| This study used Apis mellifera carnica as the research object, and gene cloning, prokaryotic expression and real-time PCR as the research methods to analyze the expression level of A. m. carnica ZnT-7-like gene in prokaryotic expression system. Rabbit anti-A m. carnica ZnT-7-like polyclonal antibody was further prepared and mRNA and protein expression levels in3,6,9,12and16days were analyzed in order to find out the expression rule of ZnT-7-like gene in different day-old hypopharyngeal glands, which would provide a theoretical basis for its functional studies In addition, we constructed the fusion expression vector pEGFP-ZnT-7-like containing EGFP reporter gene. Expression and localization of the gene was achieved at the subcellular level. Finally, tissue distribution of this gene was studied by immunohistochemistry techniques.The main results are as follows:1. In this study, a pair of primers was designed in reference to the mRNA sequence of A. m. carnica. ZnT-7-like gene in GenBank database and cDNA was obtained by reverse transcription of hypopharyngeal gland total RNA. Then the obtained cDNA was used as a template for further amplification of the full-length sequence of ZnT-7-like gene. Sequence analysis and homology comparison of CDS and amino acid sequences were conducted using bioinformatics softwares. The results showed that the cloned CDS sequence was identical to that in GenBank databse, which infers that this gene is rather conservative among different subspecies;Secondary structure prediction indicated that this protein was a a-type one; transmebrane structure prediction showed that this protein contained six transmembrane regions; prediction of disulfide bonds found that there might be two disulfide bonds and the the bonding sites might be in50,75,308and315. Phylogenetic analysis based on ZnT-7-like gene sequences showed that, Apis mellifera L. firstly clustered with Apis florea, Bombus terretris and Megachile, which were all Hymenoptera insects, and then clustered with Lepidoptera Bombyx mori, then with mammals like Homo sapiens, Mus musculus and birds like Gallus gallus. Apis mellifera L. showed the farthest kinship with Danio rerio and Brachypodium distachyon.2. Partial sequence (273bp) was chosen as peptide sequence through bioinformatics analysis, then sub-cloned into prokaryotic expression vector pGEX-4T-1and expressed in E. coli BL21(DE3) host cells. Fusion protein was purified and used to immunize New Zealand white rabbits so as to prepare polyclonal antibody. We also detected the sensitivity and specificity of the polyclonal antibody prepared through indirect ELISA and Western blot methods, respectively. The results showed the anti-ZnT-7-like polyclonal antibody showed high sensitivity (1:64,000) and specificity.3. The expression levels of this gene in different day-old hypopharyngeal glands (3,6,9,12,16) were detected by real-rime quantitative RT-PCR method. The results showed that, significant differences existed in transcriptional level among different days of age. The expression level of3-day-old bees was highly significantly higher than that of other ages (P<0.01), the12-day-old bees showed the minimum expression level, significant (P<0.05) or extremely significant differences (P<0.01) existed among6,9and12-day-old bees. Similarly, results of the protein expression level detected by Western blot was similar to that of the transcriptional level.4. The eukaryotic expression vector pEGFP-ZnT-7-like was constructed which contains EGFP reporter gene. pEGFP-ZnT-7-like was transfected into DF-1cells through Liposomes (LTX) and then observed under fluorescence inverted microscope48h later, mRNA and protein expression levels in vitro was detected by RT-PCR and Western blot. ZnT-7-like protein does not have signal peptide sequence. Observation results through fluorescence inverted microscope showed that, the fusion protein was expressed in cytoplasm, while the fluorescent protein transfected with pEGFP-Cl was evenly distributed throughout the whole cell. mRNA and protein expression levels in vitro were significant.5. Hypopharyngeal glands paraffin sections were prepaerd and immunohistochemcial analysis was conducted to detect ZnT-7-like protein positive area ratio in3,6,9,12and16day-old. The results showed that, nucleis in the sections were stained blue, ZnT-7-like protein was brown and the background was clear, which infers that the result was accurate and reliable. Meanwhile, analysis of the OD values showed that the expression rule was consistent with that of the transcription and Western blot. |
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