Yu-Juan Yao,Ning-Na Yin,Lin-Mei Pu,An-Jing Yang,Nai-Yong Liu
Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance , focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1 , CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 μM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions.
Yin-Lan Liang,Shun-Si Li,Ning-Na Yin,Shu-Lin Li,Yu-Yue Lu,Nai-Yong Liu
The cerambycid beetles are key players for the sustenance of biodiversity in the forest ecosystem, but in most cases are well known due to their harmfulness to agricultural and forest plants. Here, we characterized the odorant binding protein (OBP) gene family in Rhaphuma horsfieldi , emphasizing the roles of RhorOBP1 in odorant reception and insecticide sequestering. A homology-based search led to the identification of 35 RhorOBP genes with a major distribution in the Minus-C OBPs clade (21/35 genes). Expression profiles showed that RhorOBP1 – RhorOBP4 had the abundant expression in antennae. Binding assays revealed that the four RhorOBPs exhibited diverse odorant response profiles tuned differentially to various classes of plant odorants, comprising walnut-derived host volatiles and ordinary floral scents. Two broadly tuned RhorOBP1 and RhorOBP2 exhibited different chain length-dependent binding properties to 10C 12C alcohols, aldehydes or acetates. Compared with other three proteins, RhorOBP1 reduced the binding to ligands with high affinities at pH 5.0 (1.27–6.72-fold differences relative to pH 7.4). Molecular docking and point-mutation experiments confirmed that Ser107, Tyr118, Tyr119 and Phe120 situated in the binding pocket of RhorOBP1 were critical determinants for the recognition of 14, 15, 10 and 10 compounds, respectively. On the other hand, RhorOBP1 could strongly bind six insecticides, particularly chlorpyrifos (dissociation constant, K i = 3.69 ± 0.74 μM). This study has provided insights into different binding properties of four antenna-enriched RhorOBPs in R. horsfieldi and identifies a dual role of RhorOBP1 in the binding of odorants and insecticides.
危险品化学品经营许可证（带存储）