Topramezone is a new 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide that is widely used on corn to control annual grass weeds and broadleaf weeds. Due to its broad-spectrum weed control capacity, improved activity, excellent crop selectivity, low mammalian toxicity and high environmental safety, topramezone is considered an ideal target herbicide for transgenic engineering of herbicide tolerance. In this study, a topramezone-resistant strain, Burkholderia sp. BW-1, was screened from soil, and a novel topramezone-resistant HPPD gene (Bkhppd) was cloned from this strain. Purified BkHPPD displayed relatively high HPPD activity and topramezone resistance with a half-maximal inhibitory concentration (IC50) of 572.0 nM. Two BkHPPD mutants designated as BkHPPDt31 and BkHPPDt76 were screened through pressure acclimation. BkHPPDt31 contained three amino acid substitutions (H65D, N160 T and N258S), whereas BkHPPDt76 contained four amino acid substitutions (H65D, N160 T, N258S and N343 T). The topramezone IC50 values of BkHPPDt31 and BkHPPDt76 were 1.1- and 2.3-fold higher, respectively, than that of wild-type BkHPPD. In addition, site-directed mutagenesis indicated that the increased resistance conferred by BkHPPDt31 resulted from the synergistic effects of the three site mutations rather than a single site mutation, and that substitution of asparagine 343 with threonine significantly decreased catalytic efficiency and affinity but increased topramezone resistance. In summary, this study provides a novel topramezone-resistant HPPD gene for the engineering of genetically modified herbicide-resistant crops and facilitates further elucidation of the resistance mechanism of BkHPPD and improvement of resistance through directed evolution.

中文翻译：

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一种新型抗topramezone 4-羟基苯基丙酮酸双加氧酶基因的克隆及压力驯化提高其抗性

Topramezone 是一种新型 4-羟苯基丙酮酸双加氧酶 (HPPD) 抑制剂除草剂，广泛用于玉米防治一年生禾本科杂草和阔叶杂草。由于其广谱杂草控制能力、改进的活性、优异的作物选择性、低哺乳动物毒性和高环境安全性，topramezone被认为是除草剂耐受性转基因工程的理想靶标除草剂。在这项研究中，一种抗topramezone菌株，伯克霍尔德菌属。BW-1，是从土壤中筛选出来的，并从该菌株中克隆了一个新的抗陀螺蛉HPPD 基因（Bkhppd）。纯化的 BkHPPD 显示出相对较高的 HPPD 活性和 topamezone 抗性，半数最大抑制浓度 (IC50) 为 572.0 nM。通过压力驯化筛选了指定为 BkHPPDt31 和 BkHPPDt76 的两个 BkHPPD 突变体。BkHPPDt31 包含三个氨基酸替换（H65D、N160 T 和 N258S），而 BkHPPDt76 包含四个氨基酸替换（H65D、N160 T、N258S 和 N343 T）。BkHPPDt31 和 BkHPPDt76 的 topramezone IC50 值分别比野生型 BkHPPD 高 1.1 倍和 2.3 倍。此外，定点诱变表明 BkHPPDt31 增加的抗性是由三个位点突变的协同作用而不是单点突变引起的，用苏氨酸取代天冬酰胺 343 显着降低了催化效率和亲和力，但增加了 topramezone 抗性. 总之，