《植物生理学报》 2017, 53(11): 1971-1978

水稻根部2-酮戊二酸感应蛋白OsGlnB的铵同化碳架结合特性

郭继林¹, 曹倩文², 袁永泽^2,*, 郑勇^1,2,*

¹新疆师范大学生命科学学院, 新疆特殊环境物种保护与调控生物学重点实验室, 乌鲁木齐830054; ²华中师范大学生命科学学院, 湖北省遗传调控与整合生物学重点实验室, 武汉430079

通信作者：袁永泽；E-mail: yuan_yongze@163.com; zhengyong0720@163.com

摘　要：

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-Mg²⁺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。

关键词：OsGlnB; 碳架效应物; 等温滴定; 根部铵同化; 水稻

收稿：2017-08-09   修定：2017-09-28

资助：新疆特殊环境物种保护与调控生物学重点实验室项目(XJDX1414-2015-01)和国家自然科学基金(31101595)。

Binding characteristics of ammonium-assimilation-relating carbon skeletons with rice root 2-oxoglutarate (2-OG)-sensing protein OsGlnB

GUO Ji-Lin¹, CAO Qian-Wen², YUAN Yong-Ze^2,*, ZHENG Yong^1,2,*

¹Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology, College of Life Science, Xinjiang Normal University, Urumqi 830054, China; ²Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, China

Corresponding author: YUAN Yong-Ze; E-mail: yuan_yongze@163.com; zhengyong0720@163.com

Abstract:

To investigate the function of 2-oxoglutarate (2-OG)-sensing protein OsGlnB in the carbon-skeleton regulation of rice root ammonium assimilation, rice (Oryza sativa L. cv. 9311) seedlings were treated with 2-OG, oxaloacetate (OAA), or pyruvate (PYR) nutrient solution to determine glutamine synthetase (GS) activity and OsGlnB transcriptional abundance in roots. We further cloned root OsGlnB, expressed and purified the recombinant protein, and performed homolog modeling, molecular docking simulation and isothermal titration calorimetry (ITC) analysis to characterize molecular interaction(s) between this signaling protein and the putative carbon skeleton effectors. The results showed that among the carbon skeletons selected, 2-OG and OAA were able to synchronously increase root GS activity and OsGlnB transcriptional abundance, and 2-OG and ATP contents were also accordingly enhanced. Homolog modeling and molecular docking simulation indicated effective interactions of OsGlnB-ATP-Mg²⁺ complex with 2-OG and OAA, rather than PRY. ITC results well agreed with those of molecular docking simulation, revealing the higher binding constant (K_a) of 2-OG than OAA and no thermal effect observed from PYR-OsGlnB interaction. The present study suggested the binding of carbon skeleton effector with 2-OG-sensing protein was required for the carbon-signal regulation of rice root ammonium assimilation.

Key words: OsGlnB; carbon skeleton effectors; isothermal titration calorimetry; root ammonium assimilation; rice (Oryza sativa)