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

作　　者：潘翔[1] 李娜[1] 李印军[1] 魏弘宜[1] 张蕾[1] 陆海[1] 

机构地区：[1]北京林业大学生物科学与技术学院,林木花卉遗传育种教育部重点实验室

出　　处：《北京林业大学学报》2013年第1期36-44,共9页Journal of Beijing Forestry University

基　　金：国家自然科学基金项目(31170574);教育部新世纪优秀人才支持计划项目(NCET-08-0730);教育部科学技术研究重点项目(106039)。

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 864bp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31.78ku,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℃,pH7.0～7.4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。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 31. 78 ku. The carboxyl terminal of PtAPX2 contained a putative transmembrane domain,which might be responsible for its peroxisomal sub-cellular 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 K m values to substrate ascorbate and H2O2 were determined to be （ 1.37±0.22 ） and （0.026±0.003 ） mmol / L,respectively. The V max 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 real-time 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 tissue-specific expression of PtAPX2 together expand our knowledge on the mechanism underlying oxidative stresses resistance in woody plants.

关 键 词：毛白杨 抗坏血酸过氧化物酶 克隆 重组表达 酶学性质 表达量分析 

分 类 号：S792.119[农业科学—林木遗传育种] Q943.2[农业科学—林学]