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、�/sec> 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、�/sec> 结果从日本落叶松茎中克隆得到�?�?i>GPX基因，分别命名为 LkGPX2�?i>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（H ₂O ₂）、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�?.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�?.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、�/sec> 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、�/sec>

关键诌�
• 日本落叶杽�/a> /
• 谷胱甘肽过氧化物酵�/a> /
• 亚细胞定佌�/a> /
• 酶学性质/
• 抗逅�/a>

Abstract: ObjectiveGlutathione 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 kaempferiGPX can supply and improve genetic resources and provide theoretical basis for exploring the molecular mechanism of glutathione peroxidase stress resistance. MethodThe glutathione peroxidase genes were cloned using the stem tissue of Larix kaempferias 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. ResultTwo GPXgenes 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 H ₂O ₂, 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 colito simulated drought stress and salt stress. Conclusion Larix kaempferiglutathione peroxidase (GPX) is capable of scavenging active oxygen as well as responding to abiotic stresses.

Key words:
• Larix kaempferi/
• glutathione peroxidase/
• subcellular localization/
• enzymatic property/
• stress resistance

�?nbsp; 1日本落叶杽�i>LkGPX2不�i>LkGPX3基因克隆

M�? 000 bp DNA marker�?丹�i>LkGPX2的PCR扩增产物�?丹�i>LkGPX3的PCR扩增产物。M is 2 000 bp DNA marker; 1 is PCR amplification product ofLkGPX2; 2 is PCR amplification product ofLkGPX3.

Figure 1.LkGPX2 andLkGPX3 gene cloning ofLarix kaempferi

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�?nbsp; 2LkGPX2与LkGPX3氨基酸序列比寸�/p>

红色三角形标注的�?个Cys残基位点 。The red triangle marks the 3 Cys residue base sites.

Figure 2.Amino acid sequence alignment of LkGPX2 and LkGPX3

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�?nbsp; 3LkGPXs与其他物种GPX蛋白的系统进化树分析

At. 拟南芥；Pe. 胡杨；Pta. 油松；Pt. 毛果杨；Pm. 马尾松。At,Arabidopsis thaliana; Pe,Populus euphratica; Pta,Pinus tabuliformis; Pt,Populus trichocarpa; Pm,Pinus massoniana.

Figure 3.Phylogenetic analyses of LkGPXs and other species GPX protein

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�?nbsp; 4LkGPX2和LkGPX3亚细胞定佌�/p>

A、E、I为GFP荧光；B、F、J为荧光叠加；C、G、K为原生质体GFP荧光；D、H、L为原生质体荧光叠加。其中A、B、E、F、I、J标尺�?0 μm，C、D、K、L标尺�?5 μm，G、H标尺�?50 μm。A, E, I are GFP fluorescence; B, F, J are stacked fluorescence; C, G, K are GFP fluorescence of protoplast; D, H, L are stacked fluorescence of protoplast. The ruler of A, B, E, F, I, J is 50 μm; that of C, D, K, L is 25 μm and that of G, H is 250 μm.

Figure 4.Subcellular lolication of LkGPX2 and LkGPX3

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�?nbsp; 5LkGPX2、LkGPX3、Trx、TrxR蛋白纯化

A图中，M为标准蛋白marker�?为未诱导的pET28a空载�?为pET28a空载0.4 mmol/L IPTG诱导3 h�?为LkGPX2未诱导，4为LkGPX2 0.4 mmol/L IPTG诱导3 h�?为超声破碎后的上清液�?�?00 mmol/L咪唑洗脱的目的蛋白。B、C、D图中，M为标准蛋白marker�?为未诱导的重组载体，2�?.4 mmol/L IPTG诱导3 h�?为超声破碎后的上清液�?�?00 mmol/L咪唑洗脱的目的蛋白。In Fig. A, M is standard protein marker, 1 is uninduced pET28a blank vector, 2 is pET28a blank vector induced by 0.4 mmol/L IPTG for 3 h, 3 is uninduced LkGPX2, 4 is LkGPX2 induced by 0.4 mmol/L IPTG for 3 h, 5 is supernatant after ultrasound crushing, 6 is target protein eluted by 100 mmol/L imidazole. In Fig. B, C and D, M is standard protein marker, 1 is uninduced recombinant vector, 2 is induced by 0.4 mmol/L IPTG for 3 h, 3 is supernatant after ultrasound crushing, 4 is target protein eluted by 100 mmol/L imidazole.

Figure 5.Protein purification of LkGPX2, LkGPX3, Trx and TrxR

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�?nbsp; 6点斑试验结果

Figure 6.Experiment results of spots

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�?nbsp; 7生长曲线法测定结枛�/p>

A为pET28a空载不同条件处理；B为LkGPX2-pET28a不同条件处理；C为LkGPX3-pET28a不同条件处理；D为空白对照与0.8 mol/L山梨醇处理对比；E为空白对照与0.5 mol/L NaCl处理对比；F为空白对照与0.6 mol/L NaCl处理。A is different treatments on blank pET28a vector; B is different treatments on LkGPX2-pET28a; C is different treatments on LkGPX3-pET28a; D is comparison of blank control and0.8 mol/L sorbitol treatment; E is comparison of blank control and 0.5 mol/L NaCl treatment; F is comparison of blank control and 0.6 mol/L NaCl treatment.

Figure 7.Test results of growth curve method

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�?nbsp; 1LkGPX2不�i>LkGPX3基因的理化性质分析

Table 1.Physical and chemical property analysis ofLkGPX2 andLkGPX3

基因名称 Gene name	基因长度 Gene length/bp	编码氨基酸个�?br/>Number of coded amino acids/aa	分子野�br/>Molecular mass/kDa	等电炸�br/>Isoelectric point
LkGPX2	513	170	18.6	7.62
LkGPX3	513	170	19.0	5.34

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�?nbsp; 2LkGPX2与LkGPX3亚细胞定位预浊�/p>

Table 2.Prediction of subcellular localization of LkGPX2 and LkGPX3 protein

亚细胞定位可能�?br/>Possibility of subcellular localization	LkGPX2	LkGPX3
细胞� Cytoplasm	65.2%	60.9%
细胞� Nucleus	13.0%	13.0%
线粒� Mitochondria	8.7%	8.7%
液泡 Vacuole	4.3%	4.3%
分泌小泡 Secretory vesicle	4.3%	4.3%
高尔基体 Golgi apparatus	4.3%	4.3%
细胞骨架 Cytoskeleton		4.3%

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�?nbsp; 3LkGPX2和LkGPX3酶学活性测宙�/p>

Table 3.Enzymatic activity determination of LkGPX2 and LkGPX3

谷胱甘肽过氧化物� Glutathione peroxidase	酶学活� Enzymatic activity/（U·mg�?（�/td>
	过氧化氢 H2O2	叔丁基过氧化氡�br/> t-BHP	有机氢过氧化� Cum-OOH
LkGPX2	0.402 ± 0.037	0.419 ± 0.006	0.425 ± 0.009
LkGPX3	0.397 ± 0.027	0.449 ± 0.028	0.413 ± 0.012

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