Abstract: 【Aim】 In this study, we cloned a zinc transporter 7-like (ZnT-7-like) gene in Apis mellifera carnica in order to prepare the polyclonal antibody against recombinant ZnT-7-like, and to understand ZnT-7-like gene in hypopharyngeal glands of A. m. carnica differentially expressed in different periods. 【Methods】 The ZnT-7-like gene in A. m. carnica was amplified by RT-PCR method from the total RNA of hypopharyngeal glands. To avoid the interference of transmembrane domains, the partial sequence (273 bp) was chosen as peptide sequence through bioinformatics analysis, then sub-cloned into prokaryotic expression vector pGEX-4T-1 and 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. Finally, the expression levels of this gene in different day-old adults were detected by real-rime quantitative RT-PCR and Western blot methods. 【Results】 ZnT-7-like gene was cloned from A. m. carnica , and the obtained fragment was 1 065 bp in length. SDS-PAGE electrophoresis showed that the fusion protein was well expressed. The anti-ZnT-7-like polyclonal antibody showed high sensitivity (1∶64 000) and specificity. There was a big difference in the transcriptional level of ZnT-7-like gene in different day-old A. m. carnica adults. The expression level of 3-day-old bees was extremely significantly higher than that of other ages (P<0.01), the 12-day-old bees showed the minimum expression level, and significant (P<0.05)or extremely significant differences (P<0.01) existed among 6-day-old, 9-day-old and 12-day-old bees. The protein expression level of ZnT-7-like was generally consistent with the transcription level. 【Conclusion】 The ZnT-7-like gene was successfully cloned, the rabbit anti-A. m. carnica ZnT-7-like polyclonal antibody was prepared, and the relative expression of ZnT-7-like in hypopharyngeal glands of different day-old A. m. carnica adults were measured at the transcription and translation levels. These results lay the foundation for further studies on functions of A. m. carnica ZnT-7-like gene.

Key words: Apis mellifera carnica, hypopharyngeal gland, zinc transporter 7-like (ZnT-7-like), gene cloning, antibody preparation, differential expression