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

利用同源克隆技术得到1个毛白杨细胞质抗坏血酸过氧化物酶基因,命名为PcAPX。该基因编码249个氨基酸残基,预测分子量为33.01kD。采用原核表达技术在大肠杆菌中表达并纯化该蛋白并进行酶活性分析,结果表明:重组PcAPX蛋白对抗坏血酸(AsA)和过氧化氢(H2O2)有很高的活性,其对抗坏血酸的米氏常数(Km)和最大反应速度(Vmax)分别为(0.71±0.03)mmol·L-1和(0.41±0.02)mmol·L-1·min-1·mg-1;对过氧化氢的Km和Vmax分别为(0.60±0.21)mmol·L-1和(0.35±0.12)mmol·L-1·min-1·mg-1,表明PcAPX对AsA和H2O2拥有较高的催化底物的能力和催化效率。利用实时定量RT-PCR分析毛白杨PcAPX基因的表达模式,结果表明其在老叶中表达量高于新叶、韧皮部、形成层和根部。该研究结果将进一步促进毛白杨APX基因家族成员参与植物生长调控的研究。

Cloning and Expression of an Cytosolic Ascorbate Peroxidase Gene from Populus tomentosa

In this study,a Populus tomentosa cytosolic APX gene,named as PcAPX,was identified and characterized.The recombinant PcAPX protein,encoding a 249-residue protein with a calculated mass of 33.01 kD,showed high activity towards ascorbic acid (AsA) and H₂O₂.At fixed H₂O₂ concentration,the K_m and V_max values were (0.71±0.03) mmol·L^-1 and (0.41±0.02) mmol·L^-1·min^-1·mg^-1 for AsA.And at fixed AsA concentration,the K_m and V_max values were (0.60±0.21) mmol·L^-1 and (0.35±0.12) mmol·L^-1·min^-1·mg^-1 for H₂O₂.These data showed that the protein was active with AsA and H₂O₂ and had high catalytic efficiency.Real-time PCR analysis showed that the expression levels of PcAPX was higher in the mesophyll of aged leaves than that of other tissues.This research provided the theoretical support to investigate roles of APX gene family in plant growth regulation.