毛白杨两个Phi类GST基因的克隆及生化特性

doi:10.3724/SP.J.1259.2012.00248

植物学报 ›› 2012, Vol. 47 ›› Issue (3): 248-256.DOI: 10.3724/SP.J.1259.2012.00248

毛白杨两个Phi类GST基因的克隆及生化特性

李迪¹, 唐振鑫¹, 刘海静², 曾庆银², 杨海灵^1*

¹北京林业大学生物科学与技术学院, 北京 100083
²中国科学院植物研究所, 系统与进化植物学国家重点实验室, 北京 100093

收稿日期:2011-10-17 修回日期:2012-02-17 出版日期:2012-05-01 发布日期:2012-07-06
通讯作者: 杨海灵
基金资助:
国家重点基础研究发展计划

Molecular Characterization of Two Phi Glutathione S-transferases from Populus tomentosa

Di Li¹, Zhenxin Tang¹, Haijing Liu², Qingyin Zeng², Hailing Yang^1*

¹College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China

²State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

Received:2011-10-17 Revised:2012-02-17 Online:2012-05-01 Published:2012-07-06
Contact: Hailing Yang

摘要/Abstract

摘要： 谷胱甘肽转移酶(glutathione S-transferase, GST)在植物的抗逆反应、细胞信号转导和抗病等方面发挥着重要作用。从毛白杨(Populus tomentosa)中克隆到2个Phi类GST基因(PtoGSTF1和PtoGSTF2), 它们分别编码215和218个氨基酸残基的蛋白质。DNA序列分析显示这2个基因均含有2个内含子。组织表达模式分析表明, 这2个基因在毛白杨的根、茎、叶、韧皮部和顶芽组织中均表达, 是组成型表达基因。在大肠杆菌中表达这2个基因并纯化重组蛋白。酶活性分析显示PtoGSTF1和PtoGSTF2蛋白对底物CDNB、NBD-Cl、NBC和Cum-OOH都具有酶学活性, 但活性差异较大。动力学分析显示, PtoGSTF2对NBD-Cl的亲和力是PtoGSTF1的2.5倍, 但PtoGSTF1的催化效率是PtoGSTF2的56.8倍。热力学稳定性分析显示, PtoGSTF1比PtoGSTF2具有更高的热力学稳定性。因此, 酶学性质的差异预示着这2个Phi类GST基因可能存在功能上的分化。

Abstract: Plant glutathione S-transferases (GSTs) play important roles in stress tolerance and detoxification metabolism. We cloned two Phi GST genes (PtoGSTF1 and PtoGSTF2) from Populus tomentosa. PtoGSTF1 and PtoGSTF2 encode a protein of 215 and 218 amino acid residues, with a calculated molecular mass of 24.32 and 24.57 kDa, respectively. Genomic sequence analysis showed that PtoGSTF1 and PtoGSTF2 contained 2 introns. RT-PCR revealed PtoGSTF1 and PtoGSTF2 constitutively expressed in P. tomentosa. Recombinant PtoGSTF1 and PtoGSTF2 proteins were overexpressed in E. coli and purified by Ni-affinity chromatography. PtoGSTF1 and PtoGSTF2 showed enzymatic activities towards the substrates CDNB, NBD-Cl, NBC and Cum-OOH. The affinity to substrate NBD-Cl was 2.5-fold higher for PtoGSTF2 than PtoGSTF1, but the catalytic efficiency to NBD-Cl was 56.85-fold greater for PtoGSTF1 than PtoGSTF2. PtoGSTF1 showed greater thermal stability than PtoGSTF2. These results indicate the functional divergence between PtoGSTF1 and PtoGSTF2.

李迪, 唐振鑫, 刘海静, 曾庆银, 杨海灵. 毛白杨两个Phi类GST基因的克隆及生化特性. 植物学报, 2012, 47(3): 248-256.

Di Li, Zhenxin Tang, Haijing Liu, Qingyin Zeng, Hailing Yang. Molecular Characterization of Two Phi Glutathione S-transferases from Populus tomentosa. Chinese Bulletin of Botany, 2012, 47(3): 248-256.

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毛白杨两个Phi类GST基因的克隆及生化特性

Molecular Characterization of Two Phi Glutathione S-transferases from Populus tomentosa

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