DNA Replication Events During Larval Silk Gland Development In The Silkworm,Bombyx Mori

The silkworm is economically important due to the ability to spin silk. The silk gland is the silk protein synthesizing and secreting organ. So the researches on the silk gland have great scientific and practical value. The silk gland cells undergo about10times of division in embryonic period, and form the silk gland contains about1080cells. During larval stage silk gland cells do not divide, the number of cells remains unchanged. This relies on a special kind of cell cycle—the endomitosis. During endomitosis, the G and S cell cycle phases alternate without an intervening mitotic division, leading to an increase in DNA content. During the larval stage,17-19rounds of endomitotic DNA replication occurs in the MSG and PSG, resulting in a217-219fold increase in DNA content, this prepares genetic material for the large and rapid synthesis of silk protein. At present, however, the research about the development of larval silk gland in postembryonic periods is very little, and larval silk gland development process in postembryonic is also unclear. In this study, the DNA synthesis of larval silk gland from2nd to the5th instar larval, and the nutritional conditions and the hormones effects on the DNA synthesis were systematically analyzed. The main results are as follows:1. DNA replication in larval silk glands during the molt phase. Several BrdU positive cells were observed in the MSG and PSG of the molting larval, indicating that the silk glands of molting larval are not entirely quiescent, the endomitosis occurs at low levels during this stage of development.2. DNA replication in the silk glands of intermolt larval. At6h after molting, BrdU positive cells were identified in the MSG and PSG, but identified very few in the ASG. After24h feeding, large numbers of BrdU positive cells were detected in the MSG and PSG, several BrdU positive cells were observed in the ASG. After48h feeding, the results are similar to silk glands24h after feeding. These results show that silk gland endomitosis is gradually activated during the intermolt.3. DNA replication in the silk glands of the5th instar larval. A large number of BrdU positive cells were observed in the MSG and PSG between day1and day5of the5th instar. A few number of BrdU positive cells were observed at day6of the5th instar in the ASG, unlike the small number of BrdU positive cells in the MSG and no BrdU positive cells were detected in the PSG. During the wandering phase, BrdU positive cells were only observed in the ASG, with no positive cells in the MSG and PSG. These results indicate that silk gland cells starting to exit endomitosis during the wandering phase and the arrest of the endomitosis in the silkgland is not at the same time, it continues to the5day of the5th instar in the PSG, and the6day of the5th instar in the MSG. The PSG cells exit from the endomitotic cell cycle before MSG cells during the5th instar.4. The effect of hormones and feeding on DNA synthesis of silk glands. Silk glands of the3rd molting larval were treated with JH and MH in vitro. The results show that there was no significant difference in the number of BrdU positive cells in the JH、MH or JH and MH treated group, compared to the PBS negative control group. This result suggests that silk gland DNA synthesis is not directly regulated by JH and MH. We presume that the endomitosis during the molt is inhibited via a mechanism regulated by many different factors. The4th instar larval were starved for6h,24h,48h and fed for4h after starved for48h, the results show that DNA synthesis was detected in the ASG, MSG and PSG of starved larval6h after molting, indicating that the DNA synthesis is activated by a self-regulating process after molting and does not depend on feeding. When starved for24h, no BrdU positive cells were observed in the MSG or PSG, while a few number of BrdU positive cells were detected in the ASG. BrdU positive cells were not observed in the ASG, MSG or PSG in larval silk glands starved for48h and feeding did not revive endomitosis in starved silk glands, which suggests that feeding is required for endomitosis to continue.5. Cell cycle-related genes expression pattern in silk gland of5th instar larval. Using RT-PCR we investigated the expression pattern of cell cycle-related genes(cdt1、 pcna、cyclinE、cdk2、cdk1) during the5th instar. The results show that the mRNA expression of each cell cycle-related gene decreased and was almost undetectable in the wandering phase at6h and after24h spinning compared with4th molt and from day1to day6of the5th instar. This also indicating that silk gland development completed during this stage, and the silk gland cells start to exit endomitosis on day6of the5th instar.