| Bee venom is mainly composed of multiple proteins or peptides. With a wide range of pharmacological functions, it has great prospective in medical fields. Honeybees use their venom to defend their nest from the intruder. The Chinese honeybee Apis cerana cerana is specially distributed in most parts of China, especially in the Mountains areas of South China, and is adapted to the geographic and climate condition. About two to three millions of colonies were bred in China per years, mainly used for honey production and pollination for plants. In order to further exploit the resource of A. cerana, several main venom protein genes have been cloned and expressed in our laboratory. In this paper, we construct a venom gland cDNA library of Chinese honeybees expecting to screen some pharmacological active venom components or other biological significance venom components. At the same time, we studied the expression of melittin and phospholypase A2 gene in bee venom by integrating the gene copy numbers, transcription, post transcription, and translation/post-translation, which is biological significance in venom secretion studies.Two-Dimensional Electrophoresis Analysis of Two Bee species Venom and Construction of the venom gland cDNA library and EST analysisVenom sample from A. cerana and A. meiffera were subjected to two-dimensional electrophoresis analysis under same conditions. The results showed that only 81 identitical components were isolated from both bee species, while the other over 400 spots (components) were compleltely different between the two species. Components of the two species were significantly different. In order to futher explore the diffenence in venom components, mRNA isolated from the venom glands were used to construct a cDNA library using the cDNA library construct kit as the protocol described. The titer of the cDNA library was about 5 ×10~6 pfu/μl. Then the Lamda phage cDNA library was converted into phagemid library, so that we could deal with it in the way same as the plasmid. The clones with different length cDNA inserts were PCR amplified using the universal primer of M13+. and M13-, the amplified fragments were electrophoresed in argarose gel. Clones with different inserted cDNA fragments were selected for sequencing. The cDNA sequences were blasted in NCBI database. By this way, we screened the main venom protein cDNAsequences including phospholypase A2, melitiin, mast cell degranulating peptide, apamine and secapin, and several valuable enzyme genes of the Chinese honeybee such as phosphoglycerate mutase, thioredoxin perxidase, arginene kinase, double-strand-specific ribonuclease, ubiquitin and other structure protein genes. The cDNA library and the EST results provided a platform for the future studys on the Chinese honeybee.Transcription and expression of melittin gene in the venom gland of the Chinesehoneybee, Apis cerana ceranaMelittin is the main component of bee venom, which responsible for 50% of the dry substance. It have various pharmacological functions. The full-length of Ac-melt cDNA sequence screened from the cDNA library was 389 bp, consisting of a 47 bp 5'-UTR (untranslated region), a 213 bp Ac-melt coding region and a 129 bp 3'-UTR. Typical Poly (A) signal was not found at the 3'-UTR region. The coding region of the cDNA was highly conserved, with 93% identity to that of A. meliffera. At the protein level, three amino acid residues sited at 18th, 31st and 61st positions were different from the corresponding residues of A. meliffera, which were Phe, Ala and Gly in A. cerana, Tyr, Pro and Ser in A. meliffera, respectively. Comparison of the DNA sequence with the cDNA sequence revealed that the genomic sequence of Ac-melt consisted of two exons with 164 bp and 202 bp respectively, separated by an intron of 191 bp, which contained the consensus GT—AG borders. The intron sequence from A. cerana was not homologous to that from A meliffera, which was 1200 bp.Southern blotting analysis of the genomic DNA of worker bee was performed. The results showed that only one discrete band wa... |
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