| Bactrocera dorsalis(Hendel)is a notorious insect pest and have been widely distributed in China,especially in the area south of the Yangtze,causing serious losses to the agriculture economy every year.At present,the method of comprehensive control is adopted to control the number of B.dorsalis,and among them,traping and killing B.dorsalis using traping agents is a highly efficient and enviromentally friendly control measure,but the traping effect on female B.dorsalis is not satisfactory.Therefore,in order to analyze the function of the odorant binding protein,to provide basis for screening the female B.dorsalis traping agents,the study aimed to clone and express the odorant binding protein BdorOBP1 gene and fluorescence competitive binding experiment.The results of study are outlined below:(1)we got the BdorOBP1 a gene by PCR.The cDNA lengths of the gene is 480 bp,and code for 159 amino acids.The sequence alignment revealed 98%identity with the gene sequence in GenBank(GenBank ID:KC559112)and 99%similarity in the protein sequence.There are two amino acid differences,located at the N-terminal and C-terminal respectively.Signal peptide prediction shows that the N-terminal contain 26 amino acids,and the molecular weight of mature peptide is 15.89 KDa,the isoelectric point is 5.68.BdorOBP1a protein has 4 hydrophobic regions and a cross-membrane region.Compared with other insect sequences,the BdorOBPla protein has 4 conserved cysteines and belongs to Minus-C OBP.We constructed a Minmum-Evolution Tree using MEGA5.1 software and found that the similarity between BdorOBPla and Bctrocera latifrons BlatOBP99a is the highest.(2)We constructed the vector pET28a-BdorOBPla and made it transform into BL21(DE3).According to IPTG concentration,time and temperature optimization,the best inducing expression condition is induced at 30℃ for 6h under the condition of 0.5 mmol/L IPTG concentration,but the expression forms were all inclusion bodies.A large amount of purified target protein was obtained by nickel column after the inclusion bodies renaturation.(3)Fluorescence competitive binding experiment results showed that BdorOBP1a could bind 15 volatiles among 21 host volatiles and it has a strong binding ability with esters,aldehydes and ketones and a weak binding ability with alcohols.The binding capacity of P-ionone and Benzaldehyde is the best,and the dissociation contents is 31.1 and 31.8μmol/L respectively,followed by Ethyl caproate and Isoamyl acetate with dissociation contants of 45.3 and 47 μmol/L respectively.It is concluded that BdorOBP1a has the characteristics of selective identification and combination of various ligands and plays an important role in the localization of host plant odors.(4)Behavioral response results show that 8 single volatiles with low concentration(20 uL/mL)have some attractive effects on male and female immuture B.dorsalis(except Benzaldehyde),but the inducing effect of single volatiles with high concentrations(100 uL/mL 和 500 uL/mL)significantly decreased.8 host volatiles with different concentrations could significantly attract B.dorsalis(except benzaldehyde),and the attractiveness of these volatiles compounds to B.dorsalis female and male have no significant difference(except n-butyl acetate).Moreover,the combination of 100 uL/mL isoamyl and 20 uL/mL cis-3-hexenyl acetate significantly reduce the attractiveness to B.dorsalis female adults compared with single volatile.The combination of 100 μL/mL ethyl hexanoic and 20 μL/mL cis-3-hexenyl acetate was proved to attract the most quantity(62.4%)of mature B.dorsalis female adult compared with any single volatile.The mixture of three compounds have not notable increasing attractiveness to B.dorsalis. |
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