Molecular characterization and gene expression pattern of CRQ in Apis cerana

The aim of this study is to provide a reference and basis for the functional study of croquemort gene in Apis cerana (AcCrq) by examining the spatiotemporal expression profile of AcCrq gene and analyzing the physicochemical properties and molecular characteristics of AcCRQ protein.

The coding sequence (CDS) of AcCRQ was amplified by RT-PCR. The physicochemical and molecular properties of the AcCRQ protein were predicted using the relevant software, and the conserved motifs and structural domains of AcCrq from A. cerana and other species were identified, and the amino acid sequences were compared and a phylogenetic tree was constructed.

RT-qPCR was performed to detect the relative expression levels of AcCrq in various tissues (antennae, midgut, brain, hypopharyngeal glands, fat bodies, venom glands and epidermal tissues) of worker bees, as well as in eggs, larvae, pre-pupae, pupae and adults of different day-old worker bees of A. cerana. Successfully validated to AcCrq, CDS contained 1 719 nucleotides, encoding a total of 572 amino acids. AcCRQ protein has a molecular weight of about 65.44 kDa, a molecular formula of C₂₉₆₉H₄₅₅₄₀N₇₆₄O₈₆₄S₂₁, an isoelectric point of 5.96, an average hydrophilicity coefficient of -0.30, and contains 23 serine phosphorylation sites, 9 tyrosine phosphorylation sites, and 17 threonine phosphorylation sites, but does not have a typical signal peptide. AcCRQ has high amino acid sequence consistency in Apis laboriosa, Apis florea, Apis mellifera, Eufriesea mexicana, Habropoda laboriosa, Osmia bicornis bicornis and Osmia lignaria; A. cerana has the closest CRQ evolutionary distance from A. florea and A. mellifera. AcCrq was expressed in the antennae, midgut, brain, hypopharyngeal glands, fat body, venom glands and epidermis of adult worker bees, but there were differences in the amount of expression, and the amount of expression in the antennae was significantly higher than that in other tissues; AcCrq was expressed in the eggs, larvae, prepupae and pupae of A. cerana workers, but there were differences in the amount of expression. Compared with the expression in eggs, the expression of AcCrq in 3-day-old larvae and 8-day-old prepupae was significantly lower, while the expression of AcCrq in 7-day-old prepupae and 12-day-old pupae was down-regulated, but not significantly; AcCrq was expressed in 1-, 2-, 6-, 12-, 15-, and 17-day-old workers, but there were differences in the amount of expression. The expression of AcCrq in 1-day-old adult was significantly higher than that in other day-old adults, and the expression level of AcCrq in 2- and 17-day-old adults was lower than that in other day-old adults. AcCRQ is a putative hydrophylic but not secretory, CRQ among A. cerana and eight other bees are highly conservative. A. cerana, A. florea and A. mellifera have the closet phyletic evolution of CRQ.

AcCrq was dynamically and differentially expressed in different tissues and developmental stages of A. cerana worker bees.