Cloning, Expression And Functional Research Of GmolOBP1 And GmolOBP6 Of The Oriental Fruit Moth, Grapholita Molesta Busck(Lepidoptera:Tortricidae)

It is depends on the chemical sensor to locate the host plants and oviposition situation, select mate, avoid predators and realize chemical communications for insects. The gustatory sensilla and olfactory sensilla play vital functions on chemical communications, especially olfactory sensilla. A series of proteins involved in the process of the sense of smell, including odorant binding proteins(OBPs), olfactory receptors(ORs), odorant degradation enzymes(ODEs), sensory neuron membrane proteins(SNMPs) and ionotropic receptors(IRs). As for start proteins of odor identification, it is of great significance to determine recognition mechanism with volatiles so that we can understand physiological and behavioral responses of insects from the molecular level.This paper selected two OBP genes(GmolOBP1 and Gmol OBP6) of antennae transcriptome of Grapholita molesta(Busck). The two GmolOBPs were cloned, prokaryotic expression and protein purification as well as functional study. Physiological reactions of antennae were realized by electroantennogram response to 3-days adult. The results demonstrated that: the open reading frames of Gmol OBP1 and GmolOBP6 were 468 and 483 bp, encoding precursor proteins of 155 and 160 aa, respectively. The two proteins had 23 and 38 aa signal peptides. The isoelectric points of two mature proteins were 5.10 and 4.75, respectively. The homologous analysis and phylogenetic analysis indicated that two proteins belonged to typical odorant binding protein which both had 6 conservative cysteine residues. The identity of GmolOBP1 and AconOBP1 was 38%. The two genes were expressed abundantly in antennae of male and female. The two proteins were highly expressed at the concentration of 0.2 mmol/L IPTG and protein concentrations were 1.53 and 1.40 mg/ml, respectively. The two recombinant proteins had high binding affinities to aldehydes, especially decanal and lauraldehyde. GmolOBP1 bound dodecanol and decanal(volatile from host plants) with Ki values of 5.46 and 13.14 μM, respectively. GmolOBP6 bound Z8-12: Ac and butyl acetate with Ki values of 56.93 and 11.89μM, respectively. The strongest EAG response to female was elicited by Z8-12: Ac.It is contributed to develop new attractants and provide clue of biological control on G.molesta that two proteins have specificities to aldehydes.