Cloning And Functional Characterization Of The Olfactory Related Protein Genes Of Bactrocera Minax And Asian Citrus Psyllid

Bactrocera minax and Asian citrus psyllid（ACP）are the two important citrus pests that harm the healthy development of citrus industry.In citrus orchard,Bactrocera minax only lay eggs in citrus fruits,leading the fruit early dropping and rotting,which seriously affects the quality of citrus fruits.And ACP are important insect vectors for the transmission of citrus huanglongbing and mainly lay eggs on fresh leaves.Therefore,the study of the specific mechanism of B.minax and ACP on host volatiles recognition is conductive to exploration and develop the efficient prevention and controlling measures.Insects’recognition of host is mainly dependent on olfactory system and visual system,among which the olfactory of insects is an important factor affecting their feeding and oviposition,odorant binding protein（OBP）and chemosensory protein（CSP）are the peripheral proteins of olfactory system of insects to recognize the external environment odorants,which is the first step to activate the olfactory response of insects.The feeding or laying behavior of B.minax and ACP are affected by the volatiles of the citrus host.It is of great significance to screen and identify the functional proteins and their mechanism to volatile substances.Therefore,this paper is focus on the specificity of Bmin OBP9 protein binding to citrus volatiles and its molecular mechanism by analyzing and comparing the GOBP99a protein of B.minax with B.dorsalis,B.cucurbitae and B.tau,.At the same time,the genes of OBP and CSP were cloned and their sequence characteristics were compared.And the molecular mechanism of Dcit CSP12 protein of ACP in recognition of citrus volatiles was identified.The purpose of this paper is to explore the molecular mechanism of OBP and CSP proteins in the identification of citrus scent,and to provide theoretical guidance for the development of efficient control of the behavior of B.minax and ACP.The main results are as follows:1.Bmin OBP9 protein of B.minax in recognition of citrus volatiles was specific,and 6 amino acids（S24,L36,E53,N68,D112 and S118）were the binding sites to affecte the specificity.Bmin OBP9 was cloned by homologous comparison and uploaded to NCBI database（accession number:KY463449.1）.The results of relative expression showed that Bmin OBP9 was mainly expressed in the abdomen and ovipositor of B.minax.Phylogenetic tree results showed that Bmin OBP9 belongs to the GOBP99a family of Tephritidae,and has the highest homology with Bdor GOBP99a of B.dorsalis,Zcuc OBP99a of B.cucurbitae,and Ztau GOBP99a of B.tau,it has been reported that silencing Bdor GOBP99a can reduce the rate of oviposition of B.dorsalis.The fluorescence competitive binding assay results showed that Bmin OBP9,Bdor GOBP99a,Zcuc OBP99a and Ztau GOBP99a could bind all the 11 citrus volatiles,and the ability to citrus volatiles was ranked as:Bmin OBP9>Bdor GOBP99a>Zcuc OBP99a=Ztau GOBP99a,it is consistent with the preference of these 4 kinds of Bactrocera.The amino acid sequence alignment results showed that Bmin OBP9 had 9 specific amino acids（S24,H29,L36,E53,N68,E94,P99,D112,and A127）,these sites were mutated at site-directed mutagenesis,resulting in the decreased binding ability of Bmin OBP9 to citrus volatiles.Among them,S24,L36,E53,N68,D112 and S118 were the sites that affected the ability of Bmin OBP9 most.In conclusion,six specific amino acid sites,S24,L36,E53,N68,D112 and S118 lead to the strong binding ability of Bmin OBP9 to citrus volatiles,and is one factor influencing the specific laying of eggs in citrus fruits.2.The Dcit OBP3,Dcit OBP6 and Dcit CSP12 were the functional proteins to recognize citrus volatiles,and among which,Dcit CSP12 was the stronger one.Basic on the olfactory transcriptomics and NCBI database,9 OBP genes（Dcit OBP1～9）and 8 CSP genes（Dcit CSP2～4,Dcit CSP7,Dcit CSP8,Dcit CSP10～12）were cloned.Homologous comparison analysis results showed that the OBP sequence of citrus psyllid contains 5 conserved cysteines（Cys,C）,and is characterized by C₁-X_22-52-C₂-X₃-C₃-X₄₆-C₄-X_8-9-C₅,and CSP sequence contains 4 conservative cysteine residues,but also has a conservative arginine residues（Arg,R）,a conservative lysine residues（Lys,K）and a conservative proline residue（Pro,P）,the sequence features was R-X₇-C₁-X_6-7-C₂-X_7-8-K-X₃-P-X₆-C₃-X₂-C₄.8 Dcit OBP proteins（Dcit OBP1～3,Dcit OBP5～9）and 6 Dcit CSP proteins（Dcit CSP2,Dcit CSP3,Dcit CSP7,Dcit CSP8,Dcit CSP10 and Dcit CSP12）were purified by prokaryotic expression from the cloned genes.Dcit OBP3,Dcit OBP6 and Dcit CSP12 can bind 11volatiles of citrus in vitro（Capoaldehyde,Z-2-Hexenal,Linalool,Limonene,Myrcene,α-Terpineol,4-Carvomenthenol,Terpinolene,Ocimene,β-Caryophyllene andγ-Terpinene）from fluorescence competitive binding assay.According to the dissociation constant of receptor protein and volatiles,Dcit CSP12 had the strongest binding ability to citrus volatiles,followed by Dcit OBP6 and Dcit OBP3.Dcit CSP12has the strongest binding affinity withβ-caryophyllene among the 11 citrus volatiles.3.The L51,K67,L98 and V54 were the siginificant binding sites of Dcit CSP12 of ACP to recognize citrus volatiles.The 3D structure of Dcit CSP12was predicted by phyre2.0,an online software,and Chem3D was used to minimize the energy of 11 citrus volatiles.After molecular docking with molecular visual docking（MVD）software,it showed that Dcit CSP12 has 8 common amino acid sites（I50,V54,C55,K63,F66,K67,Y94,and L98）for 11 citrus volatiles,while it has 2specific amino acid sites（L51 and T70）in relation toβ-caryophyllene.The 10 sites were mutated by sites-directed mutagenesis with one site,double sites,three sites and four sites to identify the important amino acid sites,total 31 mutant proteins were purified and were detected by probe 1-NPN,and the results showed that the amino acid sites L51,K67 and L98 of Dcit CSP12 negatively regulated their binding ability to citrus volatiles,while V54 positively regulated its binding ability to citrus volatiles.