关键词: 东方蜜蜂, 基质金属蛋白酶, 东方蜜蜂微孢子虫, 表达模式, 分子特征

Abstract: 【Aim】 This study aims to clone the matrix metalloproteinase-14 gene AcMMP14 in Apis cerana and to analyze its molecular characteristics and expression pattern, so as to offer the reference and basis for continuous and further functional study of AcMMP14. 【Methods】 Total RNA from the 6-day-old larval gut of A. cerana workers was extracted, and the coding sequence (CDS) of AcMMP14 was amplified by PCR. Relevant bioinformatic software was employed to predict the physicochemical property and molecular characteristics of AcMMP14, to identify structural domains and conserved motifs in MMP14s from A. cerana and other bee species, followed by phylogenetic analysis. RT-qPCR was utilized to detect the relative expression levels of AcMMP14 in egg, larva, prepupa, pupa and different day-old adult workers, and antenna, brain, cuticle, fat body, venom gland, midgut and hypopharyngeal gland of the adult workers, and the relative expression levels of AcMMP14 in the midgut of the newly emerged 1-day-old adult A. cerana workers at 1-4 d post inoculation with Nosema ceranae. 【Results】 AcMMP14 has a molecular formula of  C₃₀₆₈H₄₅₈₁N₈₀₃O₉₁₅S₂₀, a molecular weight of about 68.00 kD, a liposoluble coefficient of 64.12, a theoretical isoelectric point of 5.85, and an average hydrophilic coefficient of -0.47. AcMMP14 contains one signal peptide and 36 phosphorylation sites, and can be simultaneously located in mitochondria, nucleus and cytoplasm. Three same structural domains and five same conserved motifs were included in MMP14 proteins from A. cerana and other 10 bee species. MMP14 of A. cerana and A. dorsata had the amino acid sequence identity of 94.23% and clustered into one clade on the phylogenetic tree. AcMMP14 was differentially expressed in egg, 3-day-old larva, 1- and 2-day-old prepupae and 4-day-old pupa of A. cerana workers, and the expression level of AcMMP14 was the highest in the 4-day-old pupa and significantly higher than those in the 3-day-old larva and 2-day-old prepupa. AcMMP14 was differentially expressed in different day-old adult workers, and the expression level of AcMMP14 was the highest in the 15-day-old adult and significantly higher than those in the 1-, 2-, 6-, 12- and 17-day-old adult. AcMMP14 was differentially expressed in the seven tissues of adult workers, and the expression level of AcMMP14 was the highest in the antenna and significantly higher than those in the brain, cuticle, fat body, venom gland, midgut and hypopharyngeal gland. The expression levels of AcMMP14 in workers’ midguts of adults at 1 and 2 d post inoculation with N. ceranae were significantly down-regulated, while those in workers’ midguts of adults at 3 and 4 d post inoculation with N. ceranae were down-regulated without significant difference as compared with those of the control group. 【Conclusion】 AcMMP14 is a potential hydrophilic and secretary protein. AcMMP14 plays a putative key role at different developmental stages and in different adult tissues of A. cerana workers. AcMMP14 in the adult worker’s midgut is activated and expressed at early stage of the 1st proliferation cycle of N. ceranae.

Key words: Apis cerana, matrix metalloproteinase; Nosema ceranae, expression pattern, molecular characteristics