毛白杨抗坏血酸过氧化物酶基因PtAPX2的克隆表达及分析
Cloning expression and characterization of an ascorbate peroxidase gene PtAPX2 from Populus tomentosa.
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摘要: 以毛白杨总RNA为模板反转录得到cDNA，克隆获得抗坏血酸过氧化物酶基因家族中的一个成员PtAPX2 864 bp的编码序列。该基因编码的蛋白包含287个氨基酸，理论分子质量为3178 ku，C末端包含锚定于过氧化物酶体跨膜结构域，推断其为过氧化物酶体定位蛋白。构建了PtAPX2原核表达载体，在大肠杆菌中表达获得了纯化的重组蛋白，并对其进行了酶学性质分析。PtAPX2对抗坏血酸的Km值为 (1.37±0.22) mmol/L，Vmax值为 (3.95±0.46) mmol/(L•min•mg)；对H2O2的Km值为 (0.026±0.003) mmol/L，Vmax值为 (1.27±0.03) mmol/(L•min•mg)；该酶在28 ℃，pH 7.0～7.4时活性最高。实时荧光定量PCR分析表明，PtAPX2在杨树老叶叶肉中表达量最高。对PtAPX2亚细胞定位、底物结合特征、酶学性质及组织特异性表达的分析加深了对木本植物抗氧化机理的认识。Abstract: A member of APX (ascorbate peroxidase) gene superfamily of Populus tomentosa was cloned from the cDNA of reverse transcribed RNA. The open reading frame (ORF) of PtAPX2 was 864 bp in length, and encoded a protein containing 287 amino acids with a molecular weight of 3178 ku. The carboxyl terminal of PtAPX2 contained a putative transmembrane domain, which might be responsible for its peroxisomal subcellular localization. In order to gain insights into the characteristics of this enzyme, the PtAPX2 protein was expressed in Escherichia coli and purified to homogeneity. Its enzyme activity was assayed in vitro. The Km values to substrate ascorbate and H2O2 were determined to be (1.37±0.22) and (0.026±0.003) mmol/L, respectively. The Vmax values to substrate ascorbate and H2O2 were (3.95±0.46) and (1.27±0.03) mmol/(L•min•mg), respectively. PtAPX2 exhibited the most potent activity at 28 ℃, pH 7.0-7.4. Quantitative assays using fluorescent realtime PCR revealed the highest transcription level of PtAPX2 in the mesophyll of aged leaves. The results of the prediction of subcellular localization, the pattern of substrate binding, enzyme kinetics and tissuespecific expression of PtAPX2 together expand our knowledge on the mechanism underlying oxidative stresses resistance in woody plants.
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期刊类型引用(3)
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2. 陈素素, 张嫚嫚, 于洪淼, 顾宸瑞, 魏优, 刘宇, 王朔, 陈肃, 刘桂丰. 转基因白桦不同杂交组合的种子活力测定及外源基因的遗传规律分析. 北京林业大学学报. 2016(01): 36-42 . 本站查看
3. 张蕾, 王昱堃, 刘蕾, 尹玢, 曹山, 陆海. 毛白杨细胞质抗坏血酸过氧化物酶基因的克隆及表达分析. 西北植物学报. 2014(02): 231-236 . 百度学术
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