Prototype glutathione (GSH) and oxidized glutathione (GSSG) were also tested

GSH and GSSG Reference Anderson, etc. (1992). Take 0.5 g sample, add 3 mL cold 6% partial phosphoric acid (containing 1 mmol L-1
EDTA
, pH 2.8), ice bath grinding, homogenizer at 20,000 g, 4 degrees C centrifugation 15 min, take liquid to immediately determine the GSH and GSSG content or storage at -20 degrees C waiting to be determined. The total GSH and GSSG content was measured as follows: 200 μL extract plus 1.2 mL reaction fluid containing 400 μL reaction fluid 1 (110 mmol-L-1 Na2HPO4-7H20, 40 mmol-L-1 NaH2PO4-H2O, 15 mmol-L-1EDTA, 0.3 mmol-L-1 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB, 0.04% BSA), 320 μL reactive fluid 2 (1 mmol-L-1 EDTA, 50 mmol-L-1 imidazole mizole solution and 0.02% BSA), 400 μL reactive fluid 3 (5% Na2HPO4, pH 7.5 solution dilution 50 times), 80 μL 9.0 mmol-L-1 NADPH.
to determine the absorption value under OD412. The GSSG content is measured as follows: 200 μL extract is added to 1 mL of 2-2 ethylene ethylene (dilution 50 times) at 25 degrees C in the water bath 1 h and then the absorption value under OD412 is measured. The GSH content can be obtained by subtracting the GSSG content from the total GSH and GSSG content.
GSH measurement method: take sample 0.5 g, add pre-cooled 5% sulfonyl sewer acid 2.5 ml and a little quartz sand, fully ice pre-grinding, transferred to
centrifugal tube
At 20,000×g centrifugation 20min at 4 degrees C, the liquid liquid is divided into liquids, and the liquid nitrogen is stored at -20 degrees C or directly analyzed by
antoxidants
analysis.
50 μL of liquid, with 5% sulfonyl sorghum acid to 100 μL (i.e. add 5% sulfonyl sulphate 50 μL), add 24 μL 1.84 mol L-1 triethanolaminetrieth Lene diamine with a medium sample solution, add 50 μL 10% ethylene Polyvinyl pyridine (made with 70% ethanol), 25 degrees C water bath 1 h, to remove GSH, then add 706 mL 50 mmol L-1 phosphoric acid buffer, pH 7.5, containing 2.5 mmol L-1 EDTA, added 20 μL 10 mmol L-1 NADPH and 80 μL 12.5 mmol L-1 DTNB (desulfur nitrobenzene), mixed, 25 degrees C insulation 10 min, when added 20 μL 50 U.mL-1 GR, the total volume of 1 mL, immediately mixed, read out the OD value at 3 min. This method is used to determine GSSG, the total glutathione (GSH and GSSG) assay, as long as the above-mentioned ethylene pyridine with the same volume of distilled water can be replaced. The GSSG standard curve is made in the same way with 5% sulfonyl sal fatty acid as a solvent.
the determination of the yelorotin content by reference to the arnon (1949) method. From the selection of a second fully expanded leaf, shredded, said to take 0.1 g placed in the research, plus a small amount of 80% acetone grinding into a homogenous slurry, fixed capacity to 25 mL, shake and place in the light shelter, after the residue whitening
filtration
, with 80% acetone as a control, under 663 nm and 645 nm to read the OD value. The yerotin content (mg-g-1FW) in the sample was calculated as: yerotin a content of 0.25 (12.7OD663-2.69OD645);
。