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1、MDAMalondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) reaction, as described by Heath and Packer (1968)The content of MDA was measured as described by Heath the homogenate was centrifuged at 10,000g for 15 min. The reaction mixture containing 1.5 ml of the supernatant a
2、nd 2.5 ml of 0.5% TBA in 20% TCA was incubated at 100 C for 30 min and then cooled rapidly in ice and centrifuged at 10,000g for 15 min. The absorbance of the supernatant was measured at 532 nm and corrected for nonspecific absorbance at 600 nm. The concentration of MDA was calculated by using an ex
3、tinction coefficient of 155 mM -1 cm -1 and expressed as nmol g -1 fresh weight.MDA content was determined by the thiobarbituric acid (TBA)-based colorimetric method as described by Heath EC. 1.11.1.7) activity was measured according to Ngo & Lenhoff (1980). POD activity was measured as a change in
4、absorbance at 470 nm after guaiacol oxidation according to the method described previously (Polle, Otter & Seifert 1994).Peroxidase (POD) activity was determined with guaiacol in the presence of H2O2 (Zhang et al. 1995). The oxidation of guaiacol was measured by the increase in absorbance at 470 nm
5、(e = 26.6 mm -1 cm-1 ). The POD activity was expressed as nmol guaiacol min -1 mg -1 protein.Peroxidase (POD) activity was determined with guaiacol and H2O2 (Bestwick, 1998). The reaction mixture (3mL) consisted of 50 mM phosphate buffer (pH6.1), 1% guaiacol, 0.4% H2O2 and 10 L enzyme extract. The a
6、ctivity was measured as a change in absorbance at 470 nm.Bestwick C.S., Brown I.R. Mansfield J.W. (1998). Localized changes in peroxidase activity accompany hydrogen peroxide generation during the development of a nonhost hypersensitive reaction in lettuce. Plant Physiology. 118, 10671078.蛋白质蛋白质 Pro
7、tein content was determined according to the method of Bradford (1976).蛋白质蛋白质 Soluble protein was determined as described for immunoblotting. Protein content was quantified according to the method of Bradford (1976).Chlorophyll was extracted from 0.2 g leaves by grinding them under 5 mL of ice- cold
8、 80% acetone, and the chlorophyll content per gram fresh weight (FW) of leaf was determined as described by Lichtenthaler (1987).Chlorophyll content was assayed by the method of Lichtenthaler (1987). Frozen leaves (0.1g FW) and spikes (1.0g FW) were ground with 5 mL of ice-cold 80% acetone, the abso
9、rbance of the homogenate was monitored at 663,645 and 470nm.Lichtenthaler H.K. (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology. 148, 350382.Proline and sugars were determined as described previously(Lu et al. 2007). Leaves were harvested and dried
10、 at105_C for 1 h, followed at 80_C for 12 h. The dried samples were powdered in a mortar with a pestle, and usedfor determination of proline and sugars. For measurementof proline, 50 mg dried powder samples were extractedwith 3 ml of 80% ethanol for 1 h. After filtration, the filtratewas incubated i
11、n a boiling water bath to evaporate theethanol, and distilled water was then added to make thefinal volume to 10 ml. The solution was shaken for 10 minafter addition of permutit, and then filtrated. Into 2.5 ml offiltrate, 2.5 ml of acetic acid glacial and 2.5 ml ninhydrinsolution (2.5 g ninhydrin d
12、issolved in 60 ml of acetic acidglacial and 40 ml of 6 M phosphoric acid) were added. Themixture was incubated in a boiling water bath for 1 h, andthen extracted with 2.5 ml of benzene by shaking themvigorously for 5 min. The benzene phase was used todetermine the absorbance at 515 nm (Troll and Lin
13、dsley1955). Proline concentration was calculated as to thestandard curve Proline was extracted from Frozen leaves and spikes by Spectrophotometry method at 515nm according to the method of Walter and John (1955).Walter T, John L. (1955). A photometric methods for the determination of proline. The Journal of Biological Chemistry. 215, 655-660.
