日本落叶松谷胱甘肽过氧化物酶(GPX)酶学特性与抗逆性研究  

Studies on enzymatic properties and stress resistance of glutathione peroxidase(GPX) from Larix kaempferi

作　　者：张萌 姜文婷 王雪纯 蒋湘宁[1] 盖颖[1] Zhang Meng;Jiang Wenting;Wang Xuechun;Jiang Xiangning;Gai Ying(School of Biological Sciences and Biotechnology,Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration,National Engineering Laboratory for Tree Breeding,Beijing Forestry University,Beijing 100083,China)

机构地区：[1]北京林业大学生物科学与技术学院,树木花卉育种生物工程国家林业和草原局重点实验室,林木育种国家工程实验室,北京100083

出　　处：《北京林业大学学报》2023年第2期78-86,共9页Journal of Beijing Forestry University

基　　金：国家科技专项转基因落叶松新品种培育及产业化研究(2018ZX08020003-001-002)。

摘　　要：【目的】植物谷胱甘肽过氧化物酶(GPX)是酶促活性氧清除机制中的关键酶之一,对日本落叶松GPX开展酶学特性与抗逆性研究,可以补充和完善林木改良基因资源,也为探究谷胱甘肽过氧化物酶抗逆的分子机制提供理论依据。【方法】本研究以日本落叶松茎部组织为材料克隆谷胱甘肽过氧化物酶基因,进行生物信息学分析和亚细胞定位研究。诱导、纯化目的蛋白进行体外酶学活性测定,通过点斑试验、生长曲线试验验证GPX抗逆性。【结果】从日本落叶松茎中克隆得到了2个GPX基因,分别命名为LkGPX2、LkGPX3,二者都含有完整的特征保守基序。构建系统进化树发现,LkGPXs和具有抗逆功能的PmGPX与PeGPX聚为一支。亚细胞定位显示,LkGPX2和LkGPX3蛋白在细胞核和细胞质中均有表达。以硫氧还蛋白(Trx)为电子供体进行体外酶学活性测定,LkGPX2与LkGPX3对过氧化氢(H2O2)、过氧化氢叔丁醇(t-BHP)和有机氢过氧化物(Cum-OOH)都具有催化活性,LkGPX2对于3种底物的催化活性分别为(0.402±0.037)、(0.424±0.018)、(0.425±0.009)U/mg,LkGPX3对于3种底物的催化活性分别为(0.397±0.027)、(0.449±0.028)、(0.407±0.021)U/mg。大肠杆菌体外逆境处理试验表明,LkGPX2、LkGPX3能够提高大肠杆菌对模拟干旱胁迫和盐胁迫的耐受性。【结论】日本落叶松谷胱甘肽过氧化物酶(GPX)既能起到活性氧清除作用,同时具备一定应对非生物胁迫的能力。[Objective]Glutathione peroxidase(GPX)is one of the key enzymes in the plant’s enzymatic active oxygen scavenging mechanism.Conducting enzymatic characteristics and stress resistance research on Larix kaempferi GPX can supply and improve genetic resources and provide theoretical basis for exploring the molecular mechanism of glutathione peroxidase stress resistance.[Method]The glutathione peroxidase genes were cloned using the stem tissue of Larix kaempferi as a template.In this study,bioinformatics analysis and subcellular localization research were conducted.The GPXs were induced and purified for in vitro enzymatic assays,and verified to have the stress resistance through spotting experiment and growth curve experiment.[Result]Two GPX genes were cloned from the stem of Larix kaempferi,named LkGPX2 and LkGPX3,both of which contained complete characteristic conserved motifs.LkGPXs were grouped together with the PmGPX and PeGPX in a phylogenetic tree,which were stress-resistant.Subcellular localization showed that both LkGPX2 and LkGPX3 proteins were localized in the nucleus and cytoplasm.Using thioredoxin(Trx)as an electron donor for in vitro enzymatic activity,LkGPX2 and LkGPX3 had catalytic activity on H2O2,t-BHP and Cum-OOH.The catalytic activities of LkGPX2 were(0.402±0.037)U/mg,(0.424±0.018)U/mg,(0.425±0.009)U/mg,and the catalytic activities of LkGPX3 for the three substrates were(0.397±0.027)U/mg,(0.449±0.028)U/mg,and(0.407±0.021)U/mg,respectively.Moreover,the results of stress treatment experiment indicated that LkGPX2 and LkGPX3 could improve the tolerance of Escherichia coli to simulated drought stress and salt stress.[Conclusion]Larix kaempferi glutathione peroxidase(GPX)is capable of scavenging active oxygen as well as responding to abiotic stresses.

关 键 词：日本落叶松 谷胱甘肽过氧化物酶 亚细胞定位 酶学性质 抗逆 

分 类 号：S791.223[农业科学—林木遗传育种]