| Bombyx mori is one of the important model organisms for Lepidoptera insects, and is an important economic insect due to produce silk. The silk gland is a silk-producing organ, and is a special organ of the silkworm larvae. The silk gland cells undergo about ten cycle of mitosis during embryonic phase. And the silk gland contains approximately 1080 cells and can be divided into three anatomically and physiologically distinct regions:the anterior region, the middle region and the posterior region. At larval stages, the silk gland cells do not undergo cell division but DNA replication continues and make them larger, this unique cell cycle is termed endomitosis. The gland cells undergo an accumulation of approximately 4000000 times the haploid genomic content per nuclei. In the fifth instar of silkworm larvae, silk gland nuclei filled with the cytoplasmic region, which makes the nucleus fully contact with the cytoplasm and increase their exchange. Consequently, this accumulation may be beneficial for a rapid increase in the synthesis of silk proteins. With the completion of the silkworm genome and transcriptome sequencing, many researchers have focused on the mechanism of silk protein biosynthesis. Frequent endomitosis in silk gland cells is the premise of silk protein synthesis, however, the endomitotic mechanism of silk gland cells is unclear. The time span of endomitosis and high efficient DNA synthesis in silk gland cells are most specific and efficient in the known natural organism. Elucidation of the endomitotic mechanism of silk gland cells will provide important theoretical basis for improving the yield of silk and the development in sericulture, and provide important clues for the improvement of the mechanism of cell cycle regulation.We investigated detailedly the changes of endomitotic DNA synthesis in silk gland cells. We analyzed the effects of ecdysone hormone, juvenile hormone, nutrition and insulin on DNA synthesis of silk gland cells, which further elucidate the regulation of physiological factors on endomitosis of silk gland cells, and explore the regulatory mechanism. These researches laid the foundation for the analysis of the endomitotic mechanism in silk gland cells. The main results and conclusions are as follows:1. A profile of DNA synthesis of the silk glands in the silkworm larvae phaseUsing the 5’-bromo-2’-deoxyuridine (BrdU) labeling technique, we investigated changes in endomitotic DNA synthesis in silk gland cells from the third molting stage to the spinning phase. Our results showed that endomitotic DNA synthesis of silk gland cells is fluctuated during larval development. Endomitotic DNA synthesis is self-activated in the freshly ecdysed larvae, increases during feeding phase and gradually decreases during the next molting phase. The rate of DNA synthesis swings up as larvae grow up. In particular, the DNA synthesis activity was the highest in day 3 of fifth instar. The endomitotic DNA synthesis in PSG cells is inhibited firstly, and followed by the middle silk gland and the anterior silk gland. Developmental profiles of the cell cycle-related genes are consistent with developmental change in endomitotic DNA synthesis in the silk gland cells. This trend showed that DNA synthesis of silk gland cells is related to silkworm larval development. Therefore, we hypothesized that ecdysone, juvenile hormone and nutrition may regulate endomitosis of the silk gland cells.2. Effects of hormones and starvation on endomitosis of silk gland cellsBased on the above results, the fourth larval instar is the best time to define the molecular mechanisms that regulates the endomitotic cell cycle in the silk gland cells. Using BrdU labeling technology, endomitotic DNA synthesis of the silk gland cells is inhibited by in vivo treatment with 20E, however, there are no effects of in vitro treatment with 20E on DNA synthesis of the silk gland cells. JH had no effect within a 24h period either in vivo or in vitro, sugget that JH can not activate DNA synthesis of the silk gland cells. The above results showed that DNA synthesis of the silk gland cells is inhibited by 20E in vivo, indicating that endomitotic DNA synthesis of the silk gland cells is inhibited in molt phase due to the regulation of ecdysone.Endomitotic DNA synthesis was dramatically decreased upon starvation, and was re-activated by re-feeding, and was inhibited again by starvation. After starvation, the content of trehalose and amino acids in silkworm larval haemolymph is changed. Therefore, we injected amino acids, trehalose, glucose or a mixture of the three into the silkworm larvae that starved, and found that DNA synthesis was not re-activated by them, indicating that the single nutrient factor can not stimulate DNA synthesis of silk gland cells. The above results showed that DNA synthesis of silk gland cells is greatly actived in the intermolt phase due to silkworm larvae eat a lot of mulberry leaves.3. Regulation of insulin signal on endomitosis of the silk gland cellsUsing in vivo and in vitro treatment and BrdU labeling technology, we found that bovine insulin can stimulate DNA synthesis. We ascertained that the PI3K pathway, TOR pathway and ERK pathway are involved in DNA synthesis in endomitotic silk gland cells using their specific inhibitors. However, the PI3K/Akt and TOR pathways are involved in the insulin-stimulate the DNA synthesis. The insulin can not stimulate the ERK pathway, which appears to be activated by other factor(s). Using Western blot analysis, the PI3K/Akt and TOR pathways were inhibited by injection of 20E and starvation, suggested that the insulin signaling is involved in the regulation of 20E and starvation on DNA synthesis in silk gland cells. 4. Quantitative proteomic analysis of differentially expressed proteins in silk gland between the molting phase and glutonous phaseWe identified the differentially expressed proteins between the molt phase and glutonous phase by iTRAQ labeling technology. We identified 4170 proteins, and found that 181 proteins are expressed differentially. The protein expression profile in silk gland at glutonous phase was Compared to that at the molt phase,71 proteins are higher expressed and 110 proteins are lower expressed in glutonous phase. GO analysis for the differentially expressed proteins showed that there are many changes in molecular function and biological process. KEGG Pathway enrichment analysis for the differentially expressed proteins showed that there are 115 different biochemical pathways and signaling pathway, including the insulin signaling pathway, suggested that the physiology and biochemistry of silk gland cells have been changed greatly between the molt phase and glutonous phase.We firstly screened 15 signaling pathways regarding the regulation of cell cycle and analyzed the differentially expressed genes. We further study the biggest different gene, which number is BGIBMGA005826 in the silkworm genome database. Using GO analysis and KEGG Pathway enrichment analysis, this gene is involved in the metabolism of lipids and associated with cancer. Using silkworm microarray data analysis and qPCR analysis, the expression of this gene is higher in silk gland of day 3 fifth instar, and is consistent with the changes of DNA synthesis of silk gland in silkworm larvae. Therefore, we speculate that this gene is related to the regulation of endomitosis of silk gland cells. This gene contains a conserved domain of insect type alcohol dehydrogenase (ADH)-like family, belongs to the superfamily of short chain dehydrogenase, which contain four active sites and 19 NAD(P) binding sites, so we named it BmADH and speculate that this gene is related with energy metabolism. Following, we present a detailed study of the function of BmADH. Immunofluorescent assay showed that BmADH gene localized in the cytoplasm. DNA synthesis is inhibited by Interference of BmADH in embryo cells of Bombyx mori, and the 4E-BP phosphorylation is decreased, suggested that TOR signaling pathway is involved in regulation of BmADH on DNA synthsis of silk gland. Using qPCR and Western blot analysis, the expression of BmADH is decreased by injection of 20E and starvation, suggested the expression of BmADH is regulated by ecdysone and nutrition. The above results confirmed the effects of 20E, starvation and insulin signal on DNA synthesis of silk gland, and it provides a new direction for further study of the endomitotic mechanism of silk gland cells. |
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