Preliminary Study And Application On The Sex Pheromone Recognition Mechanism Of Tirathaba Rufivena Walker

Tirathaba rufivena Walker is one of the main pests of Areca catechu.The larvae of T.rufivena eat areca nuts and flower buds.With characteristics of strong concealment,large appetite and high rate of reproduction,the damage it caused is devastating and last for a long period.The larvae are hidden in the buds of the tall areca trees,it is difficult to use chemical pesticides to control this pest effectively.The use of sex pheromones traping to control the adults is one of the feasible management strategies.In this study,the calling behavior rhythm was studied,the components of sex gland were identified by gas chromatography-mass spectrometry(GC-MS),the electroantennography(EAG)test and Y-tube olfactometer were used toscreem the active components for T.rufivena.The transcriptome of T.rufivena was constructed and odorant-binding proteins(OBPs)were identified and analyzed.The prokaryotic expression,fluorescence competitive binding assay and molecular docking experiment were used to investigate the functions of odorant-binding protein 4 of the T.rufivena(Truf OBP4).The main research results are as follows:1.The calling behaviour of male adults occurred in the dark period and the courtship rate reached the peak at 9th hours at dark period and then begins to decline.2.We totally identified 15 compounds,including nine hydrocarbons,one ketone,alcohol,terpene and ester,and two kinds of phenols.3.Based on the analysis of the transcriptome data,13 OBPs,have full open reading frames(ORF),were identified.The ORF of Truf OBP4 is 450 bp in length and the p I is4.98.The signal peptide at the N-terminus consisted of 19 amino acid residues.Truf OBP4 contains six conserved cysteine sites,which was consistent with the characteristics of the Classic OBP family.4.The expression of Truf OBP4 in vitro was performed,and the binding ability of Truf OBP4 with pheromone ligands was analyzed by fluorescence competition binding assay and molecular docking.The experimental results showed that Truf OBP4 has the strongest binding ability to 2,4-Di-tert-butylphenol and vanillin,the binding constants are7.53±1.14 μmol/L and 7.15±0.66 μmol/L,respectively,and followed by6,10,14-Trimethyl-2-pentadecanone and Heneicosane,the binding constant is 18.15±2.17μmol/L and 20.37±4.5 μmol/L.The docking results showed that the free binding energy is-5.1,-4.4,-4.8 and-6.3 kcal/mol。5.Results of EAG and Y-tube olfactory section showed that the T.rufivena had a higher responese to vanillin,6,10,14-Trimethyl-2-pentadecanone and heneicosane than other compounds,indicats the behavioural regulatory activity of sex pheromone for T.rufivena.In this thesis,the male sex pheromone of T.rufivena was extracted and identified.And the recognition mechanism of sex pheromone was also explored from the molecular biology perspective.It will provide a theoretical basis for monitoring and controlling the T.rufivena.