Cloning, analysis, and expression, of the Apis mellifera ATG5 gene
Author of the article:WU Bo-Wen1** FAN Xiao-Xue2, 3, 4** LIU Cai-Zhen1 FENG Rui-Rong1 FU Zhi-Ying1 WU Yang1 GUO Rui
Author's Workplace：1. Fujian Vocational College of Agriculture, Fuzhou 350000, China; 2. National & Local United Engineering Laboratory of Natural Biotoxin, Fuzhou 350002, China; 3. Apitherapy Research Institute of Fujian Agriculture and Forestry University, Fuzhou 350002, China; 4. College of Bee Science and Biomedicine, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Key Words：Apis mellifera; autophagy; autophagy related gene 5; molecular characteristics; phyletic evolution; expression pattern
Abstract：
[Aim] To successfully clone, and then determine the
function and expression of the autophagy related gene 5 (AmATG5)
in different tissues and developmental
stages of Apis mellifera,
thereby providing a basis for further investigation of the function of this
gene. [Methods] The CDS of AmATG5 was amplified by PCR followed by
Sanger sequencing. Relevant software was used to predict and analyze its
physicochemical properties, molecular characteristics and protein interaction
network. RT-qPCR was used to ascertain the relative expression of AmATG5
in seven diverse tissue types and seven developmental stages. [Results] The CDS of AmATG5 was
successfully cloned. AmATG5 contains 265 amino acids, has a molecular weight of
approximately 31.41 kD, and a molecular formula of C1428H2168N372O403S13.
AmATG5 includes 31 phosphorylation modification sites and 7 conserved motifs,
but there is no transmembrane structure and signal peptide. AmATG5 mRNA was mainly distributed in
the cytoplasm. AmATG5 and ATG5 in Apis florea clustered into a single
phylogenetic clade. AmATG5 contains 110 random coils, 93 alpha helices, 48
extended strands, and 14 beta turns. AmATG5 has an interaction network with 9
other proteins, such as ATG1 and ATG6. AmATG5
was differentially expressed in 7 different tissues of A. mellifera workers, including the venom gland, midgut, hypopharyngeal gland, fat body,
brain, antenna and cuticle. Its expression level was highest in the brain and
lowest in the cuticle. AmATG5 was continuously down-regulated in
eggs, 1-day-old larvae, 3-day-old larvae and 5-day-old larvae, but continuously
up-regulated in 8-day-old prepupae, 11-day-old pupae and 16-day-old pupae. Its
expression peaked in 16-day-old pupae. [Conclusion] AmATG5
is a hydrophobic, intracellular protein that potentially functions by
interacting with nine other proteins, including ATG6. Its expression varies in
different tissues and developmental stages of A. mellifera workers, and
it appears to be most highly expressed in the brain and in 16-day-old pupae.