Identification and antioxidant capacity of 4-hydroxyphenylpyruvate dioxygenase (HPPD), a new favored herbicide target, in Apis cerana cerana.

ABSTRACT

4-Hydroxyphenylpyruvate dioxygenase (HPPD), a nonheme oxygenase, catalyzes the second step of the tyrosine catabolic pathway, which is shared by almost all aerobic life forms. This demonstrates its importance in aerobic biology. We isolated an HPPD homolog from Apis cerana cerana and named it AccHPPD. AccHPPD has an open reading frame (ORF) length of 900 bp and encodes a 299 amino acid protein that has a predicted molecular weight of 34.67 kDa and an isoelectric point of 6.27. Amino acid analysis showed that AccHPPD contained three conserved metal ion active sites, H-101, H-184 and E-267. Real-time fluorescence quantitative PCR (RT-qPCR) analysis showed that AccHPPD mainly existed in specific tissue sites, mainly high in the legs and in the thorax and epidermis, and in specific developmental stages, mainly adults. Under temperature, pesticide, heavy metal and ultraviolet (UV) radiation treatments, the expression level was downregulated, but under H2O2 treatment, the expression level was upregulated. Exogenous expression of the recombinant AccHPPD plasmid in E. coli enhanced the resistance to HgCl2 and H2O2. Inhibition of AccHPPD activity was demonstrated by the upregulation of the tyrosine content after feeding with the inhibitor 2-(2-nitro-4-trifluoromethyl benzoyl)-1,3-cyclohexanedione (NTBC). After silencing of AccHPPD, the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) decreased, and the expression levels of AccBax- and AccCaspase8-related genes were upregulated. The antioxidant genes AccCAT, AccGSTZ1, AccGSTD, AccSOD2, AccTpx3, AccCYP4G11, AccGDTS4, AccGSTO2 and AccMSRA were all upregulated. These results suggest that AccHPPD may serve an integral function in the response of A. cerana cerana to oxidative stress.