SE185:/S1/M1
From Metabolonote
lower pageD1
Sample Set Information
| ID | TSE1343 |
|---|---|
| Title | Metabolic profiling analysis of genetically modified rice seedlings that overproduce tryptophan reveals the occurrence of its inter-tissue translocation |
| Description | A metabolic profiling approach using high performance liquid chromatography-mass spectrometry (LC-MS) was applied to seedlings of transgenic rice plants (Oryza sativa) that over-express the OASA1D gene encoding a feedbackinsensitive a-subunit of anthranilate synthase (AS, EC 4.1.3.27). Analysis revealed that the seedlings accumulated tryptophan (Trp) at a high concentration without marked effects on the amounts of other major metabolites. Some minor indole metabolites showed a certain degree of increase in the amounts in Trp-accumulating tissues, while no active catabolic conversion of Trp was indicated. Analysis also revealed that the distribution of Trp in the plant was uneven, with the highest level being observed in young developing tissues, despite tissue-independent expression of the OASA1D gene under the control of ubiquitin promoter. Differences in AS activity and anthranilate content among organs were small. A feeding experiment with radiolabeled Trp clearly demonstrated one-way Trp movement from old to young leaves; thus, the uneven distribution of Trp in OASA1D plants is the manifestation of Trp translocation. The negligible effects of Trp accumulation in other metabolic pathways, low metabolic activity of Trp, and efficient translocation of Trp in rice plants expressing OASA1D transgene are favorable characters from the aspect of metabolic engineering of Trp production. |
| Authors | Matsuda, F., Ishihara, A., Takanashi, K., Morino, K., Miyazawa, H., Wakasa, K., and Miyagawa, H. |
| Reference | Plant biotechnology 27(1), 17-27, 2010-03-25 |
| Comment |
Sample Information
| ID | S1 |
|---|---|
| Title | Oryza sativa L. cv. Nipponbare |
| Organism - Scientific Name | Oryza sativa |
| Organism - ID | NCBI taxonomy:4530 |
| Compound - ID | |
| Compound - Source | |
| Preparation | HW1 and HW5 lines were established from the original Agrobacterium-mediated transformation of rice calli (Oryza sativa L. cv. Nipponbare) with OASA1D (Wakasa et al. 2006). Two lines, HW1 and HW5, were grown in a non-containment greenhouse (T5). Seeds of T5 were then grown in an isolated field (T6). Seeds of both HW1 T5 generation and HW5 T6 generation were used in this study. After soaking in 5% sodium hypochlorite solution in water for 30 min, the seeds of transformed and untransformed rice were incubated at 28°C with a 16-h photoperiod per day. Rice seedlings were then grown in a pot containing Bonsol soil® (Sumitomo Chemical, Osaka) in a growth chamber under the same conditions for four weeks. |
| Sample Preparation Details ID | |
| Comment |
Analytical Method Information
| ID | M1 |
|---|---|
| Title | LC-MS |
| Method Details ID | MS1 |
| Sample Amount | 5 μL |
| Comment |
Analytical Method Details Information
| ID | MS1 |
|---|---|
| Title | Non-targeted metabolic profiling analysis using LC-MS |
| Instrument | HPLC: Shimadzu LC-10VP system, MS; Shimadzu LCMS2010 |
| Instrument Type | |
| Ionization | ESI |
| Ion Mode | Positive |
| Description | Tissue samples were cut into small pieces and extracted with 40 volumes (w/v) of extraction solvent (methanol : water (50 : 50, v/v) containing 0.2% acetic acid) at room temperature for one hour. Extracts (500 ml) were added to 2.0 ml dilution solvent (methanol : water (90 : 10, v/v) containing 0.2% acetic acid) and applied to a Sep-Pak Plus C18 Cartridge (Waters, Milford, USA) equilibrated with dilution solvent. The cartridge was washed with 2.0 ml dilution solvent and all solvent in the cartridge was eluted by aeration. The eluate was evaporated under a vacuum and the residue was dissolved in 200 ml of 20% methanol in water. Following centrifugation at 15,000 g for 10 min, supernatants were subjected to LC-ESI-Q-MS analyses. Sample extracts (5 ml) were analyzed using a LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC: Shimadzu LC-10VP system, MS; Shimadzu LCMS2010).
The analytical conditions were as follows: HPLC 18 Metabolic profiling of Trp overproducing rice Copyright © 2010 The Japanese Society for Plant Cell and Molecular Biology column: Cadenza CD-C18, Imtact Co., Kyoto, Japan, 2.075 mm; solvent system: acetonitrile (0.1% formic acid) : water (0.1% formic acid), gradient program: 5:95, v/v, at 0 min; 60:40 at 11 min; 98:2 at 12 min; 98:2 at 13 min; 5:95 at 13.1 min; 5:95 at 20 min, flow rate: 0.25 ml min-1, temperature: 35°C, MS detection; CDL temperature: 250°C, block heater temperature: 200°C, probe voltage: +4.5 kV, Q-array voltage: scanning mode, nebulizing gas flow: 1.5 l min-1, detection mode: scan mode (m/z 100–700), scan time: 2 sec. The scans were repeated for 15 min (750 times) in a single run. Data were recorded with the aid of LCMS-Solution version 2.0 software (Shimadzu, Kyoto). |
| Comment_of_details |
