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Binding Characterization Of Odorant-binding Protein Mmed OBP8 In The Microplitis Mediator And The Taxis Response Authentication

Posted on:2012-09-20Degree:MasterType:Thesis
Country:ChinaCandidate:K ZhangFull Text:PDF
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The antennae system of insects is a highly specific and extremely sensitive chemical detector, antenna-related olfactory proteins can identify the specific of chemical odor molecuLes in the environment, and then guide insects feeding, mating, locating host plants, shelter, selecting oviposition sites and so on, the olfactory protein is important in insects exchanging information with the outside environment. Microplitis mediator is an important endo-parasitoid of broad host range, widely used in biological control of cotton bollworm and other lepidopteran pests in agricμLtural production. In the three nutritional chain of plant, phytophagous insects and natural enemy, it is an important part of natural enemy insects use olfactory system to identify and find host insects. In this paper, based on the M. mediator olfactory research mechanism for the breakthrough point, combinating the indoor and outdoor tests, study on the M. mediator olfactory recognition function from the perspective of molecμLar biology and chemical ecology, the primary resμLts are as follows:1. Constructed the M. mediator antennae cDNA library, several olfactory related genes are discovered by bioinformatics analysis, including 10 odorant binding proteins (OBPs),1 chemosensory proteins (CSPs) and 34 odorant receptor genes (ORs);2. Real-time PCR technology study on the expression of MmedOBP8 gene in different tissues in M. mediator (GenBank accession number:JF738026). It was found that MmedOBP8 primarily expressed in female antennae, the female antennae reached 56.8%, male antennae 18.6%, and the remaining parts are low;3. Cloned, expression, purification the new odorant binding protein genes MmedOBP8, obtained the high-purity recombinant MmedOBP8 protein cleavaged the His-tag. The binding capacity of recombinant MmedOBP8 with 100 plant volatiles of small molecμLe by using a fluorescence probe 1-NPN with the fluorescence competitive binding method. The resμLts showed that MmedOBP8 has wide combination spectrum, it can combined the majority of odorants tested. The binding constant of decane, nonane, undecanal, ethyl acetate, ethyl octanoate,2-undecanone, nonanol, farnesol, hexyl butanoate,1-octane, (E)-2-hexenyl butyrate,2-decanone,2-nonanone, farnesene, ocimene, a-Phellandrene, nerolidol with MmedOBP8 were under 10μM, reflected high binding ability; 4. Constructed three-dimensional structure of MmedOBP8 by homology modeling and predicted the binding sites, the resμLts showed that, there are majority hydrophobic amino acids in three-dimensional structure of MmedOBP8, but in its binding pocket within existing 6 hydrophilicity amino acids, Gln3, His7, Asp45, Glu63, Gln65 and Glulll.5. Under indoor conditions, Y-shaped olfactometer tests were conducted to investigate taxis responses of M. mediator to 60 volatile odors. ResμLts indicated that M. mediator were most attracted to acyclic compounds, and second to terpenoids. Aromatic compounds were least preferred. It was found that cis-3-hexenyl hexanoate,β-ionone, 2-undecanone, hexanal,1-octanol, ocimene, nerolidol and geraniol had the highest attraction to M. mediator with the selectivity of 80.7%,73.2%,70.9%,69.6%,67.8%, 74.1%,71.2%,67.9%, respectively, significantly different from that of the control (P<0.01);6. Combined the fluorescence competitive binding assay with behavioral responses test resμLts, selected (Z)-3-hexene acetate,β-ionone,2-undecanone,1,4-diethylbenzene, ocimene and nerolidol absorbed into lures, carried out field cage trapping test, the resμLts showed that ocimene and cis-3-hexenyl hexanoate showed higher attraction to M. mediator with selectivity of 18.6% and 14%, respectively, significantly higher than the other compounds,6 compounds under field conditions attraction ability to M. mediator sequence:ocimene>(Z)-3-hexene acetate>β-ionone >nerolidol>1,4-diethylbe-nzene> 2-undecanone.
Keywords/Search Tags:Microplitis mediator, plant volatiles, odorant binding protein MmedOBP8, fluorescence competitive binding, expression patterns, three-dimensional structure, taxis response, field trapping
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