Mutations of Asn321 and Glu322 Improve Resistance of 4-Hydroxyphenylpyruvate Dioxygenase SpHPPDm to Topramezone

As a highly efficient 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, topramezone is an ideal target for herbicide-resistant genetic engineering. In this study, two mutants, K-19 (N321Y) and K-63 (Q166R/E322V), with topramezone resistance increased by 205.3 and 58.5%, respectively, were screened from the random mutation library of SpHPPDm, a topramezone-resistant HPPD mutant that we previously obtained. Sites N321 and E322 were identified as key sites for increased topramezone resistance by single-site mutation analysis. A mutant KB-145 (N321Y/E322K) was further obtained by saturation mutation at sites N321 and E322. The topramezone resistance of KB-145 increased by 955.3% compared to mutant SpHPPDm. In conclusion, this study identifies two new sites that significantly affect the topramezone resistance of SpHPPDm, which provides new insights into the molecular mechanism of herbicide resistance of HPPD, and the acquired mutants have great application potential in the construction of herbicide-resistant crops.