the growth medium. These rice phytase genes are potential used as important gene resources on improvement of organic phosphate compounds in crop production in future.