关键词: 小峰熊蜂, 磷脂酶, 基因克隆, 序列分析, 基因表达

Abstract: Phospholipase A2 (PLA2) is the major component of bee venom as well as the main allergen of venom, and plays a key role in the individual and colony defense of bumblebee. A PLA2 gene from Bombus hypocrita was cloned, identified and expressed in this study in order to clear its characteristics and function. The results indicated that the full-length cDNA of B. hypocrita PLA2 gene is 2 272 bp in length, consisting of 4 extrons and 3 introns. The coding region, 543 bp in length, encodes a 180-amino-acid protein. Comparative analysis revealed that the mature PLA2 (consisting of 136 amino acids) possesses features consistent with PLA2s from other bees, including ten conserved cysteine residues, a highly conserved Ca²⁺-binding site and one active site. Phylogenetic tree of PLA2 sequences showed that Bombus PLA2 clustered into a branch independent with Apis PLA2 which differentiated earlier. The real-time PCR analysis showed that B. hypocrita PLA2 was expressed in different day-old adults, and the expression level increased first and then decreased, with the maximum expression level in the 10 day-old adults. The PLA2 gene were highly expressed in the venom gland, ovary and midgut, less expressed in legs, antennae and esophageal glands, and not expressed in fat body, muscles, nerve, tracheae, compound eyes and brain. This study revealed the characteristics and expression of PLA2 gene in B. hypocrita, which is useful not only for further studies on the function and mechanism of Bombus PLA2, but also for the development of new reagent of bee venom.

Key words:  Bombus hypocrita, phospholipase (PLA), gene cloning, sequence analysis, gene expression