Abstract: 【Objective】The objective of this study is to clone full-length cDNA sequence of the soluble trehalase gene (Tre-1) in Bombus hypocrita, predict the physicochemical properties, clarify its expression in different tissues and developmental stages, and to understand the function of this gene in the development of B. hypocrita.【Method】 Degenerate primers were designed by using Primer Premier 5.0 software according to the conservative sequences of Tre-1 in B. terrestris and B. impatiens to get the corresponding conservative fragment in B. hypocrita. Gene-specific primers were designed according to the conserved fragment. The rapid-amplification of cDNA ends (RACE) method was used to clone B. hypocrita 5′ and 3′ sequences, then the open reading frame (ORF) was amplified by the specific primers based on the initiation codon and termination codon near 5′ and 3′, respectively. Different fragments were spliced by BioEdit to obtain the full-length cDNA. The full-length cDNA was analyzed by a variety of bioinformatics softwares such as ExPASy, SignalP 4.1, NetOGlyc 1.0 sever, ClustalW and MEGA 5.0. Finally, the relative expression of Tre-1 in different tissues and developmental stages in B. hypocrita were analyzed by real-time PCR and 2^-ΔΔCt method.【Result】The full-length cDNA of B. hypocrita Tre-1 is 3 129 bp, and designated as BhTre-1 (GenBank number: KJ025078). The bioinformatics analysis suggested that BhTre-1 has an ORF of 1 743 bp, a 5′ (untranslated region) UTR of 441 bp and a 3′ UTR of 945 bp. The ORF of BhTre-1 encodes a polypeptide of 580 amino acids with a predicted molecular weight of 67.16 kD, and an isoeletric point value of 5.95. Thededuced amino acid sequence has six predicted sequences of Asn-Xaa-Ser/Thr, a signature peptide, a highly conserved glycine-rich region (GGGGEY), and two conserved signature motifs (PGGRFKEFYYWDSY and QWDFPNAWPP), but no transmembrane domain. Homology comparison found that BhTre-1 has the greatest similarity to B【Conclusion】The cDNA sequence of BhTre-1 was successfully cloned from B. hypocrita, and the properties were similar with Tre-1 of other insects. BhTre-1 hasthe highest expression in midgut. In addition, the expression of BhTre-1 of adult worker was higher than at larva and pupa stages,and the expression of BhTre-1 increased first and then decreased. This study indicated that the expression patterns in different tissues and developmental stages of B. hypocrita, which will lay a foundation for biological functional research of BhTre-1.. terrestris BtTre-1 (99%)and B. impatiens BiTre-1 (98%), it also has greater similarity to Tre-1 of Apis mellifera and A. florea which have 78% sequence homology. The phylogenetic tree analysis showed that BhTre-1 was first clustered with BtTre-1, BiTre-1, AmTre-1 and AfTre-1. The results agreed with the sequence homology analysis. Tissue-specific expression results indicated that BhTre-1was expressed in all the major tissues, it had the highest expression in midgut, then Malpighian tubules, and lower expression in other tissues. The expression of BhTre-1 in adult worker was higher than at larva and pupa stages, and it increased from the 1st day after emergence, and had the maximum expression in the 15-day-old adults, then, it decreased with the age.

Key words: Bombus hypocrita, soluble trehalase gene, clone, sequence analysis, gene expression