| Trehalose can not only protect protein and cell molecular structure under environmental stress and resist adverse environment,but also participate in the metabolic process of organisms as an energy substance.Trehalase is an essential metabolic enzyme in insect trehalose metabolism.It specifically hydrolyzes trehalose into glucose and regulates trehalose level.Two forms of trehalase had been found in insect,namly souble trehalase（TRE1）and membrane-trehalase（TRE2）.The core function of trehalase is to degrade the extra and intercellular trehalose into glucose in order to provide energy for insects.In this study,a soluble trehalase gene（LoTRE1）of rice water weevil（Lissorhoptrus oryzophilus）was cloned and obtained and have an open reading frame of 1840 bp and encode a protein composed of 612 amino acid residues,with a molecular weight of 70833 Da and a theoretical isoelectric point（p I）of 5.38.Similarity analysis showed that LoTRE1 had the closest genetic relationship with Sitophilus oryzae TRE1,and the amino acid sequence consistency was 72%.The phylogenetic tree showed that LoTRE1 had the highest homology with Sitophilus oryzae and Rhynchophorus ferrugineus.The expression profiles of LoTRE1 in different tissues were investigated by RT-q PCR.The results showed that LoTRE1 was expressed in all tissues,including the head,midgut,haemolymph,wing,leg and fat body.But,LoTRE1 had the highest expression in the fat body which was 120 times that in wing.LoTRE1 expression vector was constructed,the recombinant protein was expressed in vitro by prokaryotic expression system,and the protein was purified by Ni-NTA gravity method.The activity of the purified LoTRE1 recombinant protein was determined with trehalose as substrate.The optimum p H of soluble trehalase in L.oryzophilus was 7.0 and the optimum temperature was 50 ℃.T The kinetic parameters（km and Vmax）of LoTRE1 were 48.6 mmol/L and 1.108 mmol/（L·min）,respectively.The protein sequence of LoTRE1 was submitted to the SWISS-MODEL online database,and the analytical crystal structure of the periplasmic trehalase of E.coli（PDB ID 2 WYN）.The reliability of the homology model was verified via the Procheck server.The molecular docking study was performed to further understand the interaction and binding position between trehalose and trehalase,Py MOL was used for protein structure visualization and further structure analysis.The results show that LoTRE1 can stably bind to the substrate trehalose molecule,and deduced that ASP 333 and Glu 299 are the necessary catalytic residues in LoTRE1 model.A three-dimensional model of LoTRE1 was predicted using the SWISS-MODEL server based on the resolved crystal structure of the periplasmic trehalase of E.coli（PDB ID 2 WYN）.The reliability of the homology model was verified via the Procheck server.The molecular docking study was performed to further understand the interaction of trehalose with trehalase and to obtain insight into the binding location of this molecule on trehalase.Py MOL was used for protein structure visualization and further structure analysis.The results showed that LoTRE1 can stably bind to the substrate trehalose molecule,and ASP 333 and Glu 299 may play an important role in the binding process with ligand molecules.After RNAi treatment of LoTRE1 gene,it was found that its expression was significantly down.After 48 hours of in terference,the mortality of the treatment group was significantly higher than that of the control group,and the trehalose content in the treatment group was increased,but trehalase activity was decreased.L.oryzophilus is an important agricultural pest,which seriously endangers the yield and quality of crops.This experiment explored the molecular characteristics and functions of soluble trehalase of L.oryzophilus,which provides a theoretical basis for the control of L.oryzophilus in the future and laid a foundation for exploring new methods of green control of L.oryzophilus. |
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