In the present study, the phosphorus-absorption capacity of transgenic Arabidopsis plants ectopically ex- pressing a novel phytase gene from Medicago truncatula Barrel Medic was evaluated. A full-length cDNA encoding ...In the present study, the phosphorus-absorption capacity of transgenic Arabidopsis plants ectopically ex- pressing a novel phytase gene from Medicago truncatula Barrel Medic was evaluated. A full-length cDNA encoding an extracellular form of phytase was isolated from the model legume M. truncatula. The phytase gene （MtPHY1） has an open reading frame of I 632 bp predicted to encode 543 amino acids, including an N- terminal signal peptide of 27 amino acids. The genomic sequence of the MtPHY1 gene is 5 151 bp, containing seven exons interrupted by six introns. Under high-Pi （2 mmol/L） growth conditions, higher levels of MtPHY1 transcripts accumulated in the leaf and stem than in the root. The transcript level was reduced in the stem and increased in the root, with no obvious changes in the hybridization signal detected in the leaf under IowPi （10 pmol/L） conditions. Chimeric transgenes were constructed by placing MtPHY1 under the control of the constitutive CaMV35S promoter and the root-specific MtPT1 promoter. Phytase activities in root apoplast of transgenic Arabidopsis were 12.3- to 16.2-fold of that in control plants. The phytase expressed was secreted into the rhizosphere, as demonstrated by HPLC analysis of phytate degradation by root exudates. Ectopic expression of MtPHY1 in Arabidopsis, leading to significant improvement in organic phosphorus absorption and plant growth, indicated that MtPHY1 has great potential for improving plant phosphorus absorption and phytoremediation.展开更多
A novel purple acid phosphatase gene （MtPAP1） was Isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp In length with an open reading frame （ORF） of 1 398 bp capable of encoding a...A novel purple acid phosphatase gene （MtPAP1） was Isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp In length with an open reading frame （ORF） of 1 398 bp capable of encoding an N-terminal signal peptlde of 23 amino acids. The transcripts of MtPAP1 were mainly detected In leaves under high-phosphate conditions, whereas under low-phosphate conditions the transcript level was reduced In leaves and Increased In roots, with the strongest hybridization signal detected In roots. A chimeric gene construct fusing MtPAP1 and GFPwas made In which the fusion was driven by the CaMV35S promoter. Transgenlc Arabidopsis plants carrying the chimeric gene constructs showed that the fusion protein was mainly located at the apoplast based on confocal mlcroecoplc analysis, showing that MtPAP1 could be secreted to the outside of the cell directed by the signal peptlde at the N-terminal. The coding region of MtPAP1 without signal peptlde was Inserted Into the prokaryotlc expression vector pET-30a （＋） and overexpressed In Escherichla coil BL21 （DE3）. The acid phosphatase （APase） proteins extracted from bacterial culture were found largely based on sodium dodecyl sulfate-polyecrylamlde gel electrophoresls. An enzyme activity assay demonstrated that the APase activity In the transformed bacteria was 3.16-fold higher than that of control. The results Imply that MtPAP1 functions to Improve phosphorus acquisition In plants under conditions of phosphorus （P） stress.展开更多
基金Supported by the Samuel Roberts Noble Foundation and the Hebei Provincial Natural Science Foundation of China （300112）.
文摘In the present study, the phosphorus-absorption capacity of transgenic Arabidopsis plants ectopically ex- pressing a novel phytase gene from Medicago truncatula Barrel Medic was evaluated. A full-length cDNA encoding an extracellular form of phytase was isolated from the model legume M. truncatula. The phytase gene （MtPHY1） has an open reading frame of I 632 bp predicted to encode 543 amino acids, including an N- terminal signal peptide of 27 amino acids. The genomic sequence of the MtPHY1 gene is 5 151 bp, containing seven exons interrupted by six introns. Under high-Pi （2 mmol/L） growth conditions, higher levels of MtPHY1 transcripts accumulated in the leaf and stem than in the root. The transcript level was reduced in the stem and increased in the root, with no obvious changes in the hybridization signal detected in the leaf under IowPi （10 pmol/L） conditions. Chimeric transgenes were constructed by placing MtPHY1 under the control of the constitutive CaMV35S promoter and the root-specific MtPT1 promoter. Phytase activities in root apoplast of transgenic Arabidopsis were 12.3- to 16.2-fold of that in control plants. The phytase expressed was secreted into the rhizosphere, as demonstrated by HPLC analysis of phytate degradation by root exudates. Ectopic expression of MtPHY1 in Arabidopsis, leading to significant improvement in organic phosphorus absorption and plant growth, indicated that MtPHY1 has great potential for improving plant phosphorus absorption and phytoremediation.
基金Supported by the Samuel Roberts Noble Foundation and the Hebei Provincial Natural Science Foundation of China （300112）.
文摘A novel purple acid phosphatase gene （MtPAP1） was Isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp In length with an open reading frame （ORF） of 1 398 bp capable of encoding an N-terminal signal peptlde of 23 amino acids. The transcripts of MtPAP1 were mainly detected In leaves under high-phosphate conditions, whereas under low-phosphate conditions the transcript level was reduced In leaves and Increased In roots, with the strongest hybridization signal detected In roots. A chimeric gene construct fusing MtPAP1 and GFPwas made In which the fusion was driven by the CaMV35S promoter. Transgenlc Arabidopsis plants carrying the chimeric gene constructs showed that the fusion protein was mainly located at the apoplast based on confocal mlcroecoplc analysis, showing that MtPAP1 could be secreted to the outside of the cell directed by the signal peptlde at the N-terminal. The coding region of MtPAP1 without signal peptlde was Inserted Into the prokaryotlc expression vector pET-30a （＋） and overexpressed In Escherichla coil BL21 （DE3）. The acid phosphatase （APase） proteins extracted from bacterial culture were found largely based on sodium dodecyl sulfate-polyecrylamlde gel electrophoresls. An enzyme activity assay demonstrated that the APase activity In the transformed bacteria was 3.16-fold higher than that of control. The results Imply that MtPAP1 functions to Improve phosphorus acquisition In plants under conditions of phosphorus （P） stress.
