[Cloning and comparative analysis of the venom prepromelittin genes from four wasp species]

The total RNAs were isolated from the venom glands of female Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax and Vespa magnifica, respectively. Using the ReverAid First Strand cDNA Synthesis Kit, the first cDNAs were synthesized from the total RNA templates. PCR was carried out for 30 cycles (40s at 94 degrees C, 40s at 52 degrees C and 1 min at 72 degrees C), with a final extension of 10 min at 72 degrees C. RT-PCR products were examined by electrophoresis in 1.5% agarose gels with ethidium bromide staining. After purification using QIAquick PCR Purification Kit, the PCR products were ligated into pGEM*-T easy vector. Positive clones were identified by double digestion of Xho I and Hind III and PCR amplification and then were sequenced. The sequencing results showed that the amplified cDNAs containing the open reading frames of prepromelittin, and their lengths were all 213 bp. The ORFs were potential to encode polypeptides of 70 amino acid residues with predicted molecular weight of 7.7 kDa, including a signal peptide of 21 residues and a promelittin of 49 residues. Comparative analysis showed that the prepromelittins from different wasp species shared more than 93% identities in nucleotide sequences and more than 95% identities in amino acid sequences with each other, respectively. The sequences of prepromelittins of Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax and Vespa magnifica share 94%, 100%, 94%, 93% homologies in nucleotide sequences, and 95.8%, 100%, 97.2% and 97.2% identities in amino acid sequences with those of the European honey bee, Apis mellifera, respectively. There are 67 common amino acid residues among the prepromelittins of the four wasps and two honey bee species, Apis mellifera and Apis cerana. Phylogenetic relationships based on the prepromelitin nucleotide sequences were also examined using the software DNAStar5.01. The result showed that a closer relationship lied in between Polistes hebraeus, Vespa velutina nigrithorax and Vespa magnifica, while Vespula maculifrons had a closer relationship with the honey bees, Apis mellifera and Apis cerana. In conclusion, the prepromelittins were very conserved in the primary structure and the wasp insects also contain the melittins in their venoms, which are very similar to that of honey bee, although they belong to different superfamilies.