根瘤菌中甲硫氨酸亚砜还原酶底物蛋白的筛选与相互作用验证

Screening and verification of substrate proteins of methionine sulfoxide reductase in rhizobia

摘要:

根瘤菌在侵染豆科植物过程中会受到活性氧的氧化胁迫,含甲硫氨酸的蛋白质易被氧化成甲硫氨酸亚砜导致蛋白结构和功能改变,甲硫氨酸亚砜还原酶(methionine sulfoxide reductases, Msrs)能将甲硫氨酸亚砜还原成甲硫氨酸,恢复蛋白的结构和功能。前期在华癸中慢生根瘤菌(Mesorhizobium huakuii) 7653R基因组中发现有4个Msrs和抗氧化压力密切相关,但其作用机制仍不清楚。【目的】通过筛选4个Msrs的相互作用底物,为阐明4个Msrs在M. huakuii 7653R中的作用机制提供证据。【方法】按照甲硫氨酸含量由高到低统计M. huakuii 7653R中所有蛋白的分布情况;利用蛋白互作网站预测获得4个Msrs的候选互作底物,将预测互作底物进行功能注释基因本体(gene ontology, GO)分析和京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)代谢通路分析;通过细菌双杂交初步验证它们之间的相互作用。【结果】甲硫氨酸含量百分数分布基本呈正态分布形式,位于中间百分数的蛋白最多,位于两边的蛋白较少;筛选获得有6个抗氧化酶和6个转录调控因子是4个Msrs的候选互作底物;细菌双杂交显示,有2个抗氧化酶和5个转录调控因子确实和4个Msrs存在不同程度的相互作用。【结论】为阐明Msrs在根瘤菌M. huakuii 7653R中抵抗氧化压力的作用机制提供了证据,为揭示根瘤菌抵抗活性氧提供了新的思路和方向。

关键词:活性氧;甲硫氨酸亚砜还原酶;根瘤菌;氧化胁迫;细菌双杂交;互作底物

Abstract:

Rhizobia would encounter oxidative stress of reactive oxygen species (ROS) in the process of infecting leguminous plants. Methionine-containing proteins are easy to be oxidized to methionine sulfoxide, leading to changes in protein structure and function. Methionine sulfoxide reductases (Msrs) can reduce methionine sulfoxide to methionine, restoring protein structure and function. We have identified four Msrs in the genome of Mesorhizobium huakuii 7653R that are involved in oxidative stress response, while the mechanism remains unclear. 【Objective】 To identify the substrates of four Msrs and elucidate the roles of the four Msrs in M. huakuii 7653R. 【Methods】 According to the methionine content, we determined the distribution of all the proteins in M. huakuii 7653R. Then, we used the online protein interaction prediction tools to predict the substrates of the four Msrs, and performed gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment for the predicted substrates. Finally, we verified the interaction between them by using the bacterial two-hybrid system. 【Results】 The methionine content followed the normal distribution, with most proteins in the middle and few proteins on both sides. Six antioxidant enzymes and six transcription factors were selected as the candidate substrates of the four Msrs. Finally, the bacterial two hybrid results showed that two antioxidant enzymes and five transcription factors can interact with the four Msrs to different degrees. 【Conclusion】 We provided the proof in illustrating the role of Msrs in the oxidative stress response to M. huakuii 7653R and provided a new idea for the research on the mechanism of rhizobia in response to ROS.

Key words:reactive oxygen species;methionine sulfoxide reductase;rhizobia;oxidative stress;bacterial two-hybrid system;interacting substrates