关键词: 中华蜜蜂, 14-3-3, 分子特征, 表达模式, 蜜蜂球囊菌

Abstract: 【Aim】 This study aims to enrich the basic information of 14-3-3ζ gene of Apis cerana cerana, so as to provide a reference and basis for its further functional study. 【Methods】 The coding sequence (CDS) of 14-3-3ζ gene was amplified by RT-PCR, followed by TA cloning and Sanger sequencing. The physicochemical properties and molecular features of 14-3-3ζ were predicted using the relevant software, and the phylogenetic analysis of 14-3-3ζ was performed. RT-qPCR was used to detect the expression levels of 14-3-3ζ gene in different developmental stages (egg, larva, pre-pupa, pupa and adult), and different tissues (antennae, midgut, fat body, hypopharyngeal gland, brain, cuticle and venom gland) of the newly emerged adult workers of Ap. cerana cerana, as well as in the guts of the 4-, 5- and 6-day-old larvae of Ap. cerana cerana after inoculating the 3-day-old larval workers with Ascosphaera apis.【Results】 The CDS of 14-3-3ζ gene of Ap. cerana cerana was successfully cloned, including 744 nucleotides and encoding 247 amino acids. 14-3-3ζ of Ap. cerana cerana had the molecular weight of about 28.0 kD, included 26 phosphorylation sites, four structural domains and one conserved motif, but had no transmembrane domains and signal peptides. The 14-3-3ζ proteins of Ap. cerana cerana, Ap. mellifera, Ap. laboriosa, Ap. florea, Ceratina calcarata, Bombus pyrosoma, B. terrestris, Megachile rotundata, Osmia lignaria and Habropoda laboriosa all contained four identical conserved motifs and one same structural domain (14-3-3_1). The 14-3-3ζ proteins of Ap. cerana cerana and Ap. mellifera clustered into a single clade on the phylogenetic tree. The expression level of 14-3-3ζ gene in Ap. cerana cerana eggs was significantly higher than those in the 3-day-old larvae, 1-day-old prepupae, 2-day-old prepupae and 4-day-old pupae. The differences in the expression level of 14-3-3ζ gene in various day-old adult workers of Ap. cerana cerana were non-significant. The expression level of 14-3-3ζ gene in the venom gland of the newly emerged adult workers of Ap. cerana cerana was the highest, significantly higher than those in the antennae, midgut, hypopharyngeal gland, brain, cuticle and fat body. Following inoculation of the 3-day-old larval workers of Ap. cerana cerana with As. apis, the expression levels of 14-3-3ζ gene in the 4-, 5- and 6-day-old larval worker guts of Ap. cerana cerana were significantly down-regulated as compared with those in the control group.【Conclusion】 Ap. cerana cerana 14-3-3ζ gene is specifically and highly expressed in the venom gland and egg of worker, and the expression of 14-3-3ζ gene in the larval guts is activated in the process of As. apis infection. 14-3-3ζ is a putative hydrophilic, non-transmembrane and intracellular protein, and highly conserved in Ap. cerana cerana and the above other ten bee species. There is the closest genetic relationship between 14-3-3ζ of Ap. cerana cerana and Ap. mellifera.
Key words: Apis cerana cerana; 14-3-3; molecular features; expression pattern; Ascospaera apis