| Ambrosia artemisiifolia L.is a worldwide invasive weed native to North America,which seriously threatens ecological security and human health.It invaded China in the 1930s and is widely distributed in China.It was included in the first list of alien invasive species in China.Ophraella communa Lesage originated in North America,is an effective biological control agent in the process of A.artemisiifolia control.The olfactory sense of O.communa dominated its recognition of the host-A.artemisiifolia.Odorant binding proteins(OBPs)is an important olfaction protein in insect odor response and plays a key role in host recognition.However,the function of OBPs in O.communa has not been reported.Therefore,this study reasearched the function of OcomOBPs in the identification of A.artemisiifolia by gene cloning,construction of prokaryotic expression vector,expression and purification of recombinant proteins,fluorescence competitive binding experiment,RNAi and other technologies.The main conclusions are as follows:1.The antennal sensilla of O.communa were observed by scanning electron microscopy.The results showed that there were six type of sensilla distributed on the antennae,inciuding sensilla trichodea,sensilla chaetica,sensilla basiconica,sensilla coeloconica,sensilla styloconica and B?hm bristles.These sensilla ensured that O.communa could receive and distinguish odors.2.Based on the antennal transcriptome of O.communa,19 coding sequences of OcomOBPs were obtained and verified through gene cloning.RT-PCR results showed that OcomOBP 1/2/3/7/8/9/10/16/18/19 were highly expressed in male and female antennae,indicating that these OcomOBPs may have olfactory-related functions.Among them,OcomOBP2/3/13/16/18/23 in male antennae were significantly higher than those in female antennae,indicating that these OcomOBPs may be related to sex pheromone recognition.3.Through the construction of prokaryotic expression vector,we constructed 12 OcomOBPs expression vectors(OcomOBP3/7/9/11/12/13/14/15/16/17/19/21)and 6 OcomOBPs(OcomOBP3/7/11/12/13/17)were purified by the immobilized metal nickel chelate affinity chromatography.All of these sample proteins were identified by LC-MS/MS.The binding characteristics of O.communa to A.artemisiifolia volatiles were investigated by fluorescence competitive binding experiments.The results showed that OcomOBP7/11/12 had a broad ligand binding affinity.OcomOBP7 could bind 14 of the 26 candidate A.artemisiifolia volatiles,including 12 alkenes,1 alkanes,and 1 esters.OcomOBP11 could bind 24 of the 26 candidate A.artemisiifolia volatiles,including 16 alkenes,1 alkanes,3 esters,2 aldehydes,and 2 alcohols.OcomOBP12 could bind 21 of the 26 candidate A.artemisiifolia volatiles,including 15 alkenes,1 alkanes,3 esters,1 aldehydes,and 1 alcohols.OcomOBP3/13/17 has almost no binding ability to A.artemisiifolia volatiles,so the functions of these three proteins need to be further explored.4.The in vivo function of OcomOBPs was verified by RNAi.The results showed that the expression levels of OcomOBP7 and OcomOBP11 were reduced by 98.4%and 97.1%,respectively,48 hours after injection of target gene dsRNA.The EAG response of O.communa antennae to α-pinene and ocimene was significantly down-regulated after silencing OcomOBP7,indicating that α-pinene and ocimene can specifically bind to OcomOBP7 and are odor ligands corresponding to OcomOBP7.After silencing OcomOBP11,the EAG response of O.communa antennae to trans-caryophyllene and humulene was significantly down-regulated,indicating that trans-caryophyllene and humulene can specifically bind to OcomOBP11 and are odor ligands corresponding to OcomOBP11.This study is the first time to express in vitor,purify and functionally verify the odorant binding protein of O.communa.Two important odorant binding proteins(OcomOBP7/OcomOBP11)were screened,which may be involved in the localization of A.artemisiifolia,and the molecular mechanism in recognition of A.artemisiifolia by OcomOBPs of O.communa was clarified.It also lays a theoretical foundation for research of O.communa attractants,which is helpful for better control of A.artemisiifolia by O.communa. |
