Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19

摘要

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21(DE3) and purified to homogeneity using Co2+-affinity chromatography. SpHPPD was found to be a monomer. The K-m and k(cat) of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 mu M and 15.0 s(-1), respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC50) and K-i values of 5.2 and 2.5 mu M, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC50: 8.7 irM; 6.0 pM) and mesotrione (IC50: 4.2 mu M; K-i 1.3 mu M) and moderate resistance to tembotrione (IC50: 2.5 mu M; K-i 1.0 mu M). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.