费氏中华根瘤菌NodD2蛋白与植物类黄酮互作的生化机制研究  

Biochemical mechanism of the interaction between Sinorhizobium fredii NodD2 and plant flavonoids

作　　者：陈敏 朱辉[1] 曹扬荣 CHEN Min;ZHU Hui;CAO Yangrong(State Key Lab of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;Hubei Hongshan Laboratory,Wuhan 430070,China)

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070 [2]湖北洪山实验室,武汉430070

出　　处：《生命的化学》2022年第7期1396-1404,共9页Chemistry of Life

基　　金：科技部重点研发计划项目(2019YFA0904703);湖北省自然基金重点项目(2020CFA008)。

摘　　要：为研究大豆根瘤菌Sinorhizobium fredii HH103 NodD2蛋白与豆科植物分泌的多种类黄酮(包括柚皮素、染料木素、大豆苷元和木犀草素)的体外相互作用,并解析NodD2蛋白与不同类黄酮结合的关键氨基酸位点,在NodD2蛋白C端选择4段氨基酸序列进行截短,利用等温滴定量热法(ITC)在体外分别测定多种截短NodD2蛋白与类黄酮小分子互作产生的热量变化,根据计算机绘制的热量图谱得到两者反应的计量系数比、平衡常数和焓变化,以此分析NodD2蛋白与类黄酮小分子互作的强弱。使用三亲本杂交技术得到敲除突变体菌株S. fredii HH103ΩnodD2基因,并将不同突变形式的NodD2进行回补,基于大豆接种并观测根瘤固氮表型。结果显示,全长NodD2蛋白与柚皮素、染料木素和大豆苷元都有较强的结合能力,与木犀草素的结合能力很弱。4个截短NodD2蛋白对不同的类黄酮的敏感性不同,截短NodD2蛋白与类黄酮结合产生的热量变化相较于全长NodD2蛋白与类黄酮结合产生的热量变化较低。以上结果表明,NodD2蛋白作为直接受体结合类黄酮化合物,其C端氨基酸序列128F-132P、146V-152P、192S-198P和246P-249L在与类黄酮的结合中起关键作用。This work aims to study the interaction between NodD2 protein from Sinorhizobium fredii HH103and several flavonoids secreted by legumes, including naringenin, genistein, daidzein and luteolin, and analyze the key residues of NodD2 required for the binding to different flavonoids. Four residues located at the Cterminus of NodD2 protein were used for creation of truncated mutations and functional study. Isothermal titration calorimetry(ITC) was used to measure the heat changes generated by the interaction of different truncated NodD2 proteins with flavonoid molecules. The stoichiometric coefficient ratio, equilibrium constant and enthalpy change from the heat map, which were used to analyze the strength of the interaction between NodD2 protein and flavonoid molecules. The S. fredii HH103ΩnodD2 mutant strain was generated using a triparental cross. All the wild type and different truncated version of NodD2 genes were expressed in S. fredii HH103ΩnodD2 for assay in soybean. The full-length NodD2 protein has strong binding ability to naringenin,genistein and daidzein, but not luteolin. Four truncated NodD2 proteins have different sensitivities to different flavonoids. Caloric change produced by the combination of truncated NodD2 protein to flavonoids is lower than that produced by the combination of full-length NodD2 protein and flavonoids. All these data strongly indicate that NodD2 is a receptor protein with high binding affinity to flavonoids, with residues at the Cterminal amino acid sequences 128F-132P, 146V-152P, 192S-198P and 246P-249L of NodD2 plays essential role for this binding.

关 键 词：共生固氮 类黄酮 等温滴定量热法 NodD蛋白 

分 类 号：Q945.13[生物学—植物学]