谷氧还蛋白(AbGrx-1)对水稻干尖线虫在氧化胁迫下的保护作用

【目的】谷氧还蛋白(glutaredoxin, Grx)作为一种抗氧化酶，在通过清除多余活性氧来维持生物细胞氧化还原平衡、降低细胞膜损伤过程中发挥重要作用。水稻干尖线虫(Aphelenchoides besseyi)能在高温、渗透及氧化胁迫等多种逆境压力中存活。本研究旨在探究Grx在水稻干尖线虫抗氧化胁迫中的作用。【方法】通过cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)，获得了一个水稻干尖线虫谷氧还蛋白基因AbGrx-1，进行了序列比对和遗传进化分析；通过qRT-PCR检测了AbGrx-1在线虫响应氧化和温度胁迫中的表达差异，通过原核表达获得了AbGrx-1的重组蛋白，并分析了AbGrx-1蛋白浸泡对水稻干尖线虫在氧化和高温胁迫下存活的影响。【结果】AbGrx-1基因全长包括90 bp的5＇非翻译区(UTR)、321 bp的编码区和97 bp的3＇UTR，开发阅读框(横跨91至411位)编码106个氨基酸。AbGrx-1蛋白的第24至27位点具有谷氧还蛋白催化残基(CPYC)，分别在第69至第72和第83至第86位点存在保守的谷胱甘肽结合位点RSVP和GGDD，归为Ⅰ类谷氧还蛋白。遗传进化树显示AbGrx-1与燕麦真滑刃线虫(Aphelenchus avenae)Grx亲缘关系最近，位于同一进化分支。AbGrx-1在H2O2处理和12℃下时显著上调表达，但在0℃, 4℃, 37℃和45℃极端温度中下调表达。高浓度H2O2和高温导致水稻干尖线虫死亡率增加，AbGrx-1重组蛋白能显著提高暴露于高浓度H2O2中线虫的存活率，但不影响高温下线虫的存活率。【结论】AbGrx-1参与调控水稻干尖线虫的抗氧化免疫反应，在抵抗氧化损伤、维持线虫生存方面具有重要功能。

Protective Effect of Glutaredoxin (AbGrx-1) on Aphelenchoides besseyi Under Oxidative Stress

【Objective】The antioxidant glutaredoxin (Grx) plays a crucial role in regulating intracellular redox homeostasis via scavenging of excess reactive oxygen species. The white-tip nematode Aphelenchoides besseyi can survive in adverse environments including high temperature, osmosis and oxidative stresses. To reveal the antioxidant function of Grx in A. besseyi, 【Method】herein the full-length cDNA of glutaredoxin (named AbGrx-1) from A. besseyi was cloned by using rapid amplification of cDNA ends (RACE), and AbGrx-1 protein and evolutionary relationship were characterized; differential gene expression level was detected in the nematodes under oxidant and temperature stresses by using quantitative real-time PCR; the effect of the recombinant AbGrx-1 protein on A.besseyi survival was also tested.【Result】The full-length AbGrx-1 cDNA contains a 5' UTR of 90, an ORF of 321 bp encoding a polypeptide of 106 amino acids and a 3' UTR of 97 bp. The deduced amino acid sequence of AbGrx-1 shares a high similarity with other nematodes’ Grxs, and the catalytic residue (CPYC) and glutathione binding sites (RSVP and GGDD) indicate that AbGrx-1 is categorized into Class I Grx. The phylogenetic tree showed AbGrx-1 is located in the same clade with the plant parasitic nematode Aphelenchus avenae. AbGrx-1 mRNA is highly induced in A. besseyi exposed to H2O2 solution and 12℃, but is suppressed in the nematodes at 0, 4, 37 and 45℃. The high concentration H2O2 solutions and high temperature are adverse to survival of A. besseyi, but the survival rate increases with the nematodes pre-soaked in AbGrx-1 recombinant protein solution. 【Conclusion】AbGrx-1 is required for A. besseyi antioxidative immunity, and plays an essential role in overcoming oxidative damage and nematode survival.