异型莎草AHAS不同位点发生突变对其功能的影响

doi:10.1016/j.jia.2023.04.009

摘要/Abstract

摘要：

异型莎草（Cyperus difformis L.）是莎草科莎草属一年生杂草，主要危害水稻田。从上世纪90年代开始，吡嘧磺隆用于防除水稻田杂草，属于乙酰羟酸合成酶（acetohydroxyacid synthase，AHAS）抑制剂中的磺酰脲类除草剂。近年来，这类除草剂连年施用引起杂草抗性问题愈发严重，其中包括异型莎草，而靶标突变是导致异型莎草对AHAS抑制剂产生抗性的主要原因。本研究以异型莎草Pro197Arg、Pro197Ser、Pro197Leu、Asp376Glu、Trp574Leu 5种AHAS突变型和1种野生型为研究对象，通过整株生物测定、AHAS离体活性、AHAS与底物亲和力及AHAS对支链氨基酸反馈抑制敏感性的测定，明确AHAS不同位点发生突变时，对吡嘧磺隆敏感性及酶功能的影响。主要研究结果为：（1）通过整株生物测定明确了不同突变型对吡嘧磺隆的抗性水平不同，其中，Trp574Leu突变型对吡嘧磺隆的抗性水平最高。（2）离体AHAS活性测定表明，突变明显降低了AHAS对吡嘧磺隆的敏感性，野生型及Asp376Glu、Pro197Leu、Pro197Arg、Pro197Ser、Trp574Leu 5种突变型AHAS的I₅₀分别为0.04、3.98、11.5、40.38、38.19及311.43 μmol L^-1，其中Trp574Leu突变型对吡嘧磺隆的敏感性最低。（3）测定了不同突变型AHAS的动力学参数，结果显示，与野生型AHAS相比，Asp376Glu、Pro197Leu、Trp574Leu及Pro197Ser突变型的Km值显著下降，表明其与底物的亲和力增加，而Pro197Arg突变型的Km值上升，表明其与底物的亲和力下降；Asp376Glu、Pro197Ser、Pro197Arg及Trp574Leu的最大反应速率Vmax与野生型AHAS相比显著增加，但Pro197Leu突变型的Vmax没有显著变化。（4）支链氨基酸对不同突变型AHAS活性的反馈抑制结果表明，与敏感型AHAS相比，Pro197Arg突变型AHAS活性受到更强的抑制，而Asp376Glu、Pro197Leu、Trp574Leu及Pro197Ser突变型AHAS活性受到的抑制作用小。

综上，本文通过AHAS催化活性、动力学及反馈抑制调节的研究，明确了异型莎草AHAS在不同位点发生突变对其功能的影响。

Abstract:

Cyperus difformis L. is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase (AHAS) inhibitors. It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis. However, the effect of different mutations on AHAS function is not clear in Cyperus difformis. To confirm the effect of mutations on AHAS function, six biotypes were collected, including Pro197Arg, Pro197Ser, Pro197Leu, Asp376Glu, Trp574Leu and wild type, from Hunan, Anhui, Jiangxi and Jiangsu provinces, China and the function of AHAS was characterized. The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl, in which the I₅₀ (the half maximal inhibitory concentration) of wild type AHAS was 0.04 μmol L^–1 and Asp376Glu, Pro197Leu, Pro197Arg, Pro197Ser and Trp574Leu mutations were 3.98, 11.50, 40.38, 38.19 and 311.43 μmol L^–1, respectively. In the determination of enzyme kinetics parameters, the Km and the maximum reaction velocity (Vmax) of the wild type were 5.18 mmol L^–1 and 0.12 nmol mg^–1 min^–1, respectively, and the Km values of AHAS with Asp376Glu, Trp574Leu, Pro197Leu and Pro197Ser mutations were 0.38–0.93 times of the wild type. The Km value of the Pro197Arg mutation was 1.14 times of the wild type, and the Vmax values of the five mutations were 1.17–3.33-fold compared to the wild type. It was found that the mutations increased the affinity of AHAS to the substrate, except for the Pro197Arg mutation. At a concentration of 0.0032–100 mmol L^–1 branched-chain amino acids (BCAAs), the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased, except for the Pro197Arg mutation. This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.

Key words: acetohydroxyacid synthase (AHAS) , mutation , enzyme function , Cyperus difformis

. 异型莎草AHAS不同位点发生突变对其功能的影响[J]. Journal of Integrative Agriculture, 2024, 23(1): 177-186.

Xiaotong Guo, Xiangju Li, Zheng Li, Licun Peng, Jingchao Chen, Haiyan Yu, Hailan Cui. Effect of mutations on acetohydroxyacid synthase (AHAS) function in Cyperus difformis L.[J]. Journal of Integrative Agriculture, 2024, 23(1): 177-186.

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