Cloning And Expression Analysis Of Odorant Binding Proteins In Aphis Gossypii Glover

Sensitive olfactory system help insect accurately sense chemical signals in the environment, directly or indirectly affect insect individual behaviors, such as host orientation, feeding, mating, searching for oviposition sites. Odorant binding proteins (OBPs) is of great significance in the insect olfactory signal transduction. The research of OBPs helps to understand the mechanism of insect olfaction recognition system. The sap-sucking insect Aphis gossypii Glover (Homoptera:Aphididae) is a cosmopolitan and destructive pest. Develop new pollution-free, effective technology for A.gossypii prevention and control is urgent. Molecular biology technologies were utilized to investigate biochemical characteristics, structure, and expression analysis of OBPs in A.gossypii olfactory recognition. Our findings lay the foundations for discovery of new A.gossypii prevention or control technology. The main results are as follows:1AgosOBPs gene cloning and sequence analysisEight AgosOBPs gene segments were identified using the A.gossypii RNA-seq transcriptome dataset constructed by our laboratory. Degenerate primers were used for cloning cDNA sequences that encoded open reading frames of AgosOBPs genes. AgosOBPs encoded141-243amino acids. Their predicted molecular mass were15.745-26.056kDa. AgosOBPs had a hydrophobic signal sequence with18-24amino acids in N-terminal region. The average hydrophobicity was0.502-0.071and all AgosOBPs belong to the hydrophilic protein. AgosOBPs was characterized by six conservative cysteine (Cys) residues. Besides AgosOBP4, the cysteine spacing pattern of seven AgosOBPs is Cl-X22-28-C2-X3-C3-X37-43-C4-X8-24-C5-X8-C6. Disulfide bonds composed of cysteine residues support the3-D structure of AgosOBPs. Both amino acid sequence homology analysis and phylogenetic relationship analysis showed that AgosOBPs have high homology among Homoptera Aphidae insects.2Validation of reference genes for the relative quantification of gene expression in A.gossypiiReal-time quantitative PCR was used to test the expression level of six traditional (18SrRNA, ACTB, RPL3, PPI, TBP, RPII) and one novel (DIMT) candidate reference genes in different morphs (alatae and apterous), hosts (Hibiscus syriacus and cotton), tissues (antenna, head, thorax, leg, and abdomen), and developmental stages (nymph and adult) of A.gossypii. Three analysis methods (geNorm, BestKeeper, and NormFinder), and a ranking system RefFinder were chosen to assess the expression stabilities. Our data indicated that PPI and DIMT were the most reliable reference genes for A.gossypii in different morphs, hosts, and tissues. PPI, RPII, and DIMT are suitable reference genes for different developmental stages samples. Even though ACTB and18SrRNA were widely used as reference genes, they were not reliable for most A.gossypii samples.3Expression analysis of six AgosOBPs by qPCRThe expression analysis of six AgosOBPs genes (AgosOBP2, AgosOBP3, AgosOBP4, AgosOBP6, AgosOBP7and AgosOBPS) was investigated by qPCR. AgosOBP2, AgosOBP3, AgosOBP6and AgosOBP8have two expression peaks in early nymphy and late adult, while AgosOBP4and AgosOBP7only appeared one expression peak. AgosOBP7has high level expression in A.gossypii antenna. AgosOBP2has the high expression level in A.gossypii head, AgosOBP3and AgosOBP4in A.gossypii abdomen have high expression level. AgosOBP6also have high expression level in A.gossypii head and antenna. AgosOBP8have high expression level in. A.gossypii apart from leg and wing. AgosOBP2, AgosOBP6and AgosOBP8have a higher expression level in alatae adult than apterous adult. AgosOBP4have a higher expression level in alatae nymphy than apterous nymphy. AgosOBP3and AgosOBP7expressed higher in apterous adult than alatae adult. AgosOBP2had a higher expression level in alatae adult from winter host hibiscus than alatae adult from summer host cotton. However AgosOBP3and AgosOBP6had a higher expression level in alatae adult from summer host cotton than alatae adult from winter host hibiscus. AgosOBP4and AgosOBP8expressed higher in alatae adult from autumn cotton and hibiscus. AgosOBP7expressed higher in alatae adult from seedling cotton and hibiscus. AgosOBP2, AgosOBP3, AgosOBP6and AgosOBP7expressed higher in apterous adult from summer host cotton than from winter host hibiscus. The expression analysis of AgosOBPs is helpful to understand their different physiological functions.4Prokaryotic expression of AgosOBPsProkaryotic expression vector of six PGEX/AgosOBPs gene were construeted and sueeessfully expressed in E.coli BL21(DE3) induced by IPTG. The expressed produets were consistent with the size of predieted reeombinant protein by SDS-PAGE. Our findings lay the foundations for understanding the structure and function of AgosOBPs.