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[目的]测定白花除虫菊四倍体株系与二倍体对照株系试管苗和田问叶片中SOD、POD及APX酶的活性,为选育抗性强的白花除虫菊优良四倍体品种提供理论依据。[方法]先加入预冷的提取缓冲液(pH=7.5,0.05 mol/L Tris-Hcl缓冲液)冷冻离心提取叶片中的各酶液。蛋白含量和SOD活性的测定按南京建成生物工程研究所试剂盒说明书操作。POD总活力测定:100μl酶液用Tris-HCl(pH=7.0)溶液稀释至1 ml,准确加入0.1%的愈创木酚1 ml,30℃水浴10 min后,立即测定溶液在470 nm处的吸光度,然后准确加入0.08%的H_2O_2 100μl,充分混匀,反应2 min时立即测定溶液在470 nm处的吸光度,求出反应前后的吸光度差值。酶的比活力用ΔOD_(470)/(mg·min)表示。APX活性测定:3 ml反应液中含50.0mmol/L PBS(pH=7.0),0.1 mmol/L EDTA,0.1 mmol/LH_2O_2,0.5 mmol/LASA,最后加入100μl酶液。在室温下记录10 s,30 s时290 nm处吸光度的变化。酶活单位定义为每小时氧化1μmol ASA的酶量为一个酶活单位。[结果]四倍体株系与二倍体对照株系比较,其保护酶系统中各种抗氧化酶的活性都能得到普遍提高,并且各株系的田间样品与试管苗样品的各抗氧化酶活性呈正相关,因此可以应用组织培养技术在育种的初期试管苗阶段进行早期筛选,缩短选育时间,节约人力物力。[结论]试管苗期间各株系的抗氧化酶活性可以作为筛选抗病抗逆性强的白花除虫菊优良株系的参考指标。
[Objective] The research aimed to determine the activities of SOD, POD and APX in vitro of Tetraploid and Tetraploid control plants of Pyrethrum pyogenes and Diplocum sp., And to provide theoretical basis for the selection of tetraploid Tetraploids . [Method] The enzyme solution in the leaves was extracted by freezing and centrifuging with precooling extraction buffer (pH = 7.5,0.05 mol / L Tris-Hcl buffer). Determination of protein content and SOD activity according to Nanjing Institute of Bioengineering kit instruction manual operation. POD total activity determination: 100μl enzyme solution was diluted to 1 ml with Tris-HCl (pH = 7.0) solution, 0.1% guaiacol accurately added 1 ml, 30 ℃ water bath for 10 min, the solution immediately measured at 470 nm Absorbance, and then accurately add 0.08% H_2O_2 100μl, and mix well, the reaction was measured immediately after 2min at 470 nm absorbance, to find the difference between the absorbance before and after the reaction. The specific activity of the enzyme is expressed as ΔOD - (470) / (mg · min). APX activity assay: 50.0 mmol / L PBS (pH = 7.0), 0.1 mmol / L EDTA, 0.1 mmol / LH 2 O 2 and 0.5 mmol / L ASA were added to 3 ml of reaction solution. The absorbance at 290 nm at 10 s was recorded at room temperature. An enzyme unit is defined as an enzyme unit that oxidizes 1 μmol of ASA per hour. [Result] Compared with the diploid control, the tetraploid and the diploid control showed that the activities of various antioxidant enzymes in the protective enzyme system were generally improved, and the antioxidant activity of each sample in field and in vitro Therefore, we can use tissue culture technology in the early stage of early breeding tube seedling stage screening, shorten the breeding time, saving manpower and resources. [Conclusion] The antioxidant enzyme activity of each line during in vitro plantlets could be used as a reference index for screening elite lines of Pyricularia oryzae with strong disease resistance and resistance.