分子对接阐明草甘膦与水稻醛酮还原酶(OsALR2)的作用及OsALR2的表达纯化

Interaction between glyphosate and OsALR2 protein in rice based on molecular docking and recombinant expression and purification of OsALR2

草甘膦是一种广谱高效的有机磷类除草剂,因其对水稻等禾本科作物缺乏选择性,导致其在水稻田中的应用受到限制。杂草中醛酮还原酶(aldo-keto reductase,AKR)具有降解小分子农药草甘膦的功能,但是水稻响应草甘膦的作用机制未知。本文通过生物信息学分析、分子对接研究草甘膦与其在水稻中的靶标AKR蛋白OsALR2的互作机制。采用TMHMM 2.0和DiANNA 1.1 web server等在线软件对OsALR2蛋白的跨膜特性和二硫键含量等生物学特性进行了分析,结果表明OsALR2分子量为35 kD,等电点为5.89;OsALR2不是膜蛋白,但是含有3组二硫键。使用AlphaFold预测OsALR2蛋白的三级结构,并利用Auto Dock Vina将草甘膦与OsALR2进行分子对接,结果显示,最低结合能为-13.4 kcal/mol,草甘膦与OsALR2的结合主要通过氢键作用。通过Ramachandran图,ERRAT和Verify 3D证明对接获得的三维结构是合理的。进一步通过分子克隆技术构建重组表达载体pET-32a-OsALR2并进行原核表达,结果显示OsALR2主要以包涵体的形式表达,对包涵体进行变复性后,蛋白表现出酶活性且蛋白结构有较好的均一性。本文的研究结果助于阐明草甘膦与水稻蛋白互作的分子机制,为研发抗草甘膦水稻新品种和新型小分子农药提供理论指导。

The use of glyphosate,a broad-spectrum and highly effective organophosphorus herbicide,is limited in paddy fields due to its lack of selectivity in killing gramineous crops such as rice.Aldo-keto reductase(AKR)in weeds can degrade glyphosate,but the mechanism by rice responds to glyphosate is unknown.The target and action mechanism of glyphosate and rice AKR protein OsALR2 were studied by bioinformatics analysis and molecular docking.The online software TMHMM 2.0 and DiANNA 1.1 web server were used to determine the physical and chemical properties,transmembrane properties and the disulfide bond content of OsALR2 protein.The results showed that the molecular weight of OsALR2 was 35 kD,with an isoelectric point of 5.89.OsALR2 was not a membrane protein but contained three disulfide bonds.AlphaFold was used to predict the tertiary structure of OsALR2 protein,and glyphosate was docked with OsALR2 by Auto Dock Vina.The results showed that the lowest binding energy was-13.4 kcal/mol and the binding of glyphosate to OsALR2 was mainly through hydrogen bonds.Analyses using Ramachandran diagram,ERRAT and Verify 3D suggested that the three-dimensional structure obtained by docking was reasonable.The recombinant expression vector pET32a-OsALR2 was constructed by molecular cloning technology.OsALR2 mainly existed in inclusion bodies.The inclusion body showed enzyme activity and homogeneity after denaturation and then renaturation.Our study helps to clarify the molecular mechanism of the interaction between glyphosate and OsALR2 protein in rice,and provides a theoretical guidance for the development of new glyphosate-resistant rice varieties and novel small molecule pesticides.