Function Alanalysis Of Olfactory- Related Gene GOBP1, CSP1 And OBP1 From The Legume Pod Borer, Maruca Vitrata Fabricius (Lepidoptera:Crambidae)

In the long history of life evolution, insects have been evolved extremely sensitive olfactory system, enabling it to perceive and discriminate a variety of chemical stimulus from surrounding, such as location of food source and habitat, sexual exchanges, mate-seeking and so on. Thus, olfactory and chemical sensory system plays important roles in the various behavior of insect. At present, more and more insect pheromone components were determined and used to interference and block insect olfactory identification, suggesting their application in the field has been one of the important measures of integrated pest prevention and biological control. The legume pod borer, Maruca vitrata Fabricius (Lepidoptera:Crambidae) is an important pest in the tropical and subtropical regions and damage cowpea. Due to the strong drill decay behavior of the larvae, prevention of this pest usually relies on chemical pesticide, which resulting in a serious threat to legumes products and security of supply. The larvae of M. vitrata like to eat young flower, leaf and pod of leguminous plants, which cause huge losses to leguminous vegetables.Based on the transcriptome sequencing results of antenna, three olfactory related genes (MvitGOBP1, MvitCSP1 and MvitOBP1) was cloned from M. vitrata. Expression pattern of MvitGOBPl, MvitCSPl and MvitOBP1 were examined by Real-Time PCR in different tissues of M. vitrata adults. Moreover, MvitGOBPl, MvitCSP1 and MvitOBP1 were expressed in E. coli and purified using by the High-Affinity Ni-NTA Resin. Subsequently, the binding affinities of three recombinant proteins (MvitGOBP1, MvitCSP1 and MvitOBP1) with floral volatiles from host-plant Vigna unguiculata were studied by fluorescence competitive binding assays. The key binding sites of MvitGOBP1 and MvitCSP1 were predicted through the three-dimensional structure modeling and molecular docking. The main results were as follows:1. Gene Cloning, sequence and phylogenetic analysis of MvitGOBP1, MvitCSPl and MvitOBP1.Three genes were cloned and named MvitGOBP1, MvitCSPl and MvitOBP1. Sequence analysis showed that the length of three ORF were 486bp,387bp and 471bp, respectively. Multiple sequence alignment demonstrated that MvitGOBPl,MvitCSP1 and MvitOBP1 amino acid sequence had significant similarities with the same sequences from other lepidopterans. The neighbor-jointing tree revealed that MvitGOBPl was clustered with CmedGOBPl of Cnaphalocrocis medinalis, MvitCSP1 was clustered with CmedCSP of Cnaphalocrocis medinalis, and MvitOBP1 was clustered with CmedOBP11 of Cnaphalocrocis medinalis.2. Tissue distribution of MvitGOBP1, MvitCSP1 and MvitOBP1Quantitative real-time PCR method was used to measure tissue-specific mRNA expression patterns of MvitGOBP1, MvitCSP1 and MvitOBP1 genes. Transcript abundance for each gene was determined for multiple tissues (antenna, heads, thoraxes, abdomens, legs and wings) from the adults of M. vitrata. The expression profiles analysis revealed that the three genes were predominantly expressed in antennae and the levels of transcripts were very low in heads, legs and wings.3. Prokaryotic expression and purification of MvitGOBP1, MvitCSP1 and MvitOBP1The recombinant plasmid was transformed into competent E. coli BL21 (DE3) cells and induced by IPTG for expression of recombinant MvitGOBP1, MvitCSP1 and MvitOBP1. After the supernatant was obtained, target protein was purified using the High-Affinity Ni-NTA Resin and His-tag was removed by enterokinase. SDS-PAGE analysis showed that the molecular weight of three recombinant proteins were about 25kD-35kD.4. Fluorescence binding assaysThe fluorescence binding results indicated that MvitGOBP1 can effectively bind with 1H-Indol-4-ol, Butanoic acid butyl ester and D-Limonene. MvitCSP1 can effectively bind with 1H-Indol-4-ol and 2-methyl-3-phenylpropanal. MvitOBP1 can effectively bind with 1H-Indol-4-ol and Butanoic acid butyl ester.5. Field trapping experimentSix effective floral volatile components from 17 volatile odorant molecules of host plant including butanoic acid butyl ester, limonene,4-ethylpropiophenone,1H-indol-4-ol, butanoic acid octyl ester and 2-methyl-3-phenylpropanal were tested in the field trapping experiment. The trapping experiment results showed that this six key floral volatile components could effectively attract female moths, which limonene had the highest trapping effect in the field.6. Three-dimensional structure modeling and molecular dockingThe results showed that the binding pocket of MvitGOBP1 and MvitCSP1 were mainly formed by its hydrophobic amino acids, which may play important roles in the process of MvitGOBP1 and MvitCSP1 binding key floral volatile components. Molecular docking results revealed that Asn83 and Arg127 may be the key binding sites of MvitGOBP1, which Thr26 and Glu63 are the key binding sites of MvitCSP1.In conclusion, the expression profiles analysis suggested that three genes were expressed in different tissues. The fluorescence competitive binding and molecular docking results indicated that MvitGOBPl, MvitCSPl and MvitOBPl play important roles in the location of suitable hosts and oviposition of M. vitrata.