Molecular Cloning And Functional Analysis Of Purple Acid Phosphatase GmPAP4 And GmPAP14 In Soybean

Organic phosphorus is a major component of phosphorus in soil, and can not be absorbed or utilized directly. Acid phosphatase is a kind of enzyme which can remobilise inorganic phosphorus (Pi) from organic phosphorus to enhance the acquisition or utilization efficiency. So it is significant important for solving the problems that lacking available Pi in soil and phosphorus deficiency in plant. In this study, we cloned two novel purple acid phosphatase genes from a high Pi-efficient soybean variety‘zhonghuang 15’. To analyze the function of the candidated gene, real-time quantitative PCR and phosphorus nutrition analysis in transgenic Arabidopsis thaliana were studied. The main results were as followed:1. Two purple acid phosphatase genes GmPAP4 and GmPAP14 were cloned. The open reading frame of GmPAP4 was 1329 bp, which encoded a polypeptide with 442 amino acid residues; and the open reading frame of GmPAP14 was 1395 bp, which encoded a polypeptide with 464 amino acid residues.2. Differential expression pattern of GmPAP4 in soybean variety‘zhong-huang15’and‘niu-mao-huang’was analyzed by real-time quantitative PCR. The results indicated that relative expression of GmPAP4 in zhong-huang 15 was significantly higher than that in niu-mao-huang from 15 d to 70 d in the condition of phytate as the sole P source.3. The prokaryotic expression of GmPAP4 was studied. The recombinant pET32a-GmPAP4 expressed a specific band of 61.2 KDa in E.coli by the induction of 0.8 mmol/L IPTG after 12 hours at 28℃, which indicated that GmPAP4 was prokaryotic expressed successfully. Enzyme activity assay of GmPAP4 showed that it has the highest activity to remobilize substrateρ-NPP in pH 5.0.4. The localization of GmPAP4 in plant cell was analyzed. The fusion vector pCamE-GmPAP4::GFP was constructed, and then was transformed into the onion epidermal cells by the gene-gun technique. The green fluorescence signals were detected on the onion cell membrane and cell wall, which indicated that the GmPAP4 might anchor on the plasma membrane or be secreted outside of the cells. 5. The localization of GmPAP4 in different tissues of transgenic Arabidopsis thaliana was studied. The fusion vector pCamE-GmPAP4::GFP was introduced into Arabidopsis thaliana genome, and the rusults showed that the green fluorescence signals were detected in plant roots vasculature and flower pollen. The results indicated that GmPAP4 might associate with phosphorus transportation in roots and the plant pollen forming.6. The function of GmPAP4 was analyzed in transgenic Arabidopsis thaliana. We constructed a over-expression vector pCamE-GmPAP4 and transformed the vector to Arabidopsis thaliana genome. The results showed that, in the condition of phytate as the sole P source, GmPAP4 can expressed efficiently in transgenic T3 plants, and the weight of transgenic plants were significantly higher than that in wild-type control.