Molecular Cloning And Functional Analysis Of Olfactory-related Gene GOBP2 And Orco From The Legume Pod Borer, Maruca Vitrata Fabricius (Lepidoptera:Crambidae)

Insect olfactory signaling system is a very important signal identification mechanism.There are lots of Protein families in this system including odorant binding proteins family(OBPs), Chemosensory proteins family(CSPs), odorant receptor proteins family(ORs) and Odor-degrading enzyme proteins family (ODEs). According to the characteristics of the amino acid sequence, the OBPs are divided into GOBPs、PBPs、 general OBPs. Odorant receptor protein family is divided into Orco and general ORs.The bean pod borer (Maruca vitrata) is a parasitic pest over the world. It is harmful mainly to cowpea, beans and other leguminous vegetables and its larvae mainly eats flowers, fruits and seeds. A large number of vegetables are damaged. It is difficult to reduce the loss in legume crops production. It is an important way to preventing and controling the pest by interfering pheromones. However, the molecular mechanism of olfaction is unclear in bean borer pheromone identification. So it is limited in application. A feature of the bean borer females laying eggs is to locate and spend eggs in flower. GOBPs protein and specific ORs mainly involved in the process of identifying flowers. We use PCR technology, race technology to clone full-length gene sequence of MvitGOBP2, and using the prokaryotic expression and fluorescence competitive binding experiment to study the characteristic of combination ability of MvitGOBP2 with 19 kinds of volatile compounds. This study aims to analyze and discuss olfactory-related gene function in host recognition and positioning spawning location. The results can reveal parts of the molecular mechanism of bean borer olfactory identification, and also provide fundamental material for the development of new and efficient bean borer attractants, repellents and behavioral disturbances drugs. In addition, we further studied the characteristics of the co-receptor(Orco) protein. The main results are as follows:(1).Functional analysis of general odorant binding protein GOBP2 in host recognition and spawning location of the bean pod borer.By designing primer and in the help of PCR and RACE technology, the full-length sequence MvitGOBP2 has been cloned. The coded boxes of MvitGOBP2 contains 486 nucleotides encoding 161 amino acids. The first twenty amino acid sequence is the signal peptide sequence. Gene expression levels of female bean borer were studied in different organizations and different developmental stages using fluorescence quantitative techniques. The results show MvitGOBP2 is mainly expressed in the female antennae, and high expressed in the adult female.By designing primers with restriction sites, the sequence MvitGOBP2 is cloned, and then builded recombinant expression vector pET32a-GOBP2, and then obtained the supernatant MvitGOBP2, and then obtained a polyclonal rabbit resistant. Fluorescence binding assays demonstrated that MvitGOBP2 had various binding affinities for 19 volatile odorant molecules, and butanoic acid octyl ester shows the highest binding affinity. MvitGOBP2 may distinguish these different odorant molecules in the progress of M. vitrata recognize and oviposit on the flower of the host plant, according to the differences of chain length, functional group and alkene geometry of volatile chemicals.(2).The full-length Cloning and Functional Analysis and of odorant receptor proteins Orco of the bean pod borer.By designing primer and in the help of PCR and RACE technology, the full-length sequence MvitOrco has been cloned. The coded boxes of MvitOrco contains 1422 nucleotides encoding 473 amino acids. It predictes that molecular weight is 53.46 KDa and the protein isoelectric point is 8.24. By the multiple sequence alignment and Phylogenetic analysis, MvitOrco gene has a very high conservative. The similarity with cucumber moth (Diaphania indica) amino acid sequence is 98%, and is higher in the same conservative families. Analyzed the expression of MvitOrco in different tissues of male and female adults and developmental stages using fluorescence quantitative techniques, MvitOrco is mainly highly expressed in the male antennae and is highly expressed in adult male.