Department of Environmental Science and Engineering, Huaqiao University, Xiamen 361021, China; Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China.
Department of Environmental Science and Engineering, Huaqiao University, Xiamen 361021, China.
Pestic Biochem Physiol. 2023 Jun;193:105464. doi: 10.1016/j.pestbp.2023.105464. Epub 2023 May 10.
Phosphinothricin (PPT) is a widely used and non-selective herbicide. PPT-resistance genes, especially PPT N-acetyltransferase genes, have been used in the development of transgenic PPT-resistant crops. However, there are only a limited number of available PPT-resistance genes for use in plant biotechnology. In this study, we found that Enterobacter LSJC7 is highly resistant to PPT and can acetylate PPT to N-acetyl phosphinothricin (Ac-PPT). Furthermore, a novel PPT N-acetyltransferase gene, named LsarsN, was identified from LSJC7. When LsarsN was expressed in E. coli AW3110, it confered resistance to PPT. Ac-PPT was detected in both the culture medium and cells of AW3110 expressing the LsarsN-pET22b plasmid. The purified LsArsN protein also showed strong N-acetylation ability in vitro, and its enzymatic kinetic curve was fitted with the Michaelis-Mentan equation. Compared with wild-type LsArsN, both R72A and R74A mutants showed significantly lower PPT N-acetylation ability. In summary, our results systematically characterized LsArsN with strong ability for PPT N-acetylation, which lays the groundwork for future research into the use of this novel gene, LsarsN, to create PPT-resistant crops.
膦丝菌素（PPT）是一种广泛使用且非选择性的除草剂。PPT 抗性基因，特别是 PPT N-乙酰转移酶基因，已被用于转基因 PPT 抗性作物的开发。然而，在植物生物技术中，可用的 PPT 抗性基因数量有限。在本研究中，我们发现肠杆菌 LSJC7 对 PPT 具有高度抗性，并且可以将 PPT 乙酰化为 N-乙酰膦丝菌素（Ac-PPT）。此外，从 LSJC7 中鉴定出一种新型的 PPT N-乙酰转移酶基因，命名为 LsarsN。当 LsarsN 在大肠杆菌 AW3110 中表达时，它赋予对 PPT 的抗性。在表达 LsarsN-pET22b 质粒的 AW3110 细胞的培养物和细胞中均检测到 Ac-PPT。纯化的 LsArsN 蛋白在体外也表现出很强的 N-乙酰化能力，其酶动力学曲线符合米氏-门坦方程。与野生型 LsArsN 相比，R72A 和 R74A 突变体的 PPT N-乙酰化能力明显降低。总之，我们的研究结果系统地描述了具有强 PPT N-乙酰化能力的 LsArsN，为未来利用该新型基因 LsarsN 创造 PPT 抗性作物奠定了基础。
