Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori

Abstract

BackgroundThrough the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated.ResultsBased on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE). In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae.ConclusionBioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland.

20-hydroxyecdysone upregulatesAtggenes to induce autophagy in the Bombyx fat body

“…Eleven putative Atg genes, including Atg1, Atg3, Atg4, Atg5, Atg6 (ortholog of yeast VPS30 and human BECN1), Atg7, Atg8, Atg9, Atg12, Atg16 and Atg18, have been identified in the Bombyx genome. 30,42 Using the same strategies used in the published reports, we found three additional Atg genes, namely Atg2, Atg11 and Atg13. All three Figure 2.…”

Autophagy and its physiological relevance in arthropods: Current knowledge and perspectives

“…Lack of identification of ATG10 in silkworm, as previously assessed in flies and bees, has been suggested to be due to a possible compensation of ATG10 function by ATG3. 51 Transcripts of the BmATG5, BmATG6 and BmATG8 genes and BmATG8 protein have also been detected in the remodeling midgut (Shi Yanxia, personal communication). The expression patterns and time course of these genes in the midgut were similar to those of BmATG8 and BmATG12 in the silk gland and they reached high levels at the 5 th instar wandering stage.…”

“…52 Expression of the genes for the ubiquitin-like conjugation systems (including BmATG3, BmATG4, BmATG8 and BmATG12), for autophagosome formation (including BmATG1, BmATG6 and BmATG18), and for upstream signal transduction of the autophagy pathway (including homologues of p70S6K, PKB and Rheb) were detected in the silk gland. 51 Full-length cDNAs of BmATG3, BmATG4, BmATG5, BmATG6, BmATG8 and BmATG12 were cloned, and the encoded proteins are homogeneous to their orthologues in other eukaryotes. Lack of identification of ATG10 in silkworm, as previously assessed in flies and bees, has been suggested to be due to a possible compensation of ATG10 function by ATG3.…”

BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation

“…14,17 To date, a number of Atg5 and Atg6 genes have been identified in insects, 28,46,47 but it has never been understood whether they play a role in regulating apoptosis in insects. In Bombyx, BmAtg5, BmAtg6 as well as BmAtg4-like have been cloned, BmAtg5, BmAtg6 but not BmAtg4-like are significantly upregulated by 20E; 28 thus, the functions of BmAtg5 and BmAtg6 in autophagy-mediated apoptosis were studied here.…”