摘要
采用盆栽方法研究了氮素形态对不同专用型小麦开花后氮素同化关键酶活性及籽粒蛋白质含量的影响。结果表明:不同专用型小麦氮素同化关键酶硝酸还原酶、谷氨酰胺合成酶和谷氨酸合酶对氮素形态的反应不同。强筋小麦豫麦34施用酰胺态氮对旗叶硝酸还原酶和谷氨酰胺合成酶活性、籽粒谷氨酰胺合成酶和谷氨酸合酶活性具有明显的促进作用,最终籽粒蛋白质含量较高;中筋小麦豫麦4 9在施用铵态氮时,3种氮素同化关键酶活性均有较大增强,籽粒蛋白质含量最高;弱筋小麦豫麦5 0硝酸还原酶活性以铵态氮处理最高,而籽粒和旗叶谷氨酰胺合成酶和谷氨酸合酶活性在酰胺态氮处理下明显增强,酰胺态氮对籽粒中蛋白质含量的增加具有明显的促进作用。相关性分析表明,籽粒蛋白质含量与旗叶GS活性和籽粒GOGAT活性呈显著或极显著正相关,与旗叶NR活性和GS活性。
In order to understand the effects of nitrogen form on key enzyme activity involved in nitrogen metabolism and grain protein content of speciality wheat cultivars, pot experiments were carried out at experimental station of Henan Agricultural University during 2000～2002. Soil containing 9.8 g/kg organic matter, 0.986 g/kg total N, 25.43 mg/kg olsen-p and 259 mg/kg NH_4OAc-K was used in the experiments. 18kg of sieved soil was placed in each 30cm×40cm pot. Three cultivars were used in experiments including Yumai34 (a strong gluten cultivar), Yumai49 (medium gluten) and Yumai50 (weak gluten). Nitrogen forms studied were NO~^-__3-N (NaNO_3), NH_4-N (NH_4HCO_3) and CONH_4-N (urea). Prior to sowing, 3.5 g N, 3.3 g K_2O and 2.9 g P_2O_5 per pot were applied and 1.6 g N was applied to each pot during the elongation stage. Seven plants from each pot were selected when plants had five leaves. The experiment was arranged in a completely random design with eight replications and all pots were managed in the same way. The NR activity of flag leaves from main stems were measured at 10、15、20、25 and 30days after flowering using the method of living body. GS and GOGAT in grains and flag leaves were extracted in the 100mmol/L Tris-HCl (pH 7.6) extraction buffer containing 1.0mmol/L EDTA, 1.0mmol/L MgCl_2·6H_2O and 10mmol/L 2-mercaptoethanol, and were used for the assay of enzyme activity. The synthetase activity of GS in extracts was determined in a reaction mixture containing imidazole-muriatic acid buffer (0.25mol/L, pH 7.0) 0.6ml,glutamic acid-Na (0.30mol/L, pH 7.0) 0.4ml,ATP-Na(30 mmol/L, pH 7.0) 0.4ml,MgSO_4 (0.5mol/L) 0.2ml and crude GS solution 1.2ml, after the mixture was incubated at 37℃ for 15min, the reaction was terminated by adding acidic FeCl_3 (2%(W/V) TCA and 3.5% (W/V) FeCl_3 in 2% HCl). Production of γ-glutamylhydroxamate was measured with a spectrophotometer at 540nm. One unit of GS activity was the enzyme catalyzing the formation of 1μmol γ-glutamylhydroxamate/min at 37℃, the whole GS activity was determined by the μ mol sum of γ- glutamylhydroxamate catalyzed by the whole enzyme per gram fresh material in 15min at present condition. The assay of GOGAT activity were carried out in reaction mixture containing L-Glutamine(20mmol/L) 0.4ml, α-ketoglutaric acid (20mmol/L) 0.5ml, KCL(10mmol/L) 0.1ml, NADH(3mmol/L) 0.2mL and crude enzyme solution 0.3ml, the total volume is 3.0ml by being added with Tris-HCl buffer(pH 7.6,1.5ml). After reaction beginning, the amount of NADH was measured with 752 ultraviolet -spectrophotometer at 340nm, the OD value was noted per 30 seconds and eleven times continually, the enzyme activity was determined with the steadily decreasing segment of the OD value. One unit of GOGAT activity was the amount of the enzyme catalyzing the decrease of 1 μ mol NADH/min in the reaction mixture at 30℃. After being milled, the grain protein was deposited in water with 5% AcCl_3 at 90℃, and the protein content in grain were measured with the way of semimicro Kjeldahl. The results showed that the key enzymes involved in nitrogen assimilation, NR, GS and GOGAT, responded differently to N forms due to the speciality wheat cultivars. The NR and GS in leaves, GS and GOGAT in grains of Yumai34 were promoted greatly by CONH_4-N, and the protein content in grains was highest at last. When NH_4-N was applied to Yumai49, the activity of key enzymes involved in nitrogen assimilation were increased evidently, and the grains kept the highest protein content. Applied with NH_4-N, NR activity of Yumai50 was the highest, but the activities of GS and GOGAT in grains and leaves were increased by CONH_4-N treatment, and applying CONH_4-N could increase the grains protein content clearly. Grain protein content was closely related to GS activity in flag leaves and GOGAT activity in grains after flowering, but not to NR and GOGAT activity in flag leaves and GS activity in grains.
出处
《生态学报》
CAS
CSCD
北大核心
2005年第4期802-807,共6页
Acta Ecologica Sinica
基金
河南省高校杰出科技人才创新工程基金资助项目 (2 0 0 2 KJCX0 5 )
教育部博士点基金资助项目 (2 0 0 40 4660 0 3 )~~
关键词
氮素形态
专用小麦
硝酸还原酶
谷氨酰胺合成酶
谷氨酸合酶
蛋白质含量
nitrogen forms
wheat cultivars with specialized end-uses
nitrate reductase
glutamine synthetase
glutamate synthase
protein content
