Abstract: 【Aim】 Aquaporin (AQP) is one of the transmembrane proteins widely existing in mammals, plants, and microorganisms, and plays an important role in the processes of cell moisture transportation, selective ion permeability and osmotic balance. This study aims to understand the gene characteristics of AQP1 from Ectropis obliqua Prout, and its subcellular localization based on polyclonal antibody  preparation.【Methods】 The complete cDNA of AQP1 was cloned from E. obliqua by RT-PCR and RACE technologies. The bioinformatics analysis was carried out by online website and edited on the bioinformatics software. The mRNA relative expression levels in different developmental stages and different tissues of the 6th instar larvae of E. obliqua were investigated by real-time quantitative PCR (qRT-PCR). The polyclonal antibody was prepared by immunizing a male New Zealand white rabbit after prokaryotic expression and Ni-sepharose purification. Subcellular localization in Drosophila melanogaster embryonic cells (S2 cells) was observed by fluorescence microscope, and Western blot analysis was performed with the polyclonal antibody of AQP1 from E. obliqua. 【Results】 The AQP1 cDNA was cloned successfully from E. obliqua and named EoAQP1 (GenBank accession no.: KT819587). It is 1 826 bp in length, including a 780 bp of open reading frame (ORF), which encodes 259 amino acids. Phylogenetic tree analysis and alignment of amino acid sequences showed that EoAQP1 is highly conserved with AQP1 proteins of other species of Lepidoptera. Transmembrane structure and water percolating simulation showed that EoAQP1 has a classical water percolation model. The qRT-PCR result revealed that EoAQP1 was expressed in all developmental stages and various tissues of the 6th instar larvae of E. obliqua at different mRNA expression levels. Subcellular localization showed that EoAQP1 aggregated around cell membrane with circular and granular models, but was not expressed in cell membrane, cytoplasm or nuclear membrane. Western blot analysis demonstrated that the obtained polyclonal antibody had high specificity and could be used for further experiments. 【Conclusion】 The nucleotide sequence, bioinformatics and expression profiling of aquaporin 1 gene EoAQP1 in E. obliqua were clarified. With the obtained polyclonal antibody, its subcellular localization of EoAQP1 was preliminarily investigated. These results provide a foundation for further research on the mechanism of water infiltration of EoAQP1.  

Key words: Ectropis obliqua, aquaporin, cloning, bioinformatics, expression patterns, subcellular localization, polyclonal antibody