Molecular cloning, characterization and expression analysis of Atg1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of
ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.
In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript
variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely
related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with
orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic
domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure
for each protein is presented.
We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and
fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last
larval instar.
Our qPCR results revealed that BmATG1 expression levels at the end of larval lifewere comparable in themidgut,
fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of
ATG1 was very different among the anterior, middle and posterior silk glands.
Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in
the fat body of silkworm, inducing an upregulation 24 h after food withdrawal, with only a slight effect in the
midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second
day of spinning phase.