小峰熊蜂蜂毒磷脂酶A2基因的克隆及表达分析

磷脂酶A2 （phospholipase A2, PLA2）是蜂毒主要成分， 也是蜂毒的主要过敏原， 在熊蜂个体和群体防御方面具有重要功能。为了探究熊蜂A2基因的生物学功能， 本研究以小峰熊蜂Bombus hypocrita为材料进行了蜂毒PLA2基因的克隆、 鉴定与表达特性分析。结果表明： 该基因全长为2 272 bp， GenBank登录号为KF214771， 由4个外显子和3个内含子组成， 编码区（CDS）长为543 bp， 共编码180个氨基酸残基。氨基酸序列相似性分析显示， 成熟的小峰熊蜂PLA2（含有136个氨基酸）与其他蜂类PLA2的氨基酸序列相似性较高， 均包含10个保守的半胱氨酸残基、 1个保守的Ca2+结合位点和1个酶活性中心。基于PLA2氨基酸序列的系统进化树分析表明， 熊蜂属Bombus与蜜蜂属Apis在不同分支上， 属单系群， 且蜜蜂属分化较早。荧光定量PCR结果表明， PLA2基因在小峰熊蜂各日龄均有表达， 且随日龄增长， 表达量呈先上升后下降的趋势， 10日龄时出现峰值， 其表达量显著高于其他日龄（P<0.05）。半定量PCR结果表明， PLA2基因在毒腺、 卵巢、 中肠中表达量较高， 在足、 触角、 食道腺中表达量较低， 在脂肪体、 肌肉、 神经、 气管、 复眼、 脑中未表达。本研究探明了小峰熊蜂PLA2的基因结构及其表达特性， 丰富了熊蜂PLA2的生物学基础， 为进一步深入研究熊蜂PLA2生物学功能和作用机制以及开发蜂毒生物制剂等鉴定了基础。

Cloning and expression analysis of a gene encoding phospholipase A2 from the venom of Bombus hypocrita (Hymenoptera: Apidae)

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.