Cloning And Expression Of Phospholipase A2(PLA2) Gene From The Venom Of Bombus Hypocrita (Hymenoptera: Apidae)

Bee venom is one of important bee products, of which many proteins were identified to play a rolein anti-inflammatory, anti-cancer, antimicrobial, anti-radiation, insect disinfestation, and so on.Phospholipase A2(PLA2), the main component of bee venom, plays a key role in the defense systemboth in individuals and colonies, and is also the major allergen that causing people allergenic. Thecloning and expression of the PLA2gene from venom of the bumblebee Bombus hypocrita has beendone. The principle results are as follows:(1) Gene cloning of PLA2from B. hypocritaThe full-length cDNA of PLA2gene from B. hypocrita venom was cloned using RT-PCR andRACE. The full-length PLA2gene was obtained by chromosome walking. The PLA2gene contains2272bp and consists of4exons and3introns. Its coding sequence (CDS) contains543bp and encodes180amino acid residues. Comparative analysis revealed that the mature B. hypocrita PLA2(136aminoacids) possesses features consistent with the PLA2s of other bee species, including ten conservedcysteine residues, as well as a highly conserved Ca2+-binding site and an active site. Phylogenetic treeanalysis indicates that B. hypocrita PLA2is an independent branch from the honeybee Apis group, andApis group divergenced early than Bombus group. Bioinformatics analysis on its sequence and structurereveals PLA2contains a unique hydrophobic N-terminal helix. A model is proposed to illustrate the roleof N-terminal helix recognizing the phospholiqid substrate and interacting with membrane.(2) Prokaryotic expression of PLA2The PLA2gene was amplified by RT-PCR using B. hypocrita venom gland cDNA as template.The PCR product was digested via enzyme BamH I and Xba I, and then inserted into pCold I vector toconstruct the recombinant expression plasmid pCold I-PLA2. The recombinant plasmid was confimedby BamH I and Xba I enzymatic digestion and then transformed into expression strain BL21competentcell. The cell was incubated and then the protein was induced to express via IPTG. The cell was thenassayed by SAS-PAGE and a17kDa band was observed, consistenting with the prediction proteinmolecular weight.(3) Gene expression of PLA2gene in the different tissues and developmental ages of B. hypocritaworkerThe expression profiles of PLA2gene in B. hypocrita’s12different tissues, including foot,antennae, esophageal gland, ovary, fat body, muscle, nerve, trachea, eyes, brain and midgut gland, weredetected by semi-quantitative PCR. The results show that no gene expression was observed in fat body,muscle, nerve, trachea, eyes and brain, little expression in foot, antennae and esophageal glands, andhigh expression observed in ovary, midgut, and venom. In conclusion, the expression of PLA2gene istissue-specific.The relative expression quantities of PLA2in the venom of B. hypocrita worker with different agesas0,5,10,15,20and25days, were detected by qRT-PCR. The results show that the expression of PLA2was observed in different ages of B. hypocrita and significantly distinct from each other day ages.The expression of PLA2in early days is of relatively low, then gradually increased during individualdevelopment, reached a peak at10day age, and then decreased.This study will be useful not only for further studies on the function of Bombus PLA2, but also forfurther studies on the mechanism of bees defense system and developing new reagent of bee venom.