Abstract: 【Objective】This study is aiming at cloning apple purple acid phosphatase gene MdPAP10 and studying its expression pattern and low phosphorus response process. The mechanism of action of MdPAP10 under low phosphorus condition was further studied. This study laid the foundation for further study of the molecular mechanism of MdPAP10 affecting phosphorus uptake in woody fruit trees. 【Method】MdPAP10 gene was cloned by homology sequence alignment and PCR technique from Malus× domestica ‘Royal Gala’ apple.The protein structure of MdPAP10 was analyzed by NCBI and the PAP10 amino acid sequences of 10 species such as white pear, peach and strawberry were obtained. The phylogenetic tree was constructed by MEGA5.0. The induced expression and tissue-specific expression profiles of MdPAP10 gene in apple with low phosphorus stress were detected by real-time fluorescent quantitative PCR (qRT-PCR). MdPAP10 was ligated into the plant overexpression vector pBI121. The resultant construct was transformed into LBA4404 Agrobacterium, for the infection of apple calli. MdPAP10 transgenic calli were obtained by screening on resistant medium and PCR identification. The acid phosphatase accumulation and the tolerance to low phosphorus stress and phosphorus content were detected by culturing MdPAP10 transgenic calli on low phosphorus medium. Finally, qRT-PCR was used to detect the expression of phosphorus-related genes in MdPAP10 transgenic calli. 【Result】An apple purple acid phosphatase gene MdPAP10 (MDP0000272096) was cloned from Malus × domestica ‘Royal Gala’. Sequence analysis showed that the open reading frame (ORF) of MdPAP10 is 1 332 bp, which encoded 443 amino acids. Protein structure analysis showed that MdPAP10 contained a signal peptide and a phosphatase domain. Gene structure analysis showed that MdPAP10 contained 5 exons and 4 introns. A phylogenetic tree indicated that the apple MdPAP10 exhibited the highest sequence similarity to Pyrus bretschneideri PbPAP10. Expression analysis showed that MdPAP10 was expressed in roots, stems, leaves, flowers and fruits, and the expression level in roots was the highest. MdPAP10 had a significant response to low phosphorus conditions, and the expression level in roots increased gradually, reached the maximum at 6 h and then decreased gradually. The expression level in leaves was always lower than that in the control group. MdPAP10 transgenic calli were obtained by Agrobacterium tumefaciens infection and verified by PCR and qRT-PCR. MdPAP10 transgenic calli can significantly advance the secretion of acid phosphatase in low phosphorus conditions. Overexpression of MdPAP10 in transgenic calli under low phosphorus conditions improved the tolerance of calli to low phosphorus stress and increased the uptake of phosphorus. The result of qRT-PCR showed that overexpression of MdPAP10 could significantly promote the expression of apple phosphorus-related genes. 【Conclusion】MdPAP10 can respond significantly to low phosphorus stress and promote phosphorus uptake and acid phosphatase secretion under low phosphorus conditions. MdPAP10 plays an important regulatory role in response to low-phosphorus stress.

Key words: apple, purple acid phosphatase, MdPAP10, expression analysis, functional identification