| Sitophilus zeamais is an important storage pest,and every year there have been a great economic losses because of the damage caused by that.The pesticides in the market such as phosphine,sulfuryl fluoride and methyl bromide have played an important role in the early prevention of Sitophilus zeamais.The long-term use of these chemical pesticides resulted in Sitophilus zeamais with a certain resistance and other unsafe factors to animals and environment,therefore,there have been some limitations in the prevention of stored grain pests.As an isothiocyanate compound extracted from plants,AITC has the advantages of safer and lower residue of plant source pesticides,and a good insecticidal effect on the fumigation of stored pests,consequently,it was considered as a kind of fumigant.It was still not clearly about the mechanism of AITC at the moment.To clarify mechanism of AITC can help to guide the rational use of highly efficient and scientific.With Sitophilus zeamais as the target organism,the study has reported AITC affect on the mitochondrial COX core subunits from the aspects of the gene cloning,vector construction and expression analysis,protein purification,homology modeling and molecular docking,and so on.The main results of this research were obtained as below.1.Gene cloning and bioinformatics analysis of COX?/?/? of Sitophilus zeamais.Total RNA was extracted from adulted Sitophilus zeamais by using CTAB method,and the first strand cDNA was synthesized by reverse transcription.The open reading frame of COX?/? was obtained by RACE and RT-PCR,and COX ? accession number was GenBank KX783028.Bioinformatics analysis showed that the ORF of COX? gene was 1542 bp,which encoded a protein of 513 amino acids with the molecular weight of 57061.12 and isoelectric point of 6.33,and the start codon of AUU,and the stop codon of UAA.The ORF of COX? gene was 684 bp,which encoded a protein of 227 amino acids with the molecular weight of 26237.88 and isoelectric point of 6.37,and the start codon of AUA,and the stop codon of UAA;The ORF of COX? gene was 792 bp,which encoded a protein of 263 amino acids with the molecular weight of 30938.1 and isoelectric point of 6.51,and the start codon of AUG,and the stop codon of UAA.2.Vector construction and prokaryotic expression of COX?/?/? gene of Sitophilus zeamais.pET28 a,pET30a,pET32 a,pET42a and pEASY-Blunt E1 recombinants with COX?/?/? were constructed,respectively,and successfully transformed into E.coli BL21?DE3?.After SDS-PAGE and Western Blot confirming,only pET42-COX?-E.coli BL21?DE3??pET32-COX?-E.coli BL21?DE3?fusion protein were expressed,with the molecular weight 44 kD and 62 kD,respectively.COX ? mainly existed in the form of inclusion bodies,while COX II was present in the supernatant.The soluble protein content of pET42-COX?-E.coli BL21?DE3?was optimized,and the quantity in the supernatant was up to the maximum under the temperature of 16 ?,1 mmol L-1 IPTG,180 rpm within 24 h.The COX? fusion protein was purified by Ni+-NTA column and the concentration was 50?g/mL.3.Expression of Sitophilus zeamais COX?/?/? gene in Pichia pastoris GS115.Primers containing EcoR I and Kpn I restriction sites were designed by using PP5 software.Then,the recombinant expression vector of pPICZ?-COX?/?/? were constructed by cloning,enzyme digestion and ligation,and confirmed by double enzyme digestion and sequencing.The three recombinants were linearized with Sac I,and successfully transformed into Pichia pastoris GS115.4.Homology modeling and molecular docking of Sitophilus zeamais COX?/?/?.The effect of AITC on the activity of COX? protein was analyzed by UV / Vis.The results showed that recombinant protein COX? had a certain catalytic activity,but the activity was weak.Combining with IR spectroscopy results,AITC might bind to COXII protein at the N-terminus or C-terminus.The protein bovine cytochrome c oxidase?PDB ID: 1V54?,which had higher homology with COX??75% homology?,COX??60% homology?and COX ??59% homology?was used as the template,Using modeller 9.17 software to construct COX?/?/? three-dimensional structure model.Based on the Autodock vina 1.1,AITC were docked with COX?/?/?,and the results showed that AITC could be bond to three subunits,respectively.Allyl group of AITC was located in the hydrophobic pockets of the COX ? amino acid residues Phe-20,Ile-73,Ile-389,Ile-390,Phe-393 and Phe-468,and the sulfur atom of AITC formed a hydrogen bond of 3.3 ? with Gly-27;Allyl group of AITC was located in the hydrophobic pockets of the COX? amino acid residues Ile-34,Pro-69,Ile-72 and Leu-73,and the sulfur atom of AITC formed a hydrogen bond of 3.4 ? with Leu-31;Allyl group of AITC was located in the hydrophobic pockets of the amino acid residues Ile-63,Leu-81,Phe-235 and Gly-236,and the sulfur atom in AITC formed a hydrogen bond of 2.8 ? with Arg-223?From the aspects of protein hydrophobicity,hydrogen bond interaction,van der Waals force and other factors,the affinity of AITC and COX?/?/? were-3.4 kcal/mol,-3.0 kcal/mol and-3.2 kcal/mol,respectively,therefore,The inhibitory activity of AITC on the core subunits were: COX I >COX III >COX?.These results were consistent with the consequences of AITC on Sitophilus zeamais using RNAi technique in early stage,which had a strong inhibitory effect on COX?.In conclusion,the three core subunits of Sitophilus zeamais were analyzed by gene cloning and bioinformatics in this paper,and ORF of COX?/? were obtained.On the basis of these,three vector subunits were constructed and were tried to express by using prokaryotic expression system and Pichia pastoris expression system.SDS-PAGE and WB test results showed that only COX ? was successfully expressed in the prokaryotic system in the form of soluble protein,and the expression conditions were optimized.The concentration and activity of the purified COX ? protein were determined.Finally,the three-dimensional structure model of three core subunits was constructed by homology modeling,and the interaction between the COX?/?/? and AITC were analyzed by using molecular docking.On one hand,the above study provides a basis for the AITC bio-fumigants in the mechanism of storage pest preservation,and on the other hand,it can provide some reference for the biosynthesis of AITC compounds. |
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