Gene Cloning, Tissue Distribution And Ligand-Binding Analysis Of Odorant Binding Proteins In The Rice Stiped Stem Borer, Chilo Suppressalis (Walker)(Lepidoptera:Pyralidae)

In the long-term evolutionary process, insects evolved a highly sensitive olfactory system to percept volatile compounds in the environments, and accordingly respond with physiological or behavioral responses such as finding mates, food, and habitat. In the process of olfaction, odorant binding proteins (OBPs) function to bind and transport odorants to odorant receptors (ORs) situated on the memberane of the receptor neurons. This interaction between OBP and OR is thought as the first biochemical step of olfaction. Chio suppressalis (Walker) is an important agricultural pest, and the study on OBPs of C. suppressalis is not only helpful for knowing more about the olfactory system, but also can provide potential targets for the development of new behavior interfering techniques so that to inhance the sustainable control of the pest. In this study,29cDNAs fragments and6full-length cDNAs of OBPs from C. suppressalis were obtained by genome data analysis and molecular cloning,5OBP genes’tissue expression patterns were determined, and finally one OBP were functionally explored by fluorescence competitive binding experiments and electroantennogram (EAG) recording assay. The main results are as follows:1. Gene cloning and sequence analysis of CsupOBPsBy genome data analysis and PCR technology,29cDNAs fragments and6full-length cDNAs of OBPs from C. suppressalis was identified, which were named as CsupOBP1, CsupOBP2, CsupOBP3, CsupOBP4, CsupOBP5and CsupABPX, respectively. Sequence analysis showed that CsupOBP3, CsupOBP4and CsupABPX beared the characteristics of typical OBPs, including six conserved cysteine sites except for CsupOBP1, CsupOBP2and CsupOBP5(only four conserved cysteine sites). The amino acid sequences of CsupOBP2, CsupOBP3, CsupOBP4and CsupABPX had high similarities of81%,66%,61%,70%, respectively, with reported OBPs of other Lepidopteran insects, while CsupOBP1and CsupOBP5shared low similarities with other OBPs, of34%and43%, respectively. 2. Expression patterns of CsupOBPs at different development stage and in different adult tissuesTo explore the functions, the expression levels of CsupOBP1, CsupOBP2, CsupOBP4, CsupOBP5and CsupABPX in different tissue of the larvae and adults were examined by RT-qPCR. The results showed that in the larval stage, four OBPs expressed mainly in the head. In comparison, CsupOBP1expressed in the highest level followed by CsupOBP5, while CsupABPX were the lowest. In the adult stage, CsupABPX mainly expressed in antennae and CsupOBP2mainly expressed in head (with antennae removed), with little or no expression in other tissues; CsupOBP1expressed in various tissues, at relatively high levels in legs, wings and male antennae and heads; CsupOBP4and CsupOBP5mainly expressed in abdomen, with little or no expression in other tissues. The highly expression in antennae and other chemosensory tissues suggests roles of these OBPs in the olfaction and gustation of C. suppressalis.3. Prokaryotic expression and ligand-binding affinity analysis of CsupOBPlAs above results showed, CsupOBP1was highly expressed in the larval heads and adult chemoreception tissues (wings, legs and male antennae and heads) thus it may play an important role in the larval olfaction. To confirm it, the CsupOBP1was in vitro expressed functionally analized. The gene was firstly fused into the pET-30a(+) expression vector, then expressed in Escherichia coli with induction of IPTG, finally purified through nickel ion affinity chromatography and enterokinase hydrolysis. The binding affinities of the CsupOBPl with38odorant compounds were tested by fluorescence competitive binding assays. Among the tested ligands, only Beta-ionone showed the highest affinity (Ki=9.53μmol/L). Since Beta-ionone is a fragrant component widerly existed in flowers and other tissues of many plants, CsupOBP1in C. suppressalis may play a role in finding host plants by recognition of Beta-ionone.4. EAG responses of C. suppressalis to four odorant compoundsTo confirm the biological activity of Beta-ionone, EAG response of C. suppressalis to Beta-ionone and other3volatile with higher binding affinity to CsupOBP1. As a result, Beta-ionone elicited significant EAG responses in both male and female antennae, supporting the result that CsupOBPl plays a role in perception of Beta-ionone. In addition, Beta-ionone elicited higher response in males than in females, which is agreeable to the difference in CsupOBP1expression levels between male and female antennae. Taken together, we propose that CsupOBP1functions to bind and transport the Beta-ionone and thus play a role in finding host plants in C. suppressalis.