Cloning And Functional Properties Of GmACP1 And GmPht1;1 Related To Phosphate Efficiency In Soybean

Inorganic phosphate （Pi） plays an essential role in plant growth and metabolism. Phosphate deficiency has an effect to all the processes of photosynthesis, respiration, carbon metabolism and energy transfer reactions within plants, and finally results in the reductions of crop yields. Plants have evolved a number of potential adaptive mechanisms to face with the low and unevenly distributed phosphate in soil solution, including changes in root morphology and architecture, improves phosphate uptake capacity through the activation of Pi transporters, increases phosphate mobilization and recycling activity through secretion of organic acids and phosphatases into the rhizosphere and enhancement of internal phosphatase activity. As one of the most important food and ecnomic crops in the world, soybean provides important source of protein for human and animal forage. Because of its unique nutritional values and health benefits, soybean has become an increasingly important agricultural commodity. Soybean is cultivated in tropical, subtropical and temperate areas, where the siols are often have a low phosphate availability. Therefore, the research on the genes related with phosphate uptake and utilization efficiency has important practical significance for revealing the mechanism and germplasm improvement.In this study, candidate genes, GmACPl （Glyma08g20820） and GmPht1;1 （Glyma10g33030）, which were related to phosphorus efficiency, were selected for further analysis in this study. For GmACP1 gene, the expression pattern and functions in E.coli、 tobacco and soybean were studied; for GmPht1;1, gene expression in response to long-term phosphate starvation, function in tobacco and eQTL mapping of GmPht1;1 in a RIL population NJRIKY were analyzed.The main results are summerized as follows:GmACP1 gene encoding a Pi starvation-induced phosphatase from soybean was isolated using a PCR cloning strategy. GmACP1 is localized on chromosome 8. The full length cDNA of GmACP1 is 1,177 bp and contains an 819bp ORF encoding a 272 amino acid polypeptide （31.01 kD）. The open reading frame is flanked by 80 bp and 278 bp of untranslated sequences at the 5’and 3’regions, respectively. Comparison of the genomic and cDNA sequences of GmACP1 found three introns existed in coding region, the sizes of these three introns were 368bp,115bp and 620bp, respectively. The cDNA encoded a water soluble protein by hydrophobicity analysis. The amino acid sequence had a number of potential phosphorylation sites including 9 serine,1 threonine and 1 tyrosine. The deduced amino acid sequence of GmACP1 showed 85% identity to Phaseolus vulgaris phosphatase （PvPS2） and 70% identity to tomato acid phosphatase （LePS2）. GmACP1 belongs to be a member of HAD superfamily. A multiple sequence alignment of the amino acid sequence of GmACP1 with other plant phosphatase sequences revealed the presence of highly conserved motifs DFDXT and GDGXXD of HAD and DDDD superfamily, and had the highest phylogenetic relationship with PvPS2;1. This result suggested that the function of plant phosphatase sequences were highly conserved.GmACP1 was highly expressed in leaves and roots, trace expression in flowers, but not expressed in stems and pods. When soybean seedlings were subjected to Pi stavation for 15 days, GmACP1 was highly induced in vaious tissues, especially in old leaves and mature leaves, while lower expression level was detected in Pi sufficient soybean seedlings. The expression patterns suggested that GmACP1 might primarily involve in the release of Pi from phosphorylated compound in shoots, and maintain Pi homeostasis within plants. The bacterially （Escherichia coli） expressed the recombinant expression plasmid pET28a-GmACP1 exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate （p-NPP）. The pH optimum of the enzyme activity suggests that GmACP1 is an acid phosphatase.In order to verify the probable function of GmACP1 in plant, the expression vector 35S::GmACP1-pMDC83 was constructed and introduced to tobacco and soybean hairy roots, respectively. The transgenic tobacco plants were generated through Agrobacterium tumefaciens-mediated transformation. In comparison with WT plants grown in +P conditions, phosphatase activity in leaves of transgenic tobacco lines was significantly improved. After grown in the nutrient solution for 20 days when phytic acid as the sole P source, the transgenic tobacco lines showed the better growth and produced more lateral roots than WT plants; in addition, the total dry weight, total phosphorus content and Pi concentration in shoot of transgenic tobacco lines were significantly improvecd. The soybean composite plants with transgenic hairy roots were obtained by Agrobacterium rhizogenes-mediated transformation. After 20 days of growth in the nutrient solution, the hairy roots overexpressing GmACPl exhibited higher phosphatase activity, root dry weight, phosphorus content and phosphorus uptake efficiency than control. These results suggested transgenic plants overexpressing GmACPl could hydrolyze organic phosphorus compounds in the extracellular nutrient solution and improve phosphorus efficiency of plants.GmPht1;1 is localized on chromosome 10, no intron exists in coding region, has a high sequence similarity with GmPht1;5. GmPht1;1 gene promoter region exists some motifs responsive to phosphorus stress, light and carbon metabolism responsive elements.GmPht1;1 had the tissue-specific expression patterns, and was highly expressed in leaf, root and flower, trace expression was detected in stem and pod. The phosphate concentrations in shoot and root were barely detected when soybean seedlings were sufferred from Pi deficiency for 15 days, however, GmPht1;1 was highly induced in various tissues, especially in vigorous metabolism tissues （young leaves and lateral roots）, the greatest expression was found in old leaves, which is 5-400 times compared with other tissues. The expression patterns suggested that GmPht1;1 might involved in Pi translocation from root to shoot and Pi remobilization in shoot. In order to further study the function of GmPht1;1, plant expression vector of 35S:: GmPht1;1-pMDC83 was constructed and transformed into tobacco plants through agrobacteriu-mediated method. The positive T1 transgenic plants were used to analysis the abality to grow in phosphate-limited nutrient solution for 20 days. Compared with wild type （WT） plants, the transgenic lines significantly increased total dry weight, root/shoot ratio, the Pi concentration in shoot, the total phosphorus centent, phosphate use efficiency and 1000-seed weights under phosphate deficiency. Under Pi stavation for 7 days, the transgenic lines have a relatively higher chlorophyll fluorescence parameters （ΦPSⅡ, ETR and qP） compared with WT plants. This result showed that a higher intracellular Pi content existed in transgenic lines, the electron transfer on the acceptor side of PSII were lightly damaged, transgenic plants with high photosynthetic activity could efficiently convert light energy into chemical energy, and finally synthesis more carbohydrate. As a result, transgenic plants had a higher dry weight under Pi-limited conditions. This result indicated GmPht1;1 involved in intracellular Pi remobilization and played a role in mataining Pi homeostasis.In order to study the regulation mechanism of GmPht1;1 and the relationship with seed yield, the expression level of GmPht1;1 in RIL population was measured by real-time PCR. Correlation analysis among the expression level of GmPhtl;1, seed yield, PN and chlorophyll fluorescence parameters were performed. The expression level of GmPht1;1 showed a significant positive correlation with seed yield, PN and ΦPSⅡ. Two eQTLs of GmPht1;1 were detected, the marker interval satt331-sat₂74 on chromosome 10 （linkage group O） for qPO.1 and AW186493-satt659 on chromosome 13 （linkage group F） for qPF.1. LOD values are 3.47 and 2.56, explained 8.08% and 7.93% of the total phenotypic variance, additive effects values indicated that the alleles of Kefeng No.l and Nannong1138-2 increased GmPht1;1 expression level at these two locuses, separately. None of the eQTLs for GmPht1;1 expression were co-localized with chlorophyll fluorescence parameters and PN. However, we found eQTL qPO.1 was located on the same marker interval with QTLs for seed yield. These results suggested that GmPht1;1 expression level might have an influence to photosynthesis and seed yield of soybean. GmPht1;1 located within the confidence interval of eQTL qPO.1 and far from qPO.2, this result showed that the expression level of GmPht1;1 might be co-regulated by cis-acting regulator and trans-acting regulation.