MtPAP1表达特性及异源表达对拟南芥有机态磷吸收的影响

高磷条件下,MtPAP1主要在叶片中表达;在低磷条件下,MtPAP1在叶片中表达水平较低,在根系中的表达量则较高,超过了其它器官中的表达量。在以植酸盐为唯一磷供源的条件下,与对照相比,超量表达MtPAP1的拟南芥转基因植株中,根系细胞间隙中的酸性磷酸化酶(APase)活性明显提高。HPLC分析表明,液体培养基中的有机态磷可被转基因拟南芥分泌的APase快速降解。在以植酸盐为唯一磷供源条件下,超量表达MtPAP1的拟南芥转基因植株的生物学产量、植株无机磷含量和全磷含量明显高于野生种。

Ectopic Expression of a Phytase Gene from Medicago truncatula Barrel Medic Enhances Phosphorus Absorption in Plants

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.

Cloning and Characterization of a Novel Purple Acid Phosphatase Gene （MtPAP1） from Medicago truncatula Barrel Medic

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.

Functional characterization of caffeic acid O-methyltransferase in internode lignification of switchgrass(Panicum virgatum)

Caffeic acid O-methyltransferase(COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1,was isolated from switchgrass(Panicum virgatum), a C4 warm-season dual-purpose forage and bioenergy crop. Our results showed that recombinant PvCOMT1 enzyme protein catalyzed the methylation of 5-OH coniferyl alcohol, 5-OH coniferaldehyde(CAld5H) and 5-OH ferulic acid. Further in vitro studies indicate that CAld5H can dominate COMT-mediated reactions by inhibiting the methylation of the other substrates. Transgenic switchgrass plants generated by an RNAi approach were further employed to study the function of COMT in internode lignification. A dramatic decrease in syringyl lignin units coupled with an obvious incorporation in 5-OH guaiacyl lignin units were observed in the COMT-RNAi transgenic plants. However, the constitutive suppression of COMT in switchgrass plants altered neither the pattern of lignin deposition along the stem nor the anatomical structure of internodes. Consistent with the biochemical characterization of PvCOMT1, a significant decrease in sinapaldehyde was found in the COMT-RNAi transgenic switchgrass plants, suggesting that CAld5H could be the optimal intermediate in the biosynthesis syringyl lignin.