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为了揭示2-酮戊二酸(2-OG)感应蛋白OsGlnB在水稻根部铵同化碳架调节中的功能,本文采用2-OG、草酰乙酸(OAA)或丙酮酸(PYR)营养液处理水稻(Oryza sativa L.cv.9311)幼苗,检测根部谷氨酰胺合成酶(GS)活性和OsGlnB基因转录量。克隆表达并分离纯化根部OsGlnB蛋白,采用同源模建、分子对接与等温滴定鉴定该信号蛋白与碳架效应物的相互作用。结果表明所选碳架分子中只有2-OG和OAA促使根部GS活性与OsGlnB基因转录量同步上升,2-OG与ATP含量也相应升高。同源模建与分子对接表明OsGlnB蛋白与ATP-Mg~(2+)形成的复合物能有效结合2-OG或OAA,但不能结合PYR。等温滴定证明2-OG与上述复合物的结合常数大于OAA,未检测到丙酮酸热效应,结果与分子对接结论一致。本研究说明碳架效应物结合2-OG感应蛋白是这类碳营养信号调节水稻根部铵同化的必要条件。
In order to reveal the function of OsGln2, a 2-ketoglutarate (2-OG) sensing protein, in the regulation of ammonium assimilation carbon in roots of rice, rice plants were treated with 2-OG, OAA or PYR (Oryza sativa L.cv.9311) seedlings, the root glutamine synthetase (GS) activity and OsGlnB gene transcription were detected. The OsGlnB protein was cloned and purified, and the interaction between the signal protein and carbon-cage effectors was identified by homology modeling, molecular docking and isothermal titration. The results showed that only 2-OG and OAA in the selected carbon-cage molecules promoted the GS activity in roots to increase synchronously with the transcription of OsGlnB gene, and the content of 2-OG and ATP increased accordingly. Homology modeling and molecular docking showed that OsGlnB protein complexed with ATP-Mg ~ (2+) could effectively bind 2-OG or OAA but not PYR. Isothermal titration demonstrated that the binding constant of 2-OG to the above complex was greater than OAA, no pyruvate thermal effect was detected, and the results were consistent with the molecular docking. This study demonstrates that carbon-cage effectors binding to 2-OG-sensing proteins are essential for the regulation of ammonium assimilation in rice roots by such carbon nutrient signals.