关键词: 西方蜜蜂, AmAGO1, 克隆, 分子特性, 时空表达谱, 原核表达, 多克隆抗体

Abstract:

【Aim】 As a highly conserved protein family in evolution, Argonaute (AGO) family mainly engages in the formation of RNA-induced silencing complex (RISC) in eukaryotes to silence gene expression and further participate in numerous biological processes. Currently, studies on AGO proteins of honey bees are lacking. This study aims to provide the reference and basis for further performing study on function and mechanism of AGO1 of Apis mellifera AmAGO1 by predicting the physicochemical and molecular properties of AmAGO1, analyzing the spatio-temporal expression profiles of AmAGO1, and preparing the polyclonal antibodies against AmAGO1.【Methods】 The coding sequence (CDS) of AmAGO1 of A. mellifera was amplified using PCR amplification followed by predicting the the physicochemical and molecular properties of AmAGO1 protein using bioinformatics. The expression levels of AmAGO1 inthe egg, 3-day-old larva, 7-day-old prepupa, 8-day-old prepupa, 12-day-old pupa, 1-, 2-, 6-, 12-, 15- and 18-day-old adult A. mellifera workers, and in the antenna, hypopharyngeal gland, brain, cuticle, midgut, fat body and venom gland of adult workers were detected by RT-qPCR. After constructing prokaryotic expression plasmid, AmAGO1 fusion protein was induced and its expression form was identified. The polyclonal antibody against AmAGO1 was prepared and the titer, sensitivity and specificity of the antibody were further detected by ELISA, Western blot and immunoprecipitation (IP), respectively. 【Results】The CDS of AmAGO1 was successfully cloned from A. mellifera. AmAGO1 contains 928 amino acids, with the molecular formula C₄₆₂₄H₇₃₃₂N₁₃₁₆O₁₃₂₅S₅₁, the molecular weight of about 104.2 kD, isoelectric point of 9.31, average hydrophilic coefficient of –0.2965, 86 phosphorylation sites, and the typical domains PAZ and PIWI, without typical signal peptides. There was a high amino acide sequence identity of AGO1 proteins of Homo sapiens, Danio rerio, Drosophila melanogaster, Bombyx mori, A. mellifera, A. cerana, and Bombus terrestris. AGO1s from A. mellifera and A. cerana were clustered into one branch, with the highest homology. AmAGO1 was differentially expressed in egg, larva, prepupa, pupa, and adult of A. mellifera. The expression levels of AmAGO1 inthe 3-day-old larva and 7-day-old prepupae were significantly lower than that in the egg, while those in the 8-day-old prepupae and 12-day-old pupae were significantly higher than that in the egg of A. mellifera workers. AmAGO1 was differentially expressed in the 1-, 2-, 6-, 12-, 15-, and 18-day-old adults. The expression levels of AmAGO1 inthe 2-, 6-, 12-, 15- and 18-day-old adults were significantly lower than that in the 1-day-old adults. AmAGO1 was differentially expressed in the antenna, venom gland, brain, midgut, hypopharyngeal gland, fat body and cuticle of worker adults. The expression level of AmAGO1 inthe antenna was significantly higher than those in venom gland, brain, midgut, fat body and cuticle but as the same as that in the hypopharyngeal gland. The expression form of AmAGO1 fusion protein was inclusion body. The prepared AmAGO1 polyclonal antibody had high titer, sensitivity and specificity. 【Conclusion】 AmAGO1 may be a hydrophilic intracellular protein which contains typical PAZ and PIWI domains. AmAGO1 plays a potentially important role in different tissues and developmental stages of A. mellifera workers. AGO1 polyclonal antibody with high titer, high sensitivity, and strong specificity was successfully prepared.

Key words: Apis mellifera, AmAGO1, cloning, molecular properties; spatio-temporal expression profile, prokaryotic expression, polyclonal antibody