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Functional Analysis Of Odorant-binding Proteins In Holotrichia Oblita Faldermann (Coleoptera: Scarabaeida)

Posted on:2012-10-23Degree:MasterType:Thesis
Country:ChinaCandidate:S S DengFull Text:PDF
GTID:2143330335479517Subject:Agricultural Entomology and Pest Control
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In nature, successful communication between insect individuals and insect with environment is the one of the important reason for these species to survival and evolution, the sensitive olfactory system of insect are play a crucial role in the communication. Insect could location food, mate, avoidance of predators and toxins in the help with the olfactory system, antenna of insect as the organ to senses the environment is the essential part of olfactory system. Moreover, different functional proteins in antenna are the basic of mataint normal olfaction. The scarab beetle Holotrichia oblita Faldermann (Coleoptera:Scarabaeidae) possess predominance in the underground pests in the northern parts of China and cause great economical loss in result of there wide host plant and covert habitat. In order to further understand the olfactory mechanism of H.oblita and control them by scientific methods, we cloned the odorant-binding proteins of H.oblita by molecular biology methods and research their function. Meanwhile, several plant volatiles were chose to electroantennogram test and bioassays for investigating the potential function of the two odorant-binding proteins from a macroscopical angle. The primary results are as follows:(1)We obtained two full-length odorant-binding proteins gene by RACE and logging in Genbank, the Genbank number are as follows: GQ856258,GQ856257.We reveal the relationship between the two HoblOBPs with other insect OBPs by particular bioinformatics analysis.(2)The two new HoblOBPs was cloned, expressed, purification and removed His-tag in laboratory. By using fluorescence assays, we tested the binding ability of the two HoblOBPs to 73 compounds with fluorescence probe 1-NPN. The results showed OBP1 and OBP2 could widely bind the plant volatile, but showed limited affinity to pheromone. The flower volatileβ-ionone was the best ligands of the two odorant-binding proteins in binding assays.OBP1 have good ability to bind several compounds comes from castor which could attract H.oblita. OBP2 showed good affinity to Eugenol which is a attractant to other scrabe beetle and the plant volatile Methyl salicylate. Moreover, the ability of the two HoblOBPs is affected by the conformation, functional group and length of carbon chain of ligands.(3)The good ligands in binding assay and several plant volatiles were chose to EAG on the antenna of H.oblita, the high compounds which can EAG value were used for behavior experiment. Trans-2-Hexenol, Linalool, cis-3-Hexenyl acetate, 1-hexanol, Methyl anthranilate and Methyl salicylate were found as more attractive in comparison to hexane and Octyl aldehyde and Phenethyl alcohol were less attractive than hexane.Combined with the results of binding assays, OBP1 and OBP2 were showed limited affinity to part compounds which can elicted the strong electrophysiological responses of the antennae of female H.oblta adults. We inferred that there are lots of OBPs exist in the genome of H.oblita and have their specifically task.(4)Polyclonal Antibody of OBP1 and OBP2 were obtained from the serum of rabbit which were injected the removed His-tag HoblOBPs mix of Freund's adjuvant. By using polyclonal antibody and electron microscope, we observed the expression of the two HoblOBPs in the sensilla of antenna. Immunohistochemistry experiment revealed anti-OBP1 and anti-OBP2 could strain the section of sensilla placodeum and basiconica in both sexes.
Keywords/Search Tags:Holotrichia oblita, Odorant-binding proteins, Fluorescence competitive binding assays, Electroantennogram, Behavioral responses, Immunohistochemistry location
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