Identification Of Odorant Degrading Enzyme Genes And Functional Analysis Of The SzeaGSTD1 From Sitophilus Zeamais

Maize weevil,Sitophilus zeamais Motschulsky,is a worldwide stored grain pest and an important pest causing huge losses to China and it belongs to order Coleoptera and family Curculionidae.With the constantly use of fumigant to prevent and control stored-grain pests,insect resistance,food safety and environmental pollution have become an ever-prominent issue now,which make it urgent to develop green environment-friendly prevention and control technologies.Insects have ability to receive the information carried by odor molecules in the environment through the developed and efficient olfactory sensing system,and then make them to respond the corresponding physiological reactions(feeding,reproduction and avoiding natural enemies).In insect olfactory sensing system,odorant degrading enzymes(ODEs)play a role to rapidly degrade and inactivate the odor molecules that have completed the information transmission,and maintain the stability and sensitivity of olfactory sensing system.Therefore,in this paper,we studied the ODEs based on the previous antennal transcriptome database of S.zeamais in our laboratory.1.Identification and sequence analysis of ODEs of S.zeamaisBased on the antennal transcriptome database of S.zeamais,we identified 30 ODE genes,including 13 glutathione S-transferases(GSTs)and 17 carboxylesterases(CXEs).The length of 13 SzeaGST genes ranged from 606 to 744 bp,and the encoding proteins length ranged from 116 to 239 amino acid residues,and all the SzeaGSTs had high homology with GSTs of other Curculionidae family insects.The predicted results of conserved domains and key amino acid sites of amino acid sequences showed that nine SzeaGSTs had GSH binding domain at N-terminal and eight SzeaGSTs had substrate binding domain at C-terminal.The length of 17 Szea CXE genes ranged from 1554 to 2535 bp,and all of them had complete open reading frames.The length of the encoding proteins ranged from 421 to 744 amino acid residues.Among the Szea CXEs,Szea CXE1,Szea CXE8 and Szea CXE17 have signal peptide at the N-terminal of the sequences,which indicate that they may be secreted outsidethe cell to perform their function.The analysis results of functional domains and conserved sites showed that these Szea CXEs had conserved insect esterase sequence characteristics(Gx Sx G version,Oxyanion hole).2.Expression pattern analysis of ODEs in different tissues of S.zeamaisAccording to the results of real-time PCR,six SzeaGSTs were highly expressed in the antennae.Among them,SzeaGSTd1 was highly expressed in the male and female antennae,but no significant difference between them;the remaining SzeaGSTs except SzeaGSTd3 were significantly higher in the male antennae as compared to the female antennae.SzeaGSTe2,SzeaGSTe5 and SzeaGSTz1 were highly expressed in the wings,while SzeaGSTt1 was highly expressed in the head.We speculate that these genes highly expressed in the non-olfactory organs may perform other functions in S.zeamais.We found six Szea CXE genes were highly expressed in antennae,seven in thorax and five in wing.Szea CXE1 is highly expressed in the head,which may play an important role in the taste system of S.zeamais.3.In vitro expression and functional analysis of SzeaGSTd1 proteinThe recombinant proteins of SzeaGSTd1 was expressed by using the Escherichia coli expression system,and the soluble protein was about 25 k Da in size.The results of GST activity show that the maximum reaction rate(Vmax)is 1047 ± 43.09 μmol/mg/min and the Michaelis constant(Km)is 0.42 ± 0.04 m M,and the optimum condition of enzyme activity is 30 ℃,p H7～7.5,which were affected by temperature and p H.The results of substrate competition test showed that Capryl alcohol,Benzaldehyde and vanillin had significant effects on the activity of SzeaGSTd1,and the relative activity decreased by47.76%,39.74% and 51.94%,respectively.Capryl alcohol,Benzaldehyde and vanillin inhibited the activity of SzeaGSTd1 with IC50 values of 0.39,0.59,and 0.43 m M.The results of degradation metabolism in vitro indicated that SzeaGSTd1 could degrade Capryl alcohol,but not benzaldehyde and vanillin.The research on ODE genes of S.zeamais and the functional study of SzeaGSTd1 will help to understandthe mechanism of these genes in the olfactory sensing system,and provide theoretical support to development the new prevention and control technologies based on the olfactory sensing system of S.zeamais.