Isolation of mesotrione-resistant strain and cloning and expression of HPPD

Abstract:

[Objective] The mesotrione-resistant strain was isolated from soil, and the resistant gene 4-Hydroxyphenylpyruvate dioxygenase was cloned and characterized. [Methods] Using tyrosine as the sole carbon source, the strain was isolated by gradient enrichment culture. The isolated strains were identified based on 16S rRNA gene sequence. The resistant gene 4-Hydroxyphenylpyruvate dioxygenase was cloned by PCR amplification using the genomic DNA as a template. The expression plasmid pETH4 was constructed and expressed in Escherichia coli BL21(DE3). The mesotrione-resistant property of E. coli BL21(DE3)-pETH4 was tested by measuring the absorbance at 440 nm. [Results] Seven bacterial strains, which could grew on MSM medium containing 10 mmol/L mesotrione and 1 g/L tyrosine, were isolated. They were identified as Acinetobacter sp., Achromobacter sp. and Pseudomonas sp.. PCR fragment of 1 056 bp was obtained from Pseudomonas sp. AM-H4, which exhibited the best resistance to mesotrione. The cloned HPPD gene shared 99% sequence identity with that from Acinetobacter baumannii, replacing aspartate-341 with alanine. The protein of HPPD was expressed in E. coli BL21(DE3) with the molecular mass of 40 kD. The absorbance of brown pigment produced by E. coli BL21(DE3)-pETH4 in tyrosine-LB culture was significantly reduced in the presence of 40 μmol/L mesotrione. In addition, the brown pigment was still visible when the concentration of mesotrione was greater than 200 μmol/L. [Conclusion] The HPPD gene with high mesotrione-resistance was obtained in this study, which maybe a novel gene source for the application in breeding of herbicide-resistant crops.