The Role Of The Non-heme Iron Oxygenases AccHPPD Of Apis Cerana Cerana In Antioxidation

Apis cerana cerana is an indigenous bee species in China that has a role in maintaining ecological stability and promoting agricultural development.In recent years,a series of abiotic stress hazards caused by climate change,misuse of pesticides and herbicides,and human destruction of vegetation have seriously affected the survival and development of Apis cerana cerana.Therefore,it is important to study the resilience and self-protection mechanism of Apis cerana cerana.4-Hydroxyphenylpyruvate dioxygenase（HPPD）is a non-heme iron oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate（HPP）to 2,5-dihydroxyphenylacetic acid（homogentisic）and regulates apoptosis and promotes the scavenging of cellular reactive oxygen species（ROS）.HPPD plays an important role in the resistance to oxidative stress in invertebrates such as spiny ginseng and earthworms,and its catalytically synthesized metabolites are also involved in resistance to abiotic stress in microorganisms and plants.Therefore,we isolated the homologous gene of HPPD from Apis cerana cerana and named it Acc HPPD.We analyzed the role of Acc HPPD in stress resistance of Apis cerana cerana and the main results were as follows.（1）We isolated Acc HPPD from Apis cerana cerana and analyzed its sequence,which showed that Acc HPPD has an open reading frame length of 900 bp and encodes a 299 amino acid protein with a predicted molecular weight of 34.67 k Da and an isoelectric point of 6.27.（2）Acc HPPD promoter analysis showed that its promoter sequence contains some sequences for embryonic or tissue development,indicating that Acc HPPD plays a role in tissue and embryonic development.Acc HPPD was also found to contain a sequence that plays a key regulator role in the ecdysteroid response.It is suggested that Acc HPPD may play a role in Apis cerana cerana molting and metamorphosis development..（3）Amino acid sequence analysis showed that Apis cerana cerana HPPD is highly homologous to Apis mellifera HPPD,and the active site of HPPD is highly conserved in different insects.And the C-terminus of HPPD is significantly more conserved than the N-terminus.Therefore,we speculate that the C-terminus is the main position where it exerts its function.The tertiary structure analysis revealed that the three conserved metal ion active sites（H-101,H-184 and E-267）of Acc HPPD form a His-X-Glu-X-His triplet face.（4）Real-time fluorescence quantitative PCR（RT-q PCR）analysis showed that Acc HPPD was highly expressed mainly in adult honeybees during different developmental stages in Apis cerana cerana.Dissection of adult honey bees to obtain different tissue sites revealed that Acc HPPD was highly expressed in the legs,abdomen and thorax.The high expression of Acc HPPD at the adult stage,the only stage when bees are in direct contact with the external environment,suggests that it may play an important role in adult bees’response to environmental stress and self-protection.（5）As detected by RT-q PCR,Acc HPPD expression levels were down-regulated in Apis cerana cerana under temperature,pesticide,heavy metal and ultraviolet（UV）radiation treatments,but up-regulated under H₂O₂ treatment.This indicates that Acc HPPD can respond to oxidative stress caused by different stresses and plays an important role in H₂O₂ stress.（6）Acc HPPD overexpression in E.coli enhanced cellular resistance to Hg Cl₂,H₂O₂ and isoproterenol hydrogen peroxide and reduced the cellular damage produced by stress.The survival curves showed that Acc HPPD overexpressed E.coli were significantly more resistant to Hg Cl₂,H₂O₂ and isoproterenol hydrogen peroxide than E.coli not transferred to Acc HPPD.（7）Feeding the HPPD inhibitor NTBC to Apis cerana cerana,we detected a reversible inhibition of Acc HPPD activity.After that,we also found that the survival ability of Apis cerana cerana was reduced.This shows that when Acc HPPD was inhibited,the survival of Apis cerana cerana became weaker.（8）After Acc HPPD silencing,antioxidant genes and apoptosis-related genes were measured in Apis cerana cerana,and the expression of Acc CAT,Acc GSTZ1,Acc GSTD,Acc SOD2,Acc Tpx3,Acc CYP4G11,Acc GDTS4,Acc GSTO2,and Acc MSRA were found to be up-regulated.This may be a remedial measure taken by the organism when the antioxidant capacity is diminished after Acc HPPD silencing.In contrast,the expression of the apoptotic genes Acc Bax and Acc Caspase8 was upregulated,indicating that apoptosis is promoted.In summary,Acc HPPD plays an important role in antioxidant in Apis cerana cerana.