Transcriptomic analysis of differentially expressed genes in the Ras1(CA)-overexpressed and wildtype posterior silk...

Using the piggyBac-mediated GAL4/UAS transgenic system established in the silkworm, Bombyx mori, we have previously reported that overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland (PSG) improved cell growth, fibroin synthesis, and thus silk yield. However, the detailed molecular mechanism remains to be fully elucidated. To achieve this goal, Illumina sequencing was used in the present study to compare the transcriptomes of the Ras1(CA)-overexpressed and wildtype PSGs.

The transcriptomic sequencing results in 56 million reads following filtering steps. Most of the reads (~70%) are successfully mapped to the Bombyx genome. The mapped reads are situated within at least 9,133 predicted genes, covering 62.46% genes of the Bombyx genome. GO annotation shows that 2512 of the 2,636 differentially expressed genes (DEGs) are mostly distributed in metabolic process, cell and cell part, and binding, and KEGG annotation shows that 1,941 DEGs are mapped into 277 pathways. Importantly, Ras1(CA) overexpression in the PSG upregulated many DEGs distributed in "pathways in cancer", "insulin signaling pathway", and "MAPK signaling pathway" as well as "purine metabolism" and "pyrimidine metabolism". Transcriptional regulation of these DEGs was verified by quantitative real-time PCR. Moreover, injection of small-molecule chemical inhibitors of the Ras1 downstream effectors into the Ras1(CA)-overexpressed silkworms revealed that both Raf-MAPK and PI3K-TORC1 pathways are required for the Ras1-induced DEG expression.

The transcriptomic analysis illustrates that, apart from phosphorylational regulation, Ras1 activates its downstream Raf-MAPK and PI3K-TORC1 pathways at the transcriptional level. Meanwhile, Ras1 increases DNA content and induces endoreplication, at least in part, by upregulating genes in "nucleotide metabolism" and "cell cycle". This study provides further insights into the molecular mechanism of how Ras1(CA) overexpression in the PSG improves silk yield.