Abstract: 【Aim】 To explore the sequence characteristics and biochemical properties of cuticular proteins from Trypoxylus dichotomus. 【Methods】 RT-PCR was used to clone cuticular protein genes of T. dichotomus, and the structural features and phylogeny of cuticular proteins were analyzed by bioinformatics methods. Recombinant cuticular proteins of T. dichotomus were expressed in Escherichia coli expression system, and purified by metal-chelating affinity chromatography. In vitro binding experiments were performed to detect the binding ability of cuticular proteins of T. dichotomus with chitins including α-chitin, β-chitin, chitosan and colloidal chitin. Liquid-liquid phase separation (LLPS) was observed and determined. 【Results】 Two cuticular protein genes TdCPR12611 (GenBank accession no.: MT813021) and TdCPR7854 (GenBank accession no.: MT813022) of T. dichotomus were cloned and obtained. Phylogenetic analysis results showed that TdCPR12611 is closely related to OtCP-1 from Onthophagus taurus, while TdCPR7854 is closely related to OtCP-acp20-1, OtCP-acp20-2, and OtCP-acp20-3 from O. taurus, all of which belong to the CPR_RR-2 family. Recombinant TdCPR12611 and TdCPR7854 proteins were expressed by prokaryotic expression and purified. The two recombinant proteins had different binding abilities with four types of chitins, among which 41.4% of TdCPR12611 could bind with chitosan, while 62.3% of TdCPR7854 could bind with β-chitin. TdCPR12611 was predicted to have a relatively disordered structure and could spontaneously coacervate at room temperature to form liquid-liquid phase separation, while TdCPR7854 could not. 【Conclusion】 In this study we assayed and analyzed the sequence characteristics and chitin binding properties of two RR-2 cuticular proteins, TdCPR7854 and TdCPR12611 of T. dichotomus. The results not only deepen our understanding of insect cuticle assembly mechanism, but also provide ideas for the development of protein biomimetic materials.

Key words: Trypoxylus dichotomus, cuticular protein, CPR protein, RR-2 family, chitin binding, liquid-liquid phase separation, bionic material