Cloning And Biological Function Of Phosphate Transporter MdPHT1;12 Gene In Apple

Apple(Malus domestica)is an important cultivated fruit tree species,which playing a crucial role in Chinese agricultural and rural economy.However,most of the orchards are suffering from soil poverty and poor ground conditions,which affects and restricts the quality and efficiency of apple production,limiting the quality and productivity of apples.Therefore,improving apple’s own absorption and utilization of nutrients is the key to improving the quality and efficiency of apples production.Phosphorus is one of the most essential nutrients in plant growth and development.Phosphorus in soil is absorbed by plant roots in the form of soluble inorganic phosphate,but it is easily fixed and adsorbed in the soil,leading to the deficiency of utilization.Phosphate transporter(PHT)is a direct phosphorus uptake by plants and plays a key role in the uptake and transport of phosphorus.In this study,the phosphate transporter gene MdPHT1;12 was isolated from apple and its function was identified using reverse genetics.The specific results were as follows:(1)The full-length c DNA of MdPHT1;12 gene is 1563 bp,encoding 521 amino acids.The phosphate transporter MdPHT1;12 is a stable hydrophobic protein.Amino acid sequence alignment shows that MdPHT1;12 has more than 80% homology with high affinity phosphate transporters from other species such as Glycine max,Arabidopsis thaliana and Solanum tuberosum.Therefore,it was hypothesized that MdPHT1;12 is a high-affinity phosphate transporter protein and may play an important function in phosphorus uptake and transport.(2)The expression pattern of MdPHT1;12 gene was analyzed by real-time fluorescence quantitative PCR.The results showed that the MdPHT1;12 gene was expressed in root,stem and leaf nutrient organs,with the expression in the root system being much higher than that in the stem and leaves.Under low phosphorus stress,the MdPHT1;12 gene was highly induced in roots,stems and leaves.Under high phosphorus stress,the relative expression of the MdPHT1;12 gene was significantly up-regulated in stems and leaves compared with the normal group,while the expression level of the MdPHT1;12 gene in roots did not change significantly.This suggests that the phosphate transporter protein may play an important role in the response to phosphorus stress.Subcellular localization analysis showed that the protein was localized to the cell membrane,which led to the speculation that the protein is a high-affinity phosphate transporter protein.(3)Genetic transformation of ’Gala’ apples using the Agrobacterium-mediated leaf disc method.A total of 18 transgenic lines were obtained and OE-2 and OE-4 were selected for phenotypic analysis.The function of the MdPHT1;12 gene was identified by comparing the differences in plant morphology and phosphorus content between the transgenic lines and the wild type at different phosphorus concentrations.The results showed that only the above-ground biomass of the transgenic plant OE-2 was slightly higher than that of the wild type at normal phosphorus concentrations,while the below-ground biomass of OE-2 and OE-4 were not significantly different from that of the wild type.At low phosphorus concentrations,the biomass of the transgenic apples was significantly higher than that of the wild-type apples and the total phosphorus content of the transgenic plants was significantly higher than that of the wild-type plants.It can be concluded that overexpression of the phosphate transporter protein gene MdPHT1;12 enhanced the phosphorus uptake capacity of apples and significantly increased the phosphorus content in the plants,enhancing the ability of apples to cope with low phosphorus stress.This study provides theoretical basis and material support for new molecular breeding methods to improve apple’s phosphorus absorption efficiency,and has important application value to improve the efficient absorption and utilization of phosphorus in apple.