Abstract: 【Objective】 The objectives of this study are to clone the ultraspiracle protein gene of Apolygus lucorum (ALUSP) and obtain the recombinant protein of prokaryotic expression, and to reveal the function of ALUSP and offer a basis for research of the ecdysis and metamorphosis of A. lucorum. 【Method】 The conserved sequence of A. lucorum was obtained by using the degenerate primers from the known USP genes of the other insects. A new set of gene-specific primers matching the primers in the 3′- and 5′-full RACE kit were designed and the original sequence files were assembled to obtain the complete cDNA sequence of the ALUSP. The characteristics of ALUSP gene sequences and which coding proteins were analyzed by using the bioinformatics methods, and the phylogenetic tree was also constructed. To construct its prokaryotic expression vector, T-vector containing ALUSP was dual-enzyme digested by EcoR I and Xho I. The expression vector was induced to express at 15, 25, 30 and 37℃. ALUSP purified protein gene in functional areas was gotten by the GST agarose affinity chromatography and molecular sieve chromatography.【Result】The open reading frame of ALUSP was 1 005 bp in length, encoding a 334 amino acid polypeptide with a predicted molecular weight of 56.63 kD and the theoretical isoelectric point of 8.42. Protein signature analysis revealed that the protein encoded by ALUSP shared typical structural features of ultraspiracle proteins with other insects, including A/B domain (249 bp), C domain (198 bp), D domain (69 bp) and E domain (489 bp), respectively. C domain was highly conserved, including two zinc fingers, a P-box, a D-box and a nuclear location signal that was constituted of eight amino acids. D domain included a T-box which could identify the DNA components and E domain, including a bag structure, was constituted of eight α-helix and β-strand. The blast results showed that the amino acids sequence of ALUSP was the highest in similarity (57.82%) with Nezada viridula, and low in similarity with other insects, such as Melipona scutellaris (46.05%), Scaptotrigona depilis (45.94%), respectively. Phylogenetic tree analysis results showed that the USP genes from Hemiptera were more differentiation between Hymenoptera, which were located in different branches, ALUSP and N. viridula USP was the nearest in genetic evolution, which may be derived from common ancestors. The recombinant plasmid pEGX6P1-ALUSP was high-efficient expression in Rosetta gami B when induced by 37℃ and 1.0 mmol•L-1 IPTG. The 65 kD target protein from the strain containing ALUSP was obtained by the GST agarose affinity chromatography and the molecular sieve chromatography.【Conclusion】The full-length cDNA of ALUSP from A. lucorum was obtained and also got the recombinant protein. The protein had typical characteristics of insect ultraspiracle protein.

Key words: Apolygus lucorum , ultraspiracle protein , gene cloning , prokaryotic expression