小麦根际土壤脱氢酶活性测定方法的改进

Improvement of determination dehydrogenase activity in wheat rhizospheric soil

为准确检测土壤脱氢酶活性,在传统TTC(氯化三苯基四氮唑)法基础上对萃取剂、测定波长、对照、培养时间、TTC浓度等条件进行了优化,并进行了标准曲线制作方法的创新。改进后的土壤脱氢酶活性测定方法为:用甲苯溶解TF(三苯基甲臜)制作标准曲线,以无土+无基质为试验对照,1.0%TTC溶液为基质,土壤中加入pH为7.4的Tris-HCl缓冲液和0.1 mol/L的葡萄糖溶液,在37℃下培养24 h,以甲苯为萃取剂萃取30 min,于492 nm波长下检测吸光值。利用优化后的方法测定转基因抗旱小麦各生育期根际土壤脱氢酶活性,结果表明:转基因抗旱小麦与对照(受体小麦)根际土壤脱氢酶活性没有显著性差异,均表现为先升高后降低趋势,于返青期达到最大值。试验结果进一步证实上述方法稳定可行。

To perform an accurate measurement of dehydrogenase activity in rhizospheric soil, four factors including extracting agent, wavelength, control, culture time and triphenyl tetrazolium chloride（TTC） concentration, were optimized based on traditional TTC method. TF dissolved in toluene was used to establish the standard curve. The optimized determination sytem for dehydrogenase activity inclues double control （no soil and no TTC, 1.0% TTC solution as reaction substrate, Tris-HCl （pH 7.4） and 0.01 mol/ L glucose solution addition in soil, culture temperature at 37 ℃for 24 h, extraction by toluene for 30 min, and detection at wavelength of 492 nm. By using the optimized determination method, the dehydrogenase activities in rhizospheric soil of a transgenic wheat cultivar with drought resistance and its receptor as control at different development stages were detected. The results showed that there was no significant difference between the two wheat cultivars at the same growth stage. The activity exhibited the same trend, being increase followed by decrease, and reaching the maximum value at seeding stage.