Functional Analysis Of Olfactory Proteins In Cnapharocrocis Medinalis

Rice leaf folder Cnaphalocrocis medinalis is one of the most important migratory pests on rice.The larvae damage plants by scraping the green leaf tissues,causing yield loss because of reducing photosynthetic activity.Normal chemical treatments are not often efficient due to the cryptic feeding habit-folding the leaves.Therefore,it is an urgent need to develop a sustainable method to control C.medinalis.is to Regulating olfactory chemoreception paves a way to help us to control target insect pests as a potential pest management measures.In this paper,we will focus on the function analysis of chemoreception proteins in C.medinalis.1.Transcriptome analysis of major chemosensory organsFirstly,we constructed six transcriptoms of major chemosensory tissues: antennae,protarsus and reproductive organs from female and male adults.67357 of unigenes were obtained and in which 36966(about 54.8%)can be matched to the known databases.Then,97 of chemoreception gens were identified from the database,containing 14 chemosensory proteins(CSPs),30 odorant binding proteins(OBPs),36 odorant receptors(ORs),15 Iontropic Receptors(IRs)and 2 Sensory Neuron Membrane Proteins(SNMPs).Expression levels in different tissues were evaluated based on the FPKM value.Some genes are proved to expressed in unique tissues or have different expression levels between males and females,providing basic information for chemosensory mechanism in rice leaf folder.2.Gene cloning and sequence analysis of three chemosensory proteins(CSPs)Three CSP genes were cloned by PCR and checked by sequencing.All of them contain 18-23 aa signal peptide,and as predicted,hydrophobic residues mainly existed in binding capacity,meanwhile the hydrophylic residues were on the surface during hydrophobic analysis.The expression levels of genes are associated with the sex,developmental stage and mate status.The results of q PCR showed that csp1 and csp2 expressed broadly in the tissues of rice leaf folder,especially in antennae,legs and wings,and the expression profile in female legs increased dramatically after mating;however,csp3 specificly expressed in antennae and the expression level in males is much higher than in females.3.Expression pattern and Functional analysis of Cmed CSP1Firstly,we expressed recombinatant protein Cmed CSP1-p ET22 b in E.coli.Polyclonal antibody Anti-CSP1 was generated from pure Cmed CSP1 and was used to test protein expression pattern in different tissues.Results showed that proteins can be detected in antennae,thorax,abdomen,legs and wings,which was consistent with the m RNA expression level.Cmed CSP1 antibody could be labeled in the sensilla basiconica,and with no significant difference between males and females.Few was labeled in female sensilla trichoidea.Next,fluorescene competitive binding assay showed that Cmed CSP1 had high binding affinities to most tested compounds,especially alkanes and terpenoids.EAG responses of Nerolidol,?-Cedrene,R-(+)-Limonene,p-Cymene,Hexadecane and Sabinene decreased dramatically after ds RNA-CSP1 treated when compared with control.In summary,Cmed CSP1 plays an important role in transporting host attractants.4.Expression pattern and Functional analysis of Cmed CSP2Recombinant protein Cmed CSP2-p GEX-6p-1 was first purified by GSTrap 4B and used as target protein for polyclonal antibody after GST tag was removed by Precission Protease.Results of Western blot showed that proteins expressed broadly in tested tissues,especially in antennae,legs and wings.CSP2-antibody was labeled in sensilla basiconica of females and males,as well as female sensilla trichodea.Binding assays displayed that Cmed CSP2 had strong binding properties with tested compounds,similar with Cmed CSP1.However,nonadecane,henicosane,nerolidol,2-heptonone,?-cedrene,and cis-?-farnesene elicited lower EAG responses to moths that treated with ds RNACmed CSP2 when compared with control,indicating that Cmed CSP2 was involved in the chemosensation of host-and oviposition site seeking.The relationship between the structure and function of CSP2 was studied by homology modeling and molecular docking as well as site-directed mutagenesis.Results showed that Arg67 and Phe73 were the key binding sites of aromatic compounds.Similar to common proteins,C-terminal of Cmed CSP2 was proved also to be playing important roles in ligand binding.5.Expression pattern and Functional analysis of Cmed CSP3Pure Cmed CSP3 were obtained from recombinant protein Cmed CSP3-p GEX-6p-1 with GST tag removed by Precission Protease.After SDS-PAGE analysis,the purified Cmed CSP3 were used for polyclonal antibody generation and subsequent experiments.Protein expression profiles showed that CSP3 only expressed in antennae and expressed more in males than in females,providing the same results as q PCR.Immuno-locaction results showed that antibodies were labeled in the lymph of male sensilla trichodea which considered to the pheromone-sensitive sensilla,and only a few was labeled in female sensilla basconica.Competitive binding assay performed in vitro exhibited that Cmed CSP3 only bound to Cyclohexanol and some terpenoids,such as ?-Cedrene,(-)-trans-Caryophyllene and so on.In addition,Cmed CSP3 had high binding affinity to two major sex pheromone components: Z11-16: Ac and Z11-16: Al.EAG responses decreased dramatically to long chain alkanes: Henicosane,Eicosane,and Terpenoids such as ?-Cedrene,Sabinene and(-)-trans-Caryophyllene,as well as Z11-16: Ac,after knocking down the target gene,which strongly supported our hypothesis.6.Functional analysis of odorant receptors in C.medinalisWe cloned six putative general Odorant receptor genes and ORco from antenna.When predicted with software online,as expected all these ORs have seven transmembranes.After that,phylogentical tree was constructed based on N-J method and results showed these Ors were highly divergent except for Cmed OR1 and Cmed OR39.Cmed OR1 and Cmed OR39 were clustered to one clade,and might have similar function.Then Occytes and voltage clamp were used to study the function of Ors/ORco complex individually.Of six Ors,only two could respond to VUAA,the specific ligand of Orco,which indicate that ORs were sucessfully formed channels with ORco.When we tested the two ORs with host volatiles,only Cmed OR1 had binding affinity to only one compound which is Phenthylacetaldehyde and further behavioral assays is needed.The identification and functional characterization of CSPs and ORs in C.medinalis will provide us with new straterges to control this pest through interring their olfaction perception and enlarge our knowledge of the molecular and cellular basis of insect chemoreception.